Science, Faculty of
Botany, Department of
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UBCV
Kuo, Chak Chung
Master of Science - MSc
University of British Columbia
Lignin is a rigid, hydrophobic polymer that strengthens the cell wall of supportive fibres and water-conducting vessels in vascular plants. Four main stages of lignification include: (1) monolignol production in the cytosol via the phenylpropanoid pathway, activated by MYB58 and MYB63 transcription factors; (2) monolignol diffusion into the cell wall; (3) monolignol radicalization by laccases (LACs) and/or peroxidases (PRXs); and (4) radical coupling of monolignols into polymeric lignin. Disrupting monolignol production or mutating oxidative enzymes often leads to improper plant development or dwarfism, suggesting that the quantity and location of lignin in the cell wall must be tightly regulated. It remains unclear how plants balance monolignol production and oxidative enzyme activity to coordinate proper lignification and plant development. In this study, Arabidopsis plants that overexpress MYB63 in xylem vessels and interfascicular fibres have increased stem lignin content without growth penalty. This suggests that a moderate increase in metabolic flux through the phenylpropanoid pathway leads to hyperlignification in lignifying cell types that possess the necessary molecular machinery for lignification. To understand how oxidative enzymes in distinct cell wall domains contribute to lignification, higher order mutants of LACs and PRXs were created with CRISPR/Cas9. Mutating genes encoding secondary cell wall-localized LAC4, LAC17, and PRX72 led to more irregular vessels and less stem lignin than the lac4-2 lac17 double mutant without additional growth penalty; mutating genes encoding cell corner/middle lamella-localized LAC4 and PRX64 led to shorter stems compared to the lac4-2 single mutant. This suggests that vessel irregularity does not contribute to lignin modification-induced dwarfism. The ability to modulate the quantity and distribution of lignin without growth penalty will provide insights into engineering low-lignin plants for more efficient conversion of cellulose to biofuels, and high-lignin plants for valuable phenolic-derived bioproducts.
DISSECTING THE ROLES OF MONOLIGNOL SUPPLY AND OXIDATIVE ENZYMES IN ARABIDOPSIS THALIANA LIGNIFICATION by Chak Chung Kuo B.Sc., The University of British Columbia, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Botany) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2023 © Chak Chung Kuo, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Dissecting the roles of monolignol supply and oxidative enzymes in Arabidopsis thaliana lignification submitted by Chak Chung Kuo in partial fulfilment of the requirements for the degree of Master of Science in Botany Examining Committee: Dr. Anne Lacey Samuels, Professor, Department of Botany, UBC Supervisor Dr. Shawn Mansfield, Professor, Departments of Botany and Wood Science, UBC Supervisory Committee Member Dr. Simone Castellarin, Professor, Wine Research Centre, UBC Supervisory Committee Member Dr. Abel Rosado, Associate Professor, Department of Botany, UBC Additional Examiner iii Abstract Lignin is a rigid, hydrophobic polymer that strengthens the cell wall of supportive fibres and water-conducting vessels in vascular plants. Four main stages of lignification include: (1) monolignol production in the cytosol via the phenylpropanoid pathway, activated by MYB58 and MYB63 transcription factors; (2) monolignol diffusion into the cell wall; (3) monolignol radicalization by laccases (LACs) and/or peroxidases (PRXs); and (4) radical coupling of monolignols into polymeric lignin. Disrupting monolignol production or mutating oxidative enzymes often leads to improper plant development or dwarfism, suggesting that the quantity and location of lignin in the cell wall must be tightly regulated. It remains unclear how plants balance monolignol production and oxidative enzyme activity to coordinate proper lignification and plant development. In this study, Arabidopsis plants that overexpress MYB63 in xylem vessels and interfascicular fibres have increased stem lignin content without growth penalty. This suggests that a moderate increase in metabolic flux through the phenylpropanoid pathway leads to hyperlignification in lignifying cell types that possess the necessary molecular machinery for lignification. To understand how oxidative enzymes in distinct cell wall domains contribute to lignification, higher order mutants of LACs and PRXs were created with CRISPR/Cas9. Mutating genes encoding secondary cell wall-localized LAC4, LAC17, and PRX72 led to more irregular vessels and less stem lignin than the lac4-2 lac17 double mutant without additional growth penalty; mutating genes encoding cell corner/middle lamella-localized LAC4 and PRX64 led to shorter stems compared to the lac4-2 single mutant. This suggests that vessel irregularity does not contribute to lignin modification-induced dwarfism. The ability to modulate the quantity and distribution of lignin without growth penalty will provide insights into engineering low-lignin plants for more efficient conversion of cellulose to biofuels, and high-lignin plants for valuable phenolic-derived bioproducts. iv Lay Summary To stand tall and absorb water efficiently from the ground, plants deposit a rigid polymer called lignin in the walls surrounding specialized cells. Lignin precursors called monolignols are first produced inside the cells and moved to the cell walls where they assemble into lignin. Plants make just enough lignin to strengthen themselves because monolignols are energy intensive to produce and toxic in high concentrations. Genetically engineered plants with increased amounts of monolignol-producing enzymes have more lignin, while plants with decreased amounts of lignin-assembling enzymes have less lignin. In both cases, the plants did not show major growth defects, demonstrating the ability to control how much lignin gets made without compromising plant growth. This lends insight into how we can engineer plants to enhance the production of renewable biofuels and/or bioproducts. v Preface Chapter 1 is modified from my Master’s thesis proposal and contains background information, motivation, and objectives of this thesis. Chapter 2 is primarily my own work. I cloned all promoter entry clones and incorporated them into the destination vector with the MYB63 entry clone prepared by Dr. Mathias Schuetz (Perkins et al., 2020). I analyzed all data. Fabrizio Chow performed qRT-PCR analysis on T1 plants and contributed to genotyping under my supervision for the BIOL 448 Directed Studies course. Dr. Faride Unda and Dr. Xinyi Huang provided training on the Agilent UHPLC system. Standards of coniferin and syringin were gifted by Dr. Sally Ralph (USDA Forest Products Laboratory, Madison, Wisconsin). Dr. Tao Huan, Alyssa Hui, and Brian Low assisted with mass spectrometry analysis of soluble phenolics. Madison Kaplen provided training on the acetyl bromide technique. Chapter 3 is primarily my own work. I cloned all Cas9 constructs using the pBEE401 destination vector modified from Wang et al. (2015), gifted by Dr. Yuelin Zhang (University of British Columbia). I created higher order mutants and screened for CRISPR/Cas9-induced mutations. I analyzed all data. The lac4-2 single mutant and the lac4-2 lac17 double mutant (Berthet et al., 2011) were provided by Dr. Richard Sibout (National Institute for Agricultural Research, France). Chantelle So and Maysa Nikzad assisted with genotyping and seed harvesting. Madison Kaplen provided training on the acetyl bromide technique. Chapter 4 summarizes major findings of my thesis work and describes future research directions. vi Table of Contents Abstract ........................................................................................................................... iii Lay Summary ..................................................................................................................iv Preface ............................................................................................................................ v Table of Contents ............................................................................................................vi List of Tables ................................................................................................................... x List of Figures ..................................................................................................................xi List of Abbreviations ...................................................................................................... xiii Acknowledgments ..........................................................................................................xv 1 Introduction .............................................................................................................. 1 1.1 Primary and secondary cell walls in land plants ................................................. 1 1.2 Biological and economic importance of lignin..................................................... 2 1.3 Biosynthesis of monolignols, sinapoyl esters, and lignin .................................... 2 1.4 Monolignol supply and export to the cell wall ..................................................... 6 1.5 Genetic regulation of monolignol biosynthesis ................................................... 7 1.6 Oxidative enzymes: fine-tuning lignification ....................................................... 7 1.7 Knowledge gap .................................................................................................. 9 1.7.1 Contributions of monolignol availability and oxidative enzymes to lignification ............................................................................................................... 9 1.7.2 Lignin modification-induced dwarfism ........................................................ 10 1.7.3 Lignin’s mechanical role in specific cell wall domains ................................ 10 1.8 Experimental system and research objectives ................................................. 11 2 MYB63 expression driven by vessel- and fibre-specific promoters increases stem lignin without growth penalty ......................................................................................... 12 2.1 Introduction ...................................................................................................... 12 vii 2.1.1 MYB58 and MYB63 transcription factors regulate the biosynthesis of phenolic compounds and the associated stress responses ................................... 12 2.1.2 MYB63 overexpression enhances plant stress and compromises growth . 13 2.1.3 Choosing cell type-specific promoters to upregulate monolignol biosynthesis in lignifying and non-lignifying cell types ............................................ 18 2.2 Results ............................................................................................................. 20 2.2.1 Cell type-specific promoters drive MYB63 overexpression in rosette leaves and inflorescence stems ........................................................................................ 20 2.2.2 Ectopic MYB63 expression in stems can lead to delayed stem growth ..... 22 2.2.3 Cell type-specific MYB63 overexpression has differential impacts on the soluble phenolic compositions in the stems ........................................................... 24 2.2.4 MYB63 expression driven by vessel- and interfascicular fibre-specific promoters led to increased lignin in stems ............................................................. 28 2.2.5 Histochemical staining of proVND7::MYB63 and proPRX64::MYB63 stem cross-sections did not reveal changes in the spatial distribution of lignin ............... 31 2.3 Discussion ........................................................................................................ 32 2.3.1 Lignifying cell types have the capacity for additional lignification ............... 32 2.3.2 Monolignol availability and oxidative enzyme activity coordinate lignification .................................................................................................................. 33 2.3.3 MYB63 overexpression in non-lignifying cell types induces dwarfism ....... 34 2.4 Methods ........................................................................................................... 35 2.4.1 Plant growth conditions.............................................................................. 35 2.4.2 Cloning cell type-specific promoter::MYB63 constructs ............................. 36 2.4.3 Arabidopsis transformation with Agrobacterium floral dip .......................... 38 2.4.4 Selection of plant transformants ................................................................ 38 2.4.5 RNA extraction, cDNA synthesis, and quantitative real-time PCR ............. 39 2.4.6 Liquid chromatography-mass spectrometry (LC-MS) ................................ 39 viii 2.4.7 Acetyl bromide soluble lignin (ABSL) quantification ................................... 41 2.4.8 Stem cross-sectioning, staining, and imaging ............................................ 41 2.4.9 Statistical analysis ..................................................................................... 42 3 Targeted lignin removal in the secondary cell wall leads to increased vessel irregularity without additional growth penalty ................................................................. 43 3.1 Introduction ...................................................................................................... 43 3.1.1 Laccases and peroxidases control lignin quantity and composition in distinct cell wall domains ........................................................................................ 43 3.1.2 Biomechanical roles of lignin in distinct cell wall domains ......................... 44 3.1.3 Genetic strategies for creating higher order oxidative enzyme mutants .... 45 3.2 Results ............................................................................................................. 46 3.2.1 CRISPR/Cas9 generates indel mutations in PRX64, PRX71, and PRX72 46 3.2.2 lac4-2 lac17 prx72 triple mutants are not phenotypically different from the lac4-2 lac17 double mutant, while the lac4-2 prx64 double mutant showed delayed stem growth ........................................................................................................... 49 3.2.3 lac4-2 lac17 prx72 triple mutants showed a slight decrease in stem lignin compared to the lac4-2 lac17 double mutant ......................................................... 52 3.2.4 Histochemical staining of lac4-2 prx64 and lac4-2 lac17 prx72 stem cross-sections showed altered lignin composition ........................................................... 54 3.2.5 Xylem vessels in lac4-2 lac17 prx72 triple mutants were more irregularly shaped than in the lac4-2 lac17 double mutant ...................................................... 56 3.3 Discussion ........................................................................................................ 58 3.3.1 Targeted lignin removal from distinct cell wall domains has unique phenotypic outcomes ............................................................................................. 58 3.3.2 PRX72 compensates for lac4 lac17 mutations in seedlings but not stems 59 3.3.3 Increased vessel irregularity and reduced lignin do not contribute directly to additional growth penalty ....................................................................................... 59 ix 3.3.4 Perturbing lignification in the interfascicular fibres may hinder plant growth and development .................................................................................................... 60 3.4 Methods ........................................................................................................... 62 3.4.1 Plant growth condition ............................................................................... 62 3.4.2 Designing and cloning CRISPR/Cas9 constructs ...................................... 62 3.4.3 Arabidopsis transformation with Agrobacterium floral dip .......................... 63 3.4.4 Selection of plant transformants and screening for gene mutations .......... 63 3.4.5 RNA extraction, cDNA synthesis, and quantitative real-time PCR ............. 64 3.4.6 Acetyl bromide soluble lignin (ABSL) quantification ................................... 64 3.4.7 Stem cross-sectioning, staining, and microscopy ...................................... 64 3.4.8 Cell wall thickness and curvature analysis................................................. 65 3.4.9 Statistical analyses .................................................................................... 65 4 Conclusion ............................................................................................................. 66 4.1 Major findings ................................................................................................... 66 4.1.1 Dwarfism due to MYB63 overexpression is triggered in non-lignifying cells .. .................................................................................................................. 66 4.1.2 Dwarfism associated with lignin deficiency is not due to vessel irregularity ... .................................................................................................................. 67 4.2 Future directions .............................................................................................. 68 4.2.1 Establishing a system in woody gymnosperms and angiosperms to study and manipulate lignification .................................................................................... 68 4.2.2 Connecting MYB63 overexpression in non-lignifying cell types and lignin modification-induced dwarfism ............................................................................... 68 4.2.3 Probing biomechanical contribution of lignin in different cell wall domains 70 References .................................................................................................................... 71 Appendices ................................................................................................................... 86 x List of Tables Table 2.1. Confirmed and putative Arabidopsis genes involved in the biosynthesis of phenylalanine (shikimate pathway), monolignols, monolignol glucosides, sinapoyl esters (phenylpropanoid and monolignol biosynthetic pathways), and lignin (oxidative enzymes). ........................................................................................... 14 Table 2.2. Arabidopsis promoter elements and the experimental evidence for their cell type-specific expression. .................................................................................... 19 Table 2.3. Quantification of sinapoyl malate in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. 27 Table 2.4. Quantification of acetyl bromide soluble lignin (ABSL) in dried, acetone-insoluble, top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. ......................................................... 30 Table 3.1. Quantification of acetyl bromide soluble lignin (ABSL) in dried, acetone-insoluble, top 20 cm primary inflorescence stems of 6-week-old T3 lac4-2 lac17 prx72 plants. ....................................................................................................... 53 Supplementary Table 1. List of primers used in this thesis work. .................................. 86 Supplementary Table 2. Thermal cycles for PCR, cloning, and qRT-PCR. ................... 89 xi List of Figures Figure 1.1. Metabolic pathways that produce phenolic compounds in Arabidopsis. ........ 5 Figure 2.1. Wild-type Arabidopsis stem cross-section stained with toluidine blue. False colours show distinct cell types that can be targeted with cell type-specific promoters. .......................................................................................................... 19 Figure 2.2. Ectopic MYB63 expression driven by cell type-specific promoters was detected in rosette leaves. .................................................................................. 20 Figure 2.3. MYB63 overexpression driven by cell type-specific promoters was detected in inflorescence stems. ....................................................................................... 22 Figure 2.4. Stem heights (in cm) of 4- and 6-week-old segregating T2 cell type-specific promoter::MYB63 plants from three independent experiments (4-5 plants per line in each experiment). ........................................................................................... 23 Figure 2.5. Correlation between mean stem heights (in cm) and relative MYB63 expression levels (fold change from wild-type Col-0) in the top 20 cm primary inflorescence stems of 6-week-old segregating T2 cell type-specific promoter::MYB63 plants. .................................................................................... 24 Figure 2.6. Quantification of sinapoyl malate in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. 26 Figure 2.7. Quantification of acetyl bromide soluble lignin (ABSL) in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. .................................................................................... 29 Figure 2.8. UV autofluorescence and histological staining of proVND7::MYB63 and proPRX64::MYB63 stem cross sections. ............................................................ 32 Figure 2.9. Construction of MYB63 plant expression clones driven by cell type-specific promoters using Invitrogen MultiSite Gateway™ BP and LR recombination reactions. Cell type-specific promoter::MYB63 expression clones created in this thesis work. ......................................................................................................... 37 Figure 3.1. Arabidopsis laccase and peroxidase mutants and predicted amino acid sequences of mutant proteins. ............................................................................ 47 Figure 3.2. Preliminary quantification of LAC4, LAC17, and PRX72 expression levels in lac4-2 lac17 prx72 plants. ................................................................................... 49 xii Figure 3.3. Phenotypes and stem heights (in cm) of 4- and 6-week-old T3 lac4-2 lac17 prx72 plants. ....................................................................................................... 50 Figure 3.4. Phenotypes and stem heights (in cm) of 4- and 6-week-old T3 lac4-2 prx64 plants. ................................................................................................................. 51 Figure 3.5. Quantification of acetyl bromide soluble lignin (ABSL) in the top 20 cm primary inflorescence stems of 6-week-old T3 lac4-2 lac17 prx72 plants. .......... 53 Figure 3.6. UV lignin autofluorescence and histological staining of lac4-2 prx64 and lac4-2 lac17 prx72 stem cross sections. ............................................................. 56 Figure 3.7. Measures of xylem vessel irregularity from stem cross-sections of lac4-2 lac17 prx72 triple mutants. .................................................................................. 57 xiii List of Abbreviations 4CL 4-coumarate:CoA ligase ABSL acetyl bromide soluble lignin ADT arogenate dehydratase APX ascorbate peroxidase BRT bright trichomes C unit (lignin) catechyl unit C3'H p-coumaroyl shikimate 3'-hydroxylase C3H p-coumarate 3-hydroxylase C4H cinnamate 4-hydroxylase CAD cinnamyl alcohol dehydrogenase Cas9 CRISPR-associated protein 9 (Streptococcus pyogenes) CCoAOMT caffeoyl-CoA 3-O-methyltransferase CCR cinnamoyl-CoA reductase cDNA complementary DNA CER eceriferum CM chorismate mutase COMT caffeic acid/5-hydroxyferulic acid O-methyltransferase CRISPR clustered regularly interspaced short palindromic repeats CS chorismate synthase CSE caffeoyl shikimate esterase CYP cytochrome P450 protein DAD diode array detector DAHPS 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase DAPI 4',6-diamidino-2-phenylindole DAP-seq DNA affinity purification sequencing DHQD-SDH dehydroquinate dehydratase-shikimate dehydrogenase DHQS 3-dehydroquinate synthase DNA deoxyribonucleic acid EF1-α elongation factor 1, alpha subunit ELI ELICITOR EPSPS 5-enolpyruvylshikimate-3-phosphate synthetase F5H/FAH ferulate 5-hydroxylase / ferulic acid 5-hydroxylase G unit (lignin) guaiacyl unit gRNA guide RNA GUS β-glucuronidase H unit (lignin) p-hydroxyphenyl unit HCALDH hydroxycinnamaldehyde dehydrogenase HCT hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase IRX irregular xylem xiv LAC laccase LMID lignin modification-induced dwarfism MED MEDIATOR MES 2-(N-morpholino)ethanesulfonic acid MYB MYELOBLASTOSIS family transcription factor NST NAC secondary wall thickening promoting factor PAL phenylalanine ammonia lyase PCR polymerase chain reaction PDT prephenate dehydratase PPA-AT prephenate aminotransferase PPY-AT phenylpyruvate aminotransferase pro promoter PRX peroxidase qRT-PCR quantitative real-time PCR REF reduced epidermal fluorescence RNA ribonucleic acid RNA-seq RNA sequencing S unit (lignin) syringyl unit S/G ratio syringyl-to-guaiacyl ratio SCR SCARECROW SD standard deviation SEM standard error of the mean SGT sinapate:UDP-glucose glucosyltransferase SK shikimate kinase SMT sinapoyl glucose:malate sinapoyltransferase SND secondary wall-associated NAC domain transcription factor SNG sinapoyl glucose accumulator SUC sucrose transporter T-DNA transfer DNA (Agrobacterium tumefaciens) TAT tyrosine aminotransferase UGT UDP-glycosyltransferase UHPLC-MS ultra-high-performance liquid chromatography-mass spectrometry UV ultraviolet VND vascular-associated NAC domain transcription factor WND wood-associated NAC domain transcription factor WRKY WRKYGQK motif transcription factor xv Acknowledgments I would like to thank Dr. Lacey Samuels, committee members Dr. Shawn Mansfield and Dr. Simone Castellarin, and Dr. Geoffrey Wasteneys who offered great mentorship and advice; previous and current members of the Samuels Lab, Wasteneys Lab, and Mansfield Lab who have supported me throughout my undergraduate and graduate studies; staff members at the Bioimaging Facility (Dr. Miki Fujita, Dr. Eun Kyoung Lee), undergraduate students (Fabrizio Chow, Chantelle So, Brianna Archibald, Maysa Nikzad), and collaborators (Dr. Tao Huan, Alyssa Hui, Brian Low) who have assisted with my research; and UBC Spring Graduate Awards for research funding. Finally, I would like to thank my family and friends for always being there for me. 1 1 Introduction This thesis investigates key factors contributing to the deposition of the lignin biopolymer during secondary cell wall formation in vascular plants. I begin by considering the importance of lignin to plant growth and development, and to humans as a renewable carbon source. A key factor in lignin production is the availability of monolignol precursors, which are made via the phenylpropanoid and monolignol biosynthetic pathways. Another important factor is the oxidative enzymes that form monolignol radicals, which then cross-link into polymeric lignin in the cell wall. Previous studies of the model plant Arabidopsis thaliana have contributed to our understanding of both factors, and I will build on this work to investigate how plants control these factors to coordinate lignification. 1.1 Primary and secondary cell walls in land plants Plant cell walls are a polysaccharide-rich extracellular matrix. In land plants, the primary cell wall forms first during cell expansion, allowing plant cells to build up the turgor pressure required for determining cell shape and directional plant growth (Cosgrove, 2022). During cell division, cytoskeletal elements organize along the division plane into a phragmoplast, guiding vesicles from the Golgi apparatus to form a cell plate where cell wall materials are deposited and synthesized (Gu & Rasmussen, 2022). Callose is first laid down in small quantities, followed by pectin, hemicelluloses, and cellulose; as the cell plate expands outwards and fuses with the mother cell wall, a new primary cell wall with a pectin-rich middle lamella between two cellulose-rich layers is created (Drakakaki, 2015). The composition of the primary cell wall is actively remodelled in response to developmental and environmental signals (Vaahtera et al., 2019). In specialized cell types, such as water-conducting tracheary elements and supportive fibres, where elongation and primary cell wall deposition are complete, a secondary cell wall is deposited underneath the primary cell wall to offer additional support to plant structures. The main components of the secondary cell wall include polysaccharides such as cellulose; hemicelluloses such as xylans and mannans; and the polyphenolic polymer lignin (Zhong et al., 2019). Biochemical and mechanical 2 properties of the primary and secondary cell walls jointly contribute to proper plant growth and development. 1.2 Biological and economic importance of lignin The rigid and hydrophobic lignin polymer confers mechanical strength to fibres, enables efficient water transport through vessels, and prevents vessel collapse under negative pressure created during transpiration water loss. This is exemplified by irregularly shaped vessel cell walls and impaired plant growth as a result of lignin deficiency (Berthet et al., 2011; Jones et al., 2001). The emergence of lignin in the green lineage allowed vascular plants to adapt to the effect of gravity and drought in terrestrial habitats (Weng & Chapple, 2010). Throughout human civilization, lignin as a carbon-based, rigid biopolymer enables wood to be used as a fuel and a construction material. In industrial settings, lignin is removed from cell wall polysaccharides to yield paper products. In the bioenergy sector, attempts to enzymatically hydrolyze cellulose for bioethanol fuel production (Pauly & Keegstra, 2008) are inefficient and economically unviable because separating lignin from the polysaccharides is energy-intensive and expensive (Rezania et al., 2020). Recently, more attention has been given to valorizing lignin, a renewable biopolymer and a carbon source that can be converted into bioproducts, biofuels, and specialty chemicals (Balakshin et al., 2021). Lignin’s biological and economic importance prompted research into the structure of lignin in the cell wall polysaccharide matrix, and the cellular mechanisms controlling the quantity, composition, and spatial distribution of lignin in different cell types and cell wall domains throughout development. 1.3 Biosynthesis of monolignols, sinapoyl esters, and lignin Lignin is a heterogeneous polymer consisting of monolignols, varying in quantity, composition, and spatial distribution across different taxa, cell types, and cell wall domains. In general, monolignol biosynthesis begins with the biosynthesis of phenylalanine via the shikimate pathway in the plastid (Maeda & Dudareva, 2012) 3 (Figure 1.1). Phenylalanine then enters the general phenylpropanoid pathway in the cytosol to generate p-coumaroyl CoA, followed by the monolignol biosynthetic pathway to produce monolignols (Bonawitz & Chapple, 2010; Dixon & Barros, 2019) (Figure 1.1). Three common types of monolignols include p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol (Figure 1.1). In Arabidopsis, a parallel pathway is the biosynthesis of sinapoyl glucose and sinapoyl malate from sinapaldehyde in the epidermis of aerial tissues as sunscreen compounds to protect plants from UV damage (Landry et al., 1995). After sinapoyl glucose is synthesized in the cytosol, it is transported into the vacuole by an unknown mechanism and converted to sinapoyl malate (Fraser & Chapple, 2011) (Figure 1.1). Once synthesized in the cytosol, monolignols move across the plasma membrane into the cell wall through simple diffusion down a concentration gradient created by monolignol polymerization into lignin in the cell wall (Perkins et al., 2022). In the cell wall, oxidative enzymes such as laccases (LACs) and class III peroxidases (PRXs) activate monolignols into radicals (Freudenberg, 1959; Shigeto & Tsutsumi, 2016) (Figure 1.1). These radicals cross-link randomly to form complex, highly branched lignin polymers (Ralph et al., 2004). In the lignin polymer, monomeric units derived from p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol are called p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively. Our current knowledge of the monolignol biosynthetic pathway came from numerous mutant studies. In Arabidopsis, mutations associated with irregular xylem (irx), reduced epidermal fluorescence (ref), and bright trichome (brt) phenotypes led to the discovery of the monolignol biosynthetic pathway genes, including CINNAMOYL CoA REDUCTASE 1 (CCR1/IRX4) (Jones et al., 2001), HYDROXYCINNAMALDEHYDE DEHYDROGENASE (HCALDH/REF1) (Nair et al., 2004), CINNAMATE 4-HYDROXYLASE (C4H/REF3) (Mizutani et al., 1997; Schilmiller et al., 2009), p-COUMAROYL SHIKIMATE 3'-HYDROXYLASE (C3'H/REF8) (Franke et al., 2002; Schoch et al., 2001), and SINAPATE:UDP-GLUCOSE GLUCOSYLTRANSFERASE (SGT/BRT1) (Ruegger & Chapple, 2001; Sinlapadech et al., 2007) (Figure 1.1). Among these mutants, ccr1/irx4, c4h/ref3, and c3'h/ref8 showed 4 lignin deficiency and impaired growth (Franke et al., 2002; Jones et al., 2001; Schilmiller et al., 2009), exemplifying the importance of lignin to plant growth and development. To this day, new pathways are still emerging, such as the recent characterization of p-COUMARATE 3-HYDROXYLASE (C3H), which encodes an intracellular peroxidase that converts p-coumaric acid into caffeic acid without the involvement of shikimate ester intermediates (Barros et al., 2019) (Figure 1.1). Mutant studies in monocots such as maize (Halpin et al., 1998); dicots such as alfalfa (Nakashima et al., 2008) and poplar (Coleman et al., 2008a; Leplé et al., 2007); and gymnosperms such as pine (Wagner et al., 2007) and spruce (Wadenbäck et al., 2008) further expanded our knowledge on how plants across different taxa regulate lignification. 5 Figure 1.1. Metabolic pathways that produce phenolic compounds in Arabidopsis: (1) biosynthesis of phenylalanine via the shikimate pathway in the plastid; (2) biosynthesis of monolignols, monolignol glucosides, and sinapoyl esters via the phenylpropanoid and monolignol pathways in the cytosol and the vacuole; and (3) monolignol oxidation and radical coupling into lignin in the cell wall. “?” indicates unknown transport mechanisms across the tonoplast. 6 1.4 Monolignol supply and export to the cell wall Lignin deposition relies on an abundant supply of monolignols, which can be produced either by the lignifying cell itself or by adjacent cells. Differences in the lignification mechanisms of Arabidopsis interfascicular fibres and xylem vessels have been demonstrated experimentally. When the expression of CCR1, a monolignol pathway gene, was suppressed in interfascicular fibres and xylem vessels, only interfascicular fibres showed a reduction in cell wall lignin (Smith et al., 2013). When the expression of the same gene was suppressed in xylem parenchyma cells that neighboured the vessels, the vessels were irregularly shaped due to reduced cell wall lignin (Smith et al., 2017). These studies showed that xylem vessels can draw monolignols from neighbouring xylem parenchyma cells (termed “good neighbours”) for lignification (Smith et al., 2013, 2017), while interfascicular fibres display cell-autonomous lignification where cells synthesize monolignols and oxidative enzymes for their own lignification (Smith et al., 2013, 2017). How do monolignols travel from their source of production inside the cell to the site of polymerization in the cell wall? In vitro, liposome-enclosed LACs led to monolignol diffusion across liposome membranes down a concentration gradient driven by monolignol coupling within liposomes (Perkins et al., 2022). In vivo, the Arabidopsis lac4 lac17 double mutant showed decreased lignin deposition in root protoxylem and monolignol accumulation in good neighbour cells, suggesting that cell wall oxidative enzymes at the site of polymerization drive monolignol diffusion away from the monolignol source inside the cell (Perkins et al., 2022). Another prevailing hypothesis of monolignol movement into the cell wall is the transport of monolignol glucosides (such as coniferin and syringin, see Figure 1.1) across the plasma membrane (Tsuyama et al., 2013; Väisänen et al., 2020), or the release of monolignol glucosides from the vacuole into the cell wall during programmed cell death (Kaneda et al., 2008). Coniferin accumulates in high amounts in cambial regions and is considered to be an intermediate of lignification in developing gymnosperm xylem (Terashima et al., 2016), but the support for this mechanism is less well established in woody angiosperms (Aoki et al., 2019; Tsuyama et al., 2013). Non-woody angiosperms such as Arabidopsis 7 normally do not accumulate monolignol glucosides, suggesting that monolignol aglycones are directly used in lignification (Perkins et al., 2022). 1.5 Genetic regulation of monolignol biosynthesis How is the phenylpropanoid pathway regulated? In Arabidopsis, upstream regulatory elements of secondary cell wall biosynthesis include NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) and VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. For example, NST1 and NST3 regulate secondary cell wall formation in interfascicular fibres (Mitsuda et al., 2007), while VND6 and VND7 regulate both secondary cell wall production and programmed cell death in xylem vessel formation (Kubo et al., 2005). These transcription factors will trigger a cascade of other transcription factors including MYB58 and MYB63, which in turn activate biosynthetic genes in the shikimate pathway and the phenylpropanoid pathway to produce monolignols (O’Malley et al., 2016; Taylor-Teeples et al., 2015; Zhou et al., 2009). RNA-seq data confirmed that in Arabidopsis plants overexpressing MYB63 (pro35S::MYB63), shikimate pathway genes such as CHORISMATE MUTASE (CM), general phenylpropanoid pathway genes such as PHENYLALANINE AMMONIA LYASE (PAL), and monolignol pathway genes such as CAFFEOYL-CoA 3-O-METHYLTRANSFERASE (CCoAOMT) were upregulated. In addition, the accumulation of monolignol glucosides and sinapoyl esters was also observed in pro35S::MYB63 (Perkins et al., 2020). Given the cytotoxicity of monolignols (Väisänen et al., 2015), monolignol glycosylation, sinapoyl ester formation, and subsequent vacuolar sequestration are likely mechanisms to prevent monolignol accumulation in the cytosol. 1.6 Oxidative enzymes: fine-tuning lignification The polymerization of monolignols typically begins in the cell corners and the middle lamella, and then in the thick secondary cell wall (Donaldson, 2001; Terashima & Fukushima, 1988). Different sets of LACs and PRXs localize to these cell wall domains where they fine-tune cell wall lignification (Hoffmann et al., 2022). LACs are multi-copper oxidases that use molecular oxygen (O2) as a co-substrate (Solomon et al., 1996), while PRXs are heme-based enzymes that use hydrogen peroxide (H2O2) as a 8 co-substrate (Hiraga et al., 2001). The Arabidopsis genome encodes 17 LACs and 73 PRXs (McCaig et al., 2005; Valério et al., 2004). Unlike LACs, the localization of PRXs must also coincide with H2O2 production to enable monolignol oxidation. Evidence has shown that PRXs were expressed ubiquitously in the Arabidopsis stem, but PRX activity was only found in lignifying cells where H2O2 co-substrates were produced (Hoffmann et al., 2020). Therefore, the regulation of reactive oxygen species also contributes to lignification. In Arabidopsis stems, LAC2, LAC4, LAC5, LAC6, LAC10, LAC11, LAC12, and LAC17 were expressed (Berthet et al., 2011). Mutant studies found the lac4 lac17 double mutant had collapsed xylem due to reduced lignin (Berthet et al., 2011), and the lac4 lac11 lac17 triple mutant had no lignin in xylem vessels, were extremely dwarf, and did not produce pollen (Zhao et al., 2013), indicating LAC4, LAC11, and LAC17 are required for lignin deposition and proper plant growth and development. Localization studies showed that both LAC4 and LAC17 localized to secondary cell walls of xylem vessels and interfascicular fibres, while LAC4 also showed transient expression in the cell corners and middle lamella of interfascicular fibres (Chou et al., 2018; Hoffmann et al., 2020). LAC11 expression was associated with tracheary elements, based on a promoter-GUS reporter assay (Turlapati et al., 2011). To identify PRXs involved in Arabidopsis stem lignification, mutant studies identified lignin defects in prx2, prx25, prx71 (Shigeto et al., 2013), prx4, prx52, and prx72 (Fernández-Pérez et al., 2015a, 2015b, 2015c; Herrero et al., 2013) mutant plants. Expression and co-expression analyses found that PRX42, PRX52, PRX64, PRX71, and PRX72 were consistently expressed in Arabidopsis stems and co-expressed with secondary cell wall-related genes (Hoffmann et al., 2020). PRX64 and PRX71 localized to the cell corners and middle lamella of interfascicular fibres with LAC4, while PRX72 localized to secondary cell walls of xylem vessels and interfascicular fibres with LAC4 and LAC17 (Chou et al., 2018; Hoffmann et al., 2020). Interestingly, PRX42 and PRX52 localized to non-lignifying cells (Hoffmann et al., 2020). It is unclear why the prx52 single mutant showed altered lignin phenotypes (Fernández-Pérez et al., 2015b), despite PRX52 localizing to non-lignifying cells. 9 The connection between PRXs and lignification is also established in root endodermis where lignin and suberin together form the Casparian strip. Mutant studies in Arabidopsis found that the prx64 single mutant had reduced lignin levels in the Casparian strip (Lee et al., 2013), while the prx3 prx9 prx39 prx64 prx72 quintuple knockout mutant had no lignin in the Casparian strip (Rojas-Murcia et al., 2020). 1.7 Knowledge gap 1.7.1 Contributions of monolignol availability and oxidative enzymes to lignification Monolignol production is controlled transcriptionally in lignifying cells and good neighbour cells. In addition, monolignols only assemble into lignin in lignifying cells where oxidative enzymes and co-substrates such as O2 and H2O2 are present (Hoffmann et al., 2020; Lee et al., 2013; Schuetz et al., 2014). Therefore, both monolignol availability and oxidative enzyme activity contribute to lignification. However, the relative contributions of these two factors to the final amount of cell wall lignin are unknown. If monolignol availability predominates, can the upregulation of the monolignol pathway in lignifying cells lead to increased lignin in the cell wall? Similarly, can the upregulation of the monolignol pathway in good neighbour cells lead to increased lignin in lignifying vessels? In both cases, what is the upper limit of lignin deposition in lignifying cell walls? What mechanisms prevent lignification in non-lignifying cells? Gene expression studies showed broad and overlapping distributions of oxidative enzymes in cells with both primary and secondary cell walls (Shigeto & Tsutsumi, 2016; Turlapati et al., 2011). When either LAC4 or LAC17 was overexpressed constitutively throughout the Arabidopsis plant, exogenously added monolignols were highly mobile in the apoplast and could be incorporated into leaf cells that do not normally lignify (Schuetz et al., 2014). These observations indicate that lignification can occur in any cell due to the ubiquitous presence of oxidative enzymes. Does this mean that monolignol availability contributes more than oxidative enzyme activity to the proper distribution of lignin in stems? If monolignols are ectopically produced in non-lignifying cells only, will these 10 cells lignify? How far will ectopically produced monolignols diffuse from the site of production? How will nearby cells react to misplaced monolignols in the apoplast? 1.7.2 Lignin modification-induced dwarfism Altering monolignol availability in plants has unexpected consequences, and mechanisms governing these consequences are complex. Dwarfism of the lignin-deficient c3'h/ref8 Arabidopsis mutant (Franke et al., 2002; Schoch et al., 2001) was rescued by mutating a mediator complex encoded by MED5a/REF4 and MED5b despite drastic changes in lignin composition (Bonawitz et al., 2014). Irregular xylem and dwarfism of the lignin-deficient ccr1 Arabidopsis mutant were rescued by restoring vessel-specific CCR1 expression (De Meester et al., 2018). Dwarfism of Arabidopsis MYB58 or MYB63 overexpression plants (pro35S::MYB58, pro35S::MYB63) was rescued by co-overexpressing LAC4 or LAC17 without increasing lignin deposition (Perkins et al., 2020). These observations exemplify lignin modification-induced dwarfism (LMID) where plant growth is inhibited by changes to the phenylpropanoid and monolignol pathways, but the unexpected phenotypic restoration after additional genetic modification suggests a complex regulatory system connecting phenylpropanoid metabolism and plant growth (Muro-Villanueva et al., 2019). Possible causes of LMID include compromised vessel integrity leading to decreased water availability, imbalanced growth-inhibiting and growth-promoting phenylpropanoid compounds, and cell wall integrity sensing (Muro-Villanueva et al., 2019). Dwarfism as a result of MYB58 or MYB63 overexpression has not been investigated in a cell type-specific manner. Would dwarfism occur if monolignol production was increased only in specific cell types? Would the consequences of increased monolignol production be the same in lignifying cell types and normally non-lignifying cell types? 1.7.3 Lignin’s mechanical role in specific cell wall domains Lignin deposition in the thick secondary cell wall, the middle lamella, and cell corners is thought to contribute unique mechanical properties to stems, but exactly how 11 do these lignified domains contribute? Eastern leatherwood (Dirca palustris) has flexible stems with no lignin in the middle lamella and cell corners of mature xylem (Mottiar et al., 2020). If lignin is not deposited into the middle lamella and cell corners, will Arabidopsis stems become more flexible? No study to date has attempted to remove lignin from specific cell wall domains, but the recent identification of LACs and PRXs localized to these regions provides an opportunity to target oxidative enzymes in a domain-specific manner (Chou et al., 2018; Hoffmann et al., 2020). 1.8 Experimental system and research objectives My research will investigate how monolignol availability and the properties of specific sets of oxidative enzymes control lignin deposition. Arabidopsis stems are chosen as the experimental system because (1) genetic tools for Arabidopsis are widely available; (2) genetic elements involved with monolignol biosynthesis and oxidative cross-linkage are well documented in the literature; and (3) Arabidopsis stem lignification resembles lignification in woody trees, such as tracheary elements receiving monolignols from neighbouring parenchyma cells (Smith et al., 2013, 2017; Blokhina et al., 2019), and the initiation of lignification at the cell corners (Hoffmann et al., 2020; Terashima & Fukushima, 1988). My research will be divided into three objectives: 1. To determine the capacity of lignification in lignifying cell types such as interfascicular fibres and xylem vessels in Arabidopsis stems. 2. To test the consequences of additional or ectopic monolignol production in lignifying and non-lignifying cell types in Arabidopsis stems. 3. To assess the role of lignin in plant growth and development through targeted lignin removal from specific cell wall domains in Arabidopsis stems. 12 2 MYB63 expression driven by vessel- and fibre-specific promoters increases stem lignin without growth penalty 2.1 Introduction 2.1.1 MYB58 and MYB63 transcription factors regulate the biosynthesis of phenolic compounds and the associated stress responses In Arabidopsis, MYB58 and MYB63 transcription factors regulate lignification (Perkins et al., 2020; Zhou et al., 2009). MYB58 was primarily expressed in the protoxylem and weakly in the interfascicular fibres, while MYB63 was primarily expressed in the interfascicular fibres, metaxylem, and secondary xylem (Zhou et al., 2009). Although the spatial distribution of MYB58 and MYB63 suggests that these transcription factors are not redundant, in vitro DNA affinity purification sequencing (DAP-seq; O’Malley et al., 2016) revealed that MYB58 and MYB63 have 1704 gene targets in common. Table 2.1 summarizes confirmed and putative genes involved in the biosynthesis of phenolic compounds via the shikimate pathway, phenylpropanoid and monolignol pathways, and genes encoding LACs and PRXs, many of which are targets of MYB58 and MYB63. This analysis also revealed previously uncharacterized regulatory targets of MYB58 and MYB63. Many LAC and PRX targets of MYB58 and MYB63 are not directly involved in lignification, but rather associated with oxidative stress based on gene ontology enrichment. Gene targets sharing sequence similarity with confirmed genes in the monolignol pathway, such as 4CL-like3 (AT1G20490), 4CL-like7 (AT4G05160), COMT-like7 (AT1G63140), COMT-like8 (AT1G76790), and COMT-like11 (AT3G53140), are involved in stress responses (Kannan et al., 2018; Koo et al., 2006; Li et al., 2017; Pfalz et al., 2011). Given that lignification requires reactive oxygen species as co-substrates for cell wall PRXs and the cytotoxic effects of monolignol aglycones (Väisänen et al., 2015), it can be speculated that MYB58 and MYB63 also regulate stress responses associated with monolignol production and lignification. 13 2.1.2 MYB63 overexpression enhances plant stress and compromises growth When MYB63 was overexpressed in all cell types (pro35S::MYB63), the shikimate pathway, the phenylpropanoid pathway, and the monolignol pathway were upregulated, resulting in the accumulation of monolignol glucosides and esters, severely dwarf plants, and a stress-associated transcriptome (Perkins et al., 2020; Zhou et al., 2009). Table 2.1 compares MYB63 DAP-seq targets and differentially expressed genes in pro35S::MYB63 compared to wild type. Upregulated genes in the shikimate pathway and phenylpropanoid pathway are mostly MYB63 DAP-seq targets, with exceptions such as COMT1, UGT84A2/BRT1, UGT72E2, and various oxidative enzyme genes. These exceptions represent genes that are upregulated as a response to MYB63 overexpression, but not due to increased MYB63 transcriptional activation. Since COMT (Chun et al., 2019; Riehl Koch et al., 1998; Vincent et al., 2005) and oxidative enzymes (produce or consume reactive oxygen species) are associated with stress signalling, and UGT84A2 (produces sinapoyl glucose; Sinlapadech et al., 2007) and UGT72E2 (produces monolignol glucosides; Lanot et al., 2006) divert aldehydes and monolignols to vacuolar sequestration, these genes are likely upregulated to signal or alleviate the stress associated with increased monolignol biosynthesis. Interestingly, some genes are downregulated in pro35S::MYB63 plants such as CAD7/ELI3-1 (AT4G37980), a defence-associated gene shown to be downregulated after salicylic acid treatment (Sørhagen et al., 2013). Together with the observation that co-overexpressing MYB63 and LAC4 or LAC17 restored normal growth and relieved stress responses (Perkins et al., 2020), it can be hypothesized that dwarfism associated with MYB63 overexpression is due to enhanced stress signalling that compromises growth. If upregulated monolignol biosynthesis in all cell types induces stress, where is the stress sensed? Lignifying cell types are likely more tolerant to monolignol-induced stress since the necessary molecular machinery to alleviate stress is probably present. If additional monolignols are produced, will cell walls be more lignified? Similarly, are non-lignifying cell types less tolerant to monolignol-induced stress? The ability to upregulate monolignol biosynthesis in a cell type-specific manner should reveal the 14 capacity of lignification in lignifying cell walls and the mechanism behind lignin modification-induced dwarfism. Table 2.1. Confirmed and putative Arabidopsis genes involved in the biosynthesis of phenylalanine (shikimate pathway), monolignols, monolignol glucosides, sinapoyl esters (phenylpropanoid and monolignol biosynthetic pathways), and lignin (oxidative enzymes). Gene targets of MYB58 and MYB63 transcription factors based on DAP-seq (O’Malley et al., 2016) are marked with solid circles, while non-targets are marked with hollow circles. The Venn diagram summarizes the number of gene targets unique or common to both transcription factors. “+” indicates upregulated genes (log2 fold change ≥ 1.5 and p < 0.05) and “-” indicates downregulated genes (log2 fold change ≤ -1.5 and p < 0.05) in pro35S::MYB63 plants relative to wild type based on RNA-seq (Perkins et al., 2020). “NS” indicates genes that are not significantly upregulated or downregulated. Gene Identifier MYB58 target MYB63 target MYB63-OE/Col-0 expression References Shikimate pathway / Phenylalanine biosynthesis 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) DAHPS1 AT4G39980 ○ ○ + (Kanaris et al., 2022) DAHPS2 AT4G33510 ○ ○ NS DAHPS3 AT1G22410 ● ● + 3-Dehydroquinate synthase (DHQS) DHQS AT5G66120 ● ● + (Tzin & Galili, 2010) Dehydroquinate dehydratase-shikimate dehydrogenase (DHQD-SDH) DHQD-SDH AT3G06350 ● ● + (Singh & Christendat, 2006) Shikimate kinase (SK) SK1 AT2G21940 ○ ○ NS (Fucile et al., 2008, 2011) SK2 AT4G39540 ● ● NS 5-Enolpyruvylshikimate-3-phosphate synthetase (EPSPS) EPSPS AT2G45300 ● ● + (Klee et al., 1987) EPSPS AT1G48860 ● ● + Chorismate synthase (CS) CS AT1G48850 ● ● + (Tzin & Galili, 2010) Chorismate mutase (CM) CM1 AT3G29200 ● ● + (Eberhard et al., 1993, 1996; Mobley et al., 1999) CM2 AT5G10870 ○ ○ NS CM3 AT1G69370 ● ○ NS Prephenate aminotransferase (PPA-AT) PPA-AT AT2G22250 ● ● + (Maeda et al., 2011) 15 Gene Identifier MYB58 target MYB63 target MYB63-OE/Col-0 expression References Arogenate dehydratase (ADT) / Prephenate dehydratase (PDT) ADT1/PDT1 AT1G11790 ● ● + (Cho et al., 2007) ADT2/PDT2 AT3G07630 ○ ○ NS ADT3 AT2G27820 ● ● NS ADT4 AT3G44720 ● ● + ADT5 AT5G22630 ● ● NS ADT6/PDT6 AT1G08250 ● ● + Phenylpyruvate aminotransferase (PPY-AT) TAT2 AT5G36160 ○ ○ NS Not well studied in Arabidopsis (Prabhu & Hudson, 2010) Phenylpropanoid pathway / Biosynthesis of monolignols, monolignol glucosides, and sinapoyl esters Phenylalanine ammonia lyase (PAL) PAL1 AT2G37040 ● ● + (Cochrane et al., 2004; Huang et al., 2010; Rohde et al., 2004) PAL2 AT3G53260 ● ● + PAL3 AT5G04230 ○ ○ NS PAL4 AT3G10340 ● ● + Cinnamate 4-hydroxylase (C4H) C4H/CYP73A5/REF3 AT2G30490 ● ● + (Mizutani et al., 1997; Schilmiller et al., 2009) 4-Coumarate:CoA ligase (4CL) 4CL1 AT1G51680 ● ● + (Costa et al., 2005; Ehlting et al., 1999; Hamberger & Hahlbrock, 2004; Li et al., 2015; Raes et al., 2003; Shockey & Browse, 2011) 4CL2 AT3G21240 ● ● + 4CL3 AT1G65060 ● ● NS 4CL4 AT3G21230 ○ ● NS 4CL-like1 AT1G20510 ○ ○ NS 4CL-like2 AT1G20500 ○ ○ NS 4CL-like3 AT1G20490 ● ● NS 4CL-like4 AT1G20480 ○ ○ NS 4CL-like5 AT1G62940 ● ● NS 4CL-like6 AT4G19010 ○ ○ + 4CL-like7 AT4G05160 ● ● NS 4CL-like8 AT5G63380 ○ ○ NS 4CL-like9 AT5G38120 ○ ○ - Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) HCT AT5G48930 ● ● + (Hoffmann et al., 2004) p-Coumaroyl shikimate 3'-hydroxylase (C3'H) C3'H/CYP98A3/REF8 AT2G40890 ● ● + (Franke et al., 2002; Matsuno et al., 2009; Schoch et al., 2001) CYP98A8 AT1G74540 ○ ○ NS CYP98A9 AT1G74550 ○ ● NS Caffeoyl shikimate esterase (CSE) CSE AT1G52760 ● ● + (Vanholme et al., 2013) p-Coumarate 3-hydroxylase (C3H) APX1 AT1G07890 ○ ● NS (Barros et al., 2019) Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) CCoAOMT1 AT4G34050 ● ● + (Do et al., 2007; Raes et al., 2003; Wils et al., 2013; Yang et al., 2019) CCoAOMT2 AT1G24735 ○ ○ NS CCoAOMT3 AT3G61990 ○ ○ NS CCoAOMT4 AT3G62000 ○ ○ NS 16 Gene Identifier MYB58 target MYB63 target MYB63-OE/Col-0 expression References CCoAOMT5 AT1G67990 ○ ○ NS CCoAOMT6 AT1G67980 ○ ○ NS CCoAOMT7 AT4G26220 ● ○ NS Cinnamoyl-CoA reductase (CCR) CCR1/IRX4 AT1G15950 ● ● NS (Jones et al., 2001; Lauvergeat et al., 2001; Raes et al., 2003) CCR2 AT1G80820 ○ ● NS CCR-like1 AT1G76470 ○ ○ NS CCR-like2 AT2G02400 ○ ○ NS CCR-like3 AT2G33590 ○ ○ NS CCR-like4 AT2G33600 ○ ● NS CCR-like5 AT5G58490 ○ ○ NS Ferulate 5-hydroxylase (F5H) F5H/CYP84A1/FAH1 AT4G36220 ○ ○ NS (Meyer et al., 1996; Raes et al., 2003; Weng et al., 2012) CYP84A4 AT5G04330 ○ ○ - Caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) COMT1/OMT1 AT5G54160 ○ ○ + (Goujon et al., 2003; Raes et al., 2003) COMT-like1 AT1G21100 ○ ○ NS COMT-like2 AT1G21110 ○ ○ NS COMT-like3 AT1G21120 ○ ○ NS COMT-like4 AT1G21130 ○ ○ NS COMT-like5 AT1G33030 ○ ○ NS COMT-like6 AT1G51990 ○ ○ NS COMT-like7 AT1G63140 ● ○ NS COMT-like8 AT1G76790 ● ○ NS COMT-like9 AT1G77520 ● ○ NS COMT-like10 AT1G77530 ○ ○ NS COMT-like11 AT3G53140 ○ ● NS COMT-like12 AT5G37170 ○ ○ NS COMT-like13 AT5G53810 ○ ○ NS Cinnamyl alcohol dehydrogenase (CAD) CAD1 AT1G72680 ○ ○ NS (Raes et al., 2003; Sibout et al., 2005; Somssich et al., 1996); CAD numbering based on (Kim et al., 2004) CAD2 AT2G21730 ○ ○ NS CAD3 AT2G21890 ○ ○ NS CAD4/CAD-C AT3G19450 ● ● NS CAD5/CAD-D AT4G34230 ● ○ NS CAD6 AT4G37970 ○ ○ NS CAD7/ELI3-1 AT4G37980 ● ○ - CAD8/ELI3-2 AT4G37990 ○ ○ - CAD9 AT4G39330 ● ○ NS Hydroxycinnamaldehyde dehydrogenase (HCALDH) ALDH1A/REF1 AT3G24503 ○ ○ NS (Nair et al., 2004) Sinapate:UDP-glucose glucosyltransferase (SGT) UGT84A2/BRT1 AT3G21560 ○ ○ + (Ruegger & Chapple, 2001; Sinlapadech et al., 2007) Sinapoyl glucose:malate sinapoyltransferase (SMT) SNG1 AT2G22990 ○ ○ NS (Lehfeldt et al., 2000) Monolignol UDP-glycosyltransferse (UGT) UGT72B1 AT4G01070 ○ ○ NS UGT72B2 AT1G01390 ○ ○ NS 17 Gene Identifier MYB58 target MYB63 target MYB63-OE/Col-0 expression References UGT72B3 AT1G01420 ○ ○ NS (Baldacci-Cresp et al., 2020a; Lanot et al., 2006; Lim et al., 2005; Lin et al., 2016) UGT72E1 AT3G50740 ○ ○ NS UGT72E2 AT5G66690 ○ ○ + UGT72E3 AT5G26310 ○ ○ NS Oxidative enzymes LAC1 AT1G18140 ○ ● NS (Brown et al., 2005; Turlapati et al., 2011; Valério et al., 2004) LAC2 AT2G29130 ● ● NS LAC3 AT2G30210 ○ ● NS LAC4/IRX12 AT2G38080 ○ ○ - LAC5 AT2G40370 ○ ○ NS LAC6 AT2G46570 ○ ○ NS LAC7 AT3G09220 ○ ○ NS LAC8 AT5G01040 ● ● NS LAC9 AT5G01050 ○ ○ NS LAC10 AT5G01190 ○ ○ NS LAC11 AT5G03260 ● ● NS LAC12 AT5G05390 ● ○ NS LAC13 AT5G07130 ● ○ NS LAC14 AT5G09360 ○ ○ NS LAC15 AT5G48100 ○ ○ NS LAC16 AT5G58910 ○ ○ NS LAC17 AT5G60020 ● ○ NS PRX1 AT1G05240 ○ ○ NS PRX2 AT1G05250 ○ ○ NS PRX3 AT1G05260 ● ● NS PRX4 AT1G14540 ○ ○ - PRX5 AT1G14550 ○ ○ NS PRX6 AT1G24110 ○ ○ NS PRX7 AT1G30870 ○ ○ NS PRX8 AT1G34510 ○ ○ NS PRX9 AT1G44970 ● ● NS PRX10 AT1G49570 ○ ○ NS PRX11 AT1G68850 ● ● NS PRX12 AT1G71695 ○ ● NS PRX13 AT1G77100 ○ ○ NS PRX14 AT2G18140 ○ ○ NS PRX15 AT2G18150 ○ ○ + PRX16 AT2G18980 ○ ○ NS PRX17 AT2G22420 ● ● NS PRX18 AT2G24800 ○ ○ NS PRX19 AT2G34060 ○ ○ NS PRX20 AT2G35380 ○ ○ NS PRX21 AT2G37130 ○ ○ NS PRX22 AT2G38380 ○ ○ NS PRX23 AT2G38390 ○ ○ + PRX24 AT2G39040 ○ ○ NS PRX25 AT2G41480 ○ ○ NS PRX26 AT2G43480 ○ ○ NS PRX27 AT3G01190 ○ ○ NS PRX28 AT3G03670 ● ○ NS PRX29 AT3G17070 ○ ○ NS PRX30 AT3G21770 ○ ○ + PRX31 AT3G28200 ○ ○ NS PRX32 AT3G32980 ○ ○ NS PRX33 AT3G49110 ○ ○ NS PRX34 AT3G49120 ○ ○ NS PRX35 AT3G49960 ○ ○ NS PRX36 AT3G50990 ● ○ NS 18 Gene Identifier MYB58 target MYB63 target MYB63-OE/Col-0 expression References PRX37 AT4G08770 ○ ○ NS PRX38 AT4G08780 ○ ○ NS PRX39 AT4G11290 ● ○ NS PRX40 AT4G16270 ○ ○ NS PRX41 AT4G17690 ● ○ NS PRX42 AT4G21960 ● ● NS PRX43 AT4G25980 ○ ○ NS PRX44 AT4G26010 ○ ○ NS PRX45 AT4G30170 ○ ○ NS PRX46 AT4G31760 ● ● NS PRX47 AT4G33420 ○ ○ NS PRX48 AT4G33870 ○ ○ NS PRX49 AT4G36430 ● ○ + PRX50 AT4G37520 ○ ○ + PRX51 AT4G37530 ○ ○ NS PRX52 AT5G05340 ○ ○ NS PRX53 AT5G06720 ○ ○ NS PRX54 AT5G06730 ○ ○ NS PRX55 AT5G14130 ○ ○ NS PRX56 AT5G15180 ○ ○ NS PRX57 AT5G17820 ● ○ NS PRX58 AT5G19880 ○ ○ NS PRX59 AT5G19890 ● ○ NS PRX60 AT5G22410 ○ ○ NS PRX61 AT5G24070 ○ ○ NS PRX62 AT5G39580 ○ ○ NS PRX63 AT5G40150 ● ● NS PRX64 AT5G42180 ● ○ NS PRX65 AT5G47000 ● ○ NS PRX66 AT5G51890 ○ ○ NS PRX67 AT5G58390 ○ ○ NS PRX68 AT5G58400 ○ ○ NS PRX69 AT5G64100 ○ ○ NS PRX70 AT5G64110 ○ ○ + PRX71 AT5G64120 ○ ○ NS PRX72 AT5G66390 ○ ○ NS PRX73 AT5G67400 ○ ○ NS 2.1.3 Choosing cell type-specific promoters to upregulate monolignol biosynthesis in lignifying and non-lignifying cell types To upregulate monolignol biosynthesis in lignifying and non-lignifying cell types, the MYB63 genomic sequence can be paired with cell type-specific promoters. Figure 2.1 highlights candidate cell types for monolignol upregulation in Arabidopsis stems, including xylem vessels, xylem parenchyma cells, interfascicular fibre cells, epidermal cells, starch sheath cells, and phloem companion cells. Table 2.2 summarizes the cell type-specific promoters used in this study and the literature that provides experimental evidence for their expression patterns. Xylem vessels and interfascicular fibre cells are 19 the main lignifying cell types in Arabidopsis stems, while xylem parenchyma cells contribute monolignols to the lignification of neighbouring vessel cell walls. Epidermal cells, starch sheath cells, and phloem companion cells are not lignified. Figure 2.1. Wild-type Arabidopsis stem cross-section stained with toluidine blue. False colours show distinct cell types that can be targeted with cell type-specific promoters. Scale bar = 50 µm. Table 2.2. Arabidopsis promoter elements and the experimental evidence for their cell type-specific expression. Promoter Length (bp upstream) Cell type Function of encoded protein Evidence of cell type-specific expression References proVND7 (AT1G71930) 2000 Xylem vessels (lignified) Transcription factor for vascular secondary cell wall formation proVND7::GUS (Kubo et al., 2005; Zhong et al., 2008) proPRX47 (AT4G33420) 1593 Xylem parenchyma (produces monolignol but not lignified) Peroxidase proPRX47::GUS and proPRX47::GFP (Smith et al., 2017; Tokunaga et al., 2009) proPRX64 (AT5G42180) 900 Interfascicular fibres (lignified) Peroxidase proPRX64::GUS and proPRX64:: PRX64-mCherry (Chou et al., 2018; Smith et al., 2017) proCER6 (AT1G68530) 1209 Epidermis (not lignified) Very-long-chain fatty acid condensing enzyme for epidermal wax production proCER6::GUS (Hooker et al., 2002) proSCR (AT3G54220) 2468 Root endodermis (lignified) and quiescent centre (not lignified); stem starch sheath (not lignified) Establishes cortex-endodermis patterning in the root proSCR::GUS (Malamy & Benfey, 1997) proSUC2 (AT1G22710) 945 Phloem companion cells (not lignified) Sucrose-H+ symporter proSUC2::GUS (Imlau et al., 1999; Truernit & Sauer, 1995) pro ylem vessels (v)pro ylem parenchyma (xp)pro 6 nterfascicular fibres (if)pro 6 pidermis (ep)proS Starch sheath (ss)proS Phloem companion cells (ph) 20 2.2 Results 2.2.1 Cell type-specific promoters drive MYB63 overexpression in rosette leaves and inflorescence stems MYB63 is expressed in Arabidopsis rosette leaves at low levels (Zhou et al., 2009), making them an ideal tissue for detecting MYB63 overexpression in T1 plants transformed with MYB63 constructs driven by cell type-specific promoters. Qualitatively, MYB63 transcripts were barely detected in wild-type rosette leaves, while amplicons were detected in all cell type-specific lines (Figure 2.2B). Each cell type-specific line showed varying degrees of MYB63 overexpression. MYB63 transcript abundance was 4-fold above wild-type levels when driven by proVND7, 3-fold by proPRX47, 6-fold by proPRX64, 30-fold by proCER6, 36-fold by proSCR, and 213-fold by proSUC2 (Figure 2.2A). As a positive control, constitutive, ubiquitous MYB63 overexpression driven by pro35S was 1765-fold above wild-type levels, the highest among all plant lines in the analysis (Figure 2.2A). Figure 2.2. ctopic MYB63 expression driven by cell type-specific promoters was detected in rosette leaves. (A) qRT-PCR quantifies MYB63 expression (log2-transformed) in the rosette leaves of 4-5 pooled T1 cell type-specific promoter::MYB63 024681012MYB63 300 200 100A Relative expression(log2 fold change)MYB63bp21 plants relative to wild-type Col-0 plants. Wild-type MYB63 expression is normalized to log2(1) = 0. pro35S::MYB63 (Perkins et al., 2020; Zhou et al., 2009) is used as a positive control. Bars and error bars represent mean relative expression from six technical replicates ± SD. ( ) qRT-PCR amplicons of MYB63 (target) and α (reference) are visualized with agarose gel electrophoresis. After confirming that cell type-specific promoters confer MYB63 overexpression in rosette leaves, three independent transformants of each cell type-specific promoter::MYB63 line were taken to the T2 generation for further analyses. The top 20 cm of 40-50 cm mature primary inflorescence stems of T2 homozygous or hemizygous cell type-specific promoter::MYB63 plants were sampled, because this part of the stem includes the native lignification of tracheary elements and interfascicular fibres, and any potential ectopic lignification. qRT-PCR analysis on cDNA prepared from top 20 cm stems revealed that proVND7::MYB63 lines did not show MYB63 overexpression above wild-type levels, while all other cell type-specific promoter::MYB63 lines showed MYB63 overexpression in at least two out of three independent transformant lines (Figure 2.3). Consistent MYB63 overexpression above wild-type levels was observed in all three independent transformants of proPRX47::MYB63 and proPRX64::MYB63 (Figure 2.3). proSUC2::MYB63 line #4 showed the highest average MYB63 expression (14-fold above wild-type levels) among all cell type-specific promoter::MYB63 lines (Figure 2.3). As a positive control, pro35S::MYB63 showed 118-fold MYB63 expression above wild-type levels (Figure 2.3). 22 Figure 2.3. MYB63 overexpression driven by cell type-specific promoters was detected in inflorescence stems. qRT-PCR quantified MYB63 expression in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants relative to wild-type Col-0 plants. Wild-type MYB63 expression was normalized to 1. Three independent transformant lines of each cell type-specific promoter::MYB63 line were analyzed. 4-5 plants were pooled per line in each independent experiment. pro35S::MYB63 (Perkins et al., 2020; Zhou et al., 2009) was used as a positive control. Bars and error bars represent mean relative expression from three independent experiments ± SD, with three technical replicates per sample in each independent experiment. 2.2.2 Ectopic MYB63 expression in stems can lead to delayed stem growth A previous study found a negative correlation between MYB63 expression levels in Arabidopsis rosette leaves and rosette diameter (Perkins et al., 2020). To test this correlation in Arabidopsis stems, stem heights of 4- and 6-week-old T2 cell type-specific promoter::MYB63 plants were measured. At week 4, all three independent transformant lines of proPRX47::MYB63 (lines #1, #2, #3) and proSUC2::MYB63 (lines #4, #5, #7), and two independent transformant lines of proCER6::MYB63 (lines #14, #18) and 1051151251350510152025MYB63Relative expression (fold change)23 proSCR::MYB63 (lines #1, #2) were shorter than wild type (Figure 2.4). At week 6, only two lines of proPRX47::MYB63 (lines #2, #3), and one line of proCER6::MYB63 (line #18), proSCR::MYB63 (line #1), and proSUC2::MYB63 (line #4) were shorter than wild type (Figure 2.4). As a control, pro35S::MYB63 displayed the most severe dwarf phenotype (Figure 2.4). Figure 2.4. Stem heights (in cm) of 4- and 6-week-old segregating T2 cell type-specific promoter::MYB63 plants from three independent experiments (4-5 plants per line in each experiment). Kruskal-Wallis χ2(Week 4) = 132.37, p(Week 4) < 2.2e-16; χ2(Week 6) = 98.597, p(Week 6) = 9.6e-13. Asterisks denote significant statistical difference from wild-type Col-0 plants: * (p < 0.05), ** (p < 0.01), *** (p < 0.001), calculated with post-hoc Dunn’s test and Benjamini-Hochberg adjustments. MYB63 expression levels and stem heights in 6-week-old cell type-specific promoter::MYB63 plants showed a negative correlation (Pearson’s r = -0.64, p = 0.0034) (Figure 2.5). All lines with more than 10-fold MYB63 expression above wild type, including proPRX47::MYB63 (lines #2, #3) and proSUC2::MYB63 (line #4), had shorter stems than wild type (Figure 2.5). proSCR::MYB63 line #1, the highest MYB63-expressing line (4.3-fold) among three independent transformant lines, also had shorter stems than wild type. proCER6::MYB63 line #18 did not show MYB63 overexpression 02040600204060********** *** ******** *********** * ** **Stem height (cm)Week 4Week 624 (0.73-fold) but still had shorter stems than wild type, indicating that other factors such as the genomic location of the inserted transgene, rather than MYB63 overexpression, may have contributed to the phenotype. The data point representing pro35S::MYB63 was not included in the analysis because extreme height measurements or expression values skewed the correlation. Figure 2.5. Correlation between mean stem heights (in cm) and relative MYB63 expression levels (fold change from wild-type Col-0) in the top 20 cm primary inflorescence stems of 6-week-old segregating T2 cell type-specific promoter::MYB63 plants. ach independent transformant line is shown as one data point. ach data point represents mean values of three independent experiments, with 4-5 plants per line in each independent experiment. Asterisks indicate plant lines that had significantly shorter stems than wild-type plants at 6 weeks (Figure 2.4). Pearson’s r = -0.64, p = 0.0034. 2.2.3 Cell type-specific MYB63 overexpression has differential impacts on the soluble phenolic compositions in the stems Given that the pro35S::MYB63 overexpression plant accumulated monolignol glucosides and sinapoyl esters in rosette leaves (Perkins et al., 2020), I hypothesized that cell type-specific promoter::MYB63 plants would accumulate these compounds as a result of MYB63 overexpression. Methanol extracts of soluble phenolics from top inflorescence stems of cell type-specific promoter::MYB63 plants were analyzed with UHPLC (collaboration with Dr. Shawn Mansfield’s Lab, UBC Depts. of Botany and 024681012141637 39 41 43 45 47 49Relative MYB63expression(Fold change)Mean stem height (cm)r = -0.64p = 0.0034 Col-0 pro ::MYB63 pro ::MYB63 pro 6 ::MYB63 pro 6::MYB63 proS ::MYB63 proS ::MYB63 25 Wood Science), and peak identities were confirmed with external standards of coniferin and syringin, as well as mass spectrometry (collaboration with Dr. Tao Huan’s Lab, UBC Dept. of Chemistry). As previously reported, the positive control pro35S::MYB63 stems accumulated higher amounts of monolignol glucosides (coniferin and syringin; Figure 2.6A) and sinapoyl malate (Figure 2.6B, C, D; Table 2.3), but lower amounts of flavonol glycosides than wild-type stems (Figure 2.6B). This confirms that the upregulated monolignol pathway diverts the metabolic flux away from the parallel flavonoid pathway. In both wild-type stems and cell type-specific promoter::MYB63 stems, coniferin and syringin were not present at detectable levels (Figure 2.6A) while sinapoyl malate and flavonol glycosides were always present (Figure 2.6B). This suggests that cell type-specific MYB63 overexpression is moderate, permitting flavonol biosynthesis. Although the monolignol-related metabolites were not increased, the hydroxycinnamate esters, sunscreen compounds that are typically the dominant soluble phenolics in wild-type stems, were altered. In the epidermis where sinapoyl malate typically accumulates, MYB63 overexpression line #11 and #14 driven by proCER6 (Figure 2.3) showed a 40-43% increase in sinapoyl malate (Figure 2.6C, D; Table 2.3). proVND7::MYB63 lines accumulated 35-80% more sinapoyl malate (Figure 2.6B, C, D; Table 2.3) than wild-type plants, despite MYB63 overexpression not being detected in the stem (Figure 2.3). Lines with the highest MYB63 expression in the stem, including proPRX47::MYB63 (lines #2, #3) and proSUC2::MYB63 (line #4) (Figure 2.3), accumulated more sinapoyl malate than wild-type plants, but not more than proVND7::MYB63 or proCER6::MYB63 lines (Figure 2.6B, C, D; Table 2.3). proSCR::MYB63 did not accumulate more sinapoyl malate than wild type (Figure 2.6B, C, D; Table 2.3), despite MYB63 overexpression (Figure 2.3). These data suggest that MYB63 overexpression within the distinctive metabolic environments of each cell type has differential impacts on the soluble phenolic composition in the stem. 26 Figure 2.6. Quantification of sinapoyl malate in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. Selected UHPLC chromatograms showed absorbance values at (A) 260 nm to detect monolignol glucosides and ( ) 330 nm to detect sinapoyl esters and flavonol (kaempferol) 0.7 1.1 1.50.00.51.01.50246Relative sinapoylmalate(Fold change)mg sinapoylmalate g stem A Retention time (min)Normalized A260proS ::MYB63proS ::MYB63pro 6::MYB63pro 6 ::MYB63pro ::MYB63pro ::MYB63pro35S::MYB63Col-01.5 1.9 2.3 2.7 3.1 3.5 3.9 4.3 4.7 5.1Normalized A33027 glycosides. Absorbance values were normalized to the peak area of morin hydrate (internal standard). Compound identities were confirmed by mass spectrometry analysis. ( ) Quantity of sinapoyl malate (in miligrams) per gram of fresh stem tissues, calculated from the sinapic acid standard. ( ) Quantity of sinapoyl malate relative to wild-type plants. Three independent transformant lines of each cell type-specific promoter::MYB63 line were analyzed. 4-5 plants were pooled per line in each independent experiment. pro35S::MYB63 (Perkins et al., 2020; Zhou et al., 2009) was used as a control. Bars and error bars represent mean quantity from three independent experiments ± SD, with three technical replicates per sample in each independent experiment. Table 2.3. Quantification of sinapoyl malate in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. Three independent transformant lines of each cell type-specific promoter::MYB63 line were analyzed. 4-5 plants were pooled per line in each independent experiment. Mean values are from three independent experiments ± S of the mean, with three technical replicates per sample in each independent experiment. Genotype Line Sinapoyl malate (mg / g fresh stem) (± SEM) Fold change (± SEM) Col-0 0.44 ± 0.06 1.00 ± 0.07 pro35S::MYB63 0.96 ± 0.18 3.05 ± 1.31 proVND7::MYB63 2 0.70 ± 0.17 1.80 ± 0.11 3 0.69 ± 0.27 1.66 ± 0.27 6 0.55 ± 0.18 1.35 ± 0.15 proPRX47::MYB63 1 0.49 ± 0.16 1.27 ± 0.19 2 0.42 ± 0.12 1.08 ± 0.10 3 0.45 ± 0.05 1.25 ± 0.19 proPRX64::MYB63 2 0.39 ± 0.05 1.07 ± 0.16 3 0.45 ± 0.02 1.37 ± 0.45 4 0.41 ± 0.13 1.01 ± 0.17 proCER6::MYB63 11 0.49 ± 0.14 1.40 ± 0.42 14 0.47 ± 0.11 1.43 ± 0.57 18 0.31 ± 0.08 0.79 ± 0.05 proSCR::MYB63 1 0.33 ± 0.05 0.95 ± 0.21 2 0.33 ± 0.09 0.84 ± 0.13 3 0.39 ± 0.11 0.97 ± 0.11 proSUC2::MYB63 4 0.36 ± 0.09 1.20 ± 0.63 5 0.32 ± 0.06 0.85 ± 0.07 7 0.42 ± 0.10 1.10 ± 0.04 28 2.2.4 MYB63 expression driven by vessel- and interfascicular fibre-specific promoters led to increased lignin in stems Given that many cell type-specific promoter::MYB63 lines accumulated sinapoyl esters (Figure 2.6, Table 2.3), the phenylpropanoid and monolignol biosynthetic pathways were likely upregulated, and additional monolignols may have diffused to the cell wall to be cross-linked into lignin. I hypothesized that lignifying cells became more lignified, while ectopic lignin formed in non-lignifying cell walls as a result of cell type-specific MYB63 overexpression. To quantify total lignin in cell type-specific promoter::MYB63 lines, acetone-insoluble stem tissues were treated with acetyl bromide, and the amount of acetyl bromide soluble lignin (ABSL) was quantified with UV-Visible spectrophotometry (Chang et al., 2008). To account for the variation observed between independent experiments, quantities of ABSL relative to wild-type plants in each independent experiment were also calculated. proVND7::MYB63 and proPRX64::MYB63 plants had 9-26% and 2-20% more ABSL than wild-type plants, respectively (Figure 2.7, Table 2.4). Compared to wild-type plants, proCER6::MYB63 and proSCR::MYB63 plants had similar amounts of ABSL, while proPRX47::MYB63 and proSUC2::MYB63 plants showed 6-15% and 11-16% reduction in ABSL, respectively (Figure 2.7, Table 2.4). In contrast to earlier findings where Klason lignin was increased in 8- to 10-week-old pro35S::MYB63 plants (Perkins et al., 2020), 6-week-old pro35S::MYB63 plants in this study had 22% less ABSL than wild type (Figure 2.7, Table 2.4) despite high MYB63 expression (Figure 2.3) and monolignol accumulation (Figure 2.6A, C, D). Overall, these results indicate that lignin levels only increased when MYB63 was expressed in lignifying cell types, while high levels of ectopic MYB63 expression in xylem parenchyma, phloem companion cells, or all cell types led to delayed stem development (Figure 2.4, Figure 2.5) and decreased lignin deposition (Figure 2.7, Table 2.4). 29 Figure 2.7. Quantification of acetyl bromide soluble lignin (ABSL) in the top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. (A) Quantity of ABSL (in grams) per gram of dried, acetone-insoluble stem tissues. ( ) Quantity of ABSL relative to wild-type plants. Three independent transformant lines of each cell type-specific promoter::MYB63 line were analyzed. 4-5 plants were pooled per line in each independent experiment. pro35S::MYB63 (Perkins et al., 2020; Zhou et al., 2009) was used as a control. Bars and error bars represent mean quantity from three independent experiments ± SD, with three technical replicates per sample in each independent experiment. Relative ABSL (Fold change)g ABSL g stemA 0.000.050.100.150.00.51.01.530 Table 2.4. Quantification of acetyl bromide soluble lignin (ABSL) in dried, acetone-insoluble, top 20 cm primary inflorescence stems of 6-week-old T2 segregating cell type-specific promoter::MYB63 plants. Three independent transformant lines of each cell type-specific promoter::MYB63 line were analyzed. 4-5 plants were pooled per line in each independent experiment. Mean values are from three independent experiments ± S of the mean, with three technical replicates per sample in each independent experiment. Genotype Line ABSL (g / g stem) (± SEM) ABSL Fold change (± SEM) Col-0 0.127 ± 0.007 1.00 ± 0.03 pro35S::MYB63 0.096 ± 0.006 0.78 ± 0.08 proVND7::MYB63 2 0.139 ± 0.012 1.17 ± 0.07 3 0.148 ± 0.004 1.26 ± 0.14 6 0.131 ± 0.016 1.09 ± 0.02 proPRX47::MYB63 1 0.109 ± 0.020 0.90 ± 0.10 2 0.112 ± 0.009 0.94 ± 0.05 3 0.104 ± 0.020 0.85 ± 0.08 proPRX64::MYB63 2 0.129 ± 0.016 1.07 ± 0.02 3 0.143 ± 0.012 1.20 ± 0.05 4 0.124 ± 0.019 1.02 ± 0.06 proCER6::MYB63 11 0.121 ± 0.017 1.00 ± 0.06 14 0.111 ± 0.022 0.91 ± 0.10 18 0.106 ± 0.016 0.87 ± 0.05 proSCR::MYB63 1 0.120 ± 0.014 1.00 ± 0.06 2 0.118 ± 0.014 0.98 ± 0.01 3 0.115 ± 0.019 0.94 ± 0.07 proSUC2::MYB63 4 0.106 ± 0.018 0.87 ± 0.07 5 0.110 ± 0.025 0.89 ± 0.12 7 0.105 ± 0.027 0.84 ± 0.16 31 2.2.5 Histochemical staining of proVND7::MYB63 and proPRX64::MYB63 stem cross-sections did not reveal changes in the spatial distribution of lignin Given more lignin was detected in the stems of proVND7::MYB63 and proPRX64::MYB63 plants, I hypothesized that the lignin was deposited in vessels or interfascicular fibres specified by proVND7 and proPRX64. To visualize lignin in stem cross-sections, UV autofluorescence was used to detect total lignin, Wiesner staining was used to detect coniferaldehyde as a proxy for G-rich lignin (Blaschek et al., 2020), and Mäule staining was used to detect S-rich lignin (Iiyama & Pant, 1988). In all cross-sections of proVND7::MYB63 and proPRX64::MYB63 plants, UV autofluorescence patterns were similar to wild-type plants, confirming that there was no ectopic lignification (Figure 2.8A, D, G); Wiesner staining produced bright red signals in G-rich vessels (Figure 2.8B, E, H), and Mäule staining produced red signals in S-rich fibres and brown signals in G-rich vessels (Figure 2.8C, F, I), suggesting no qualitative difference in lignin distribution from wild-type plants. Additional lignification can therefore occur in lignifying cell walls without altering lignin distribution in the stem. 32 Figure 2.8. UV autofluorescence and histological staining of pro ::MYB63 and pro 6 ::MYB63 stem cross sections. Serial cross-sections were taken 20 cm from the shoot apical meristem of the primary inflorescence stem. Cross-sections were stained with the Wiesner method (phloroglucinol-HCl) to detect coniferaldehyde or the Mäule method (KMnO4, HCl, Na2CO3) to detect lignin enriched in syringyl units. Wild-type Col-0 stem cross-sections were imaged as controls. Scale bar = 50 µm. 2.3 Discussion 2.3.1 Lignifying cell types have the capacity for additional lignification What determines the quantity of lignin deposited in cell walls? It is reasonable to speculate that plants will not produce more lignin than necessary to support plant structures as monolignol production requires phenylalanine, an essential amino acid, and energy input. In addition, lignification involves redox reactions and reactive oxygen UV Wiesner MäuleCol-0pro ::MYB63pro 6 ::MYB63A 33 species, requiring additional energy to alleviate the associated oxidative stress. When nutrients are abundant and monolignol production is only increased in lignifying cells that already have mechanisms to alleviate oxidative stress, more lignin may be deposited into lignifying cell walls. This study showed that more lignin could be deposited when MYB63 was expressed under the vessel-specific proVND7 and the interfascicular-specific proPRX64 (Figure 2.7, Table 2.4) without qualitative changes in lignin composition or distribution (Figure 2.8). Therefore, lignifying cell walls have the capacity for additional lignification, and the level of MYB63 expression, a proxy for monolignol supply, can be a factor in determining lignin quantities in Arabidopsis stems. 2.3.2 Monolignol availability and oxidative enzyme activity coordinate lignification What mechanisms prevent lignification in non-lignifying cell types? Mutations in the Arabidopsis transcription factor WRKY12, a repressor of the fibre-specific NST2 transcriptional activator of secondary cell wall biosynthesis (Mitsuda et al., 2005; Zhong & Ye, 2015), led to ectopic lignification in pith cells (Wang et al., 2010). This suggests that lignification is actively repressed in non-lignifying cell types. Given monolignols can diffuse within the apoplast, it suggests that despite the presence of monolignols, non-lignifying cell types do not have the necessary apoplastic environment for lignification. It can be speculated that PRXs, which are ubiquitously present in non-lignifying cell types (Hoffmann et al., 2020), do not normally incorporate monolignols into lignin, unless the production of H2O2 is induced by wounding (Denness et al., 2011). Lignifying cell types possess a unique set of LACs and PRXs that specialize in oxidizing monolignols by consuming O2 and H2O2, respectively (Hoffmann et al., 2020). In this study, no additional lignin was detected when MYB63 was overexpressed in non-lignifying cell types with proPRX47, proCER6, proSCR, and proSUC2 (Figure 2.7, Table 2.4), despite the accumulation of sinapoyl malate as an indication of the upregulated phenylpropanoid pathway (Figure 2.6, Table 2.3). This suggests that even if monolignols are ectopically produced, lignin will not be deposited in non-lignifying cell walls in the absence of specific oxidative enzymes and H2O2 co-substrates. Taken together, the data suggest that monolignol availability must be coupled with oxidative 34 enzyme activity in the appropriate cell types to coordinate cell wall lignification. The role of oxidative enzymes in cell wall lignification will be further discussed in Chapter 3. 2.3.3 MYB63 overexpression in non-lignifying cell types induces dwarfism Lignin is a renewable polyphenolic biopolymer that can be converted into valuable aromatic hydrocarbon compounds and bioproducts (Balakshin et al., 2021). Attempts to increase lignin deposition by constitutively overexpressing MYB63 in plants led to severe growth penalty and reduced biomass yield (Perkins et al., 2020; Zhou et al., 2009), making it undesirable for engineering high-lignin plants. This study showed that MYB63 overexpression in lignifying vessels and interfascicular fibres resulted in more lignin (Figure 2.7, Table 2.4) without affecting growth (Figure 2.3, Figure 2.4, Figure 2.5). This suggests that lignifying cell types can tolerate additional monolignol production, as they likely contain protective mechanisms against the oxidative stress associated with lignification (sections 2.1.1, 2.1.2). A recent study in Populus also reported increased lignin without growth penalty by overexpressing WND1B, which encodes a fibre-specific transcriptional activator of secondary cell wall biosynthesis, with a vasculature-specific 4CL promoter (Payyavula et al., 2022). These data present a strategy for increasing lignin deposition in herbaceous and woody species. In addition, the presence of highly-expressed cell wall-bound oxidative enzymes in lignifying cells can prevent the accumulation of cytotoxic monolignols in the cytosol, because monolignol oxidation leads to lignin polymer formation and a monolignol concentration gradient for monolignol diffusion out of the cell (Perkins et al., 2022). Epidermal cells may have their own mechanism to prevent monolignol cytotoxicity, as upregulating the phenylpropanoid pathway in epidermal cells did not affect plant growth (Figure 2.3, Figure 2.4, Figure 2.5). These cells typically accumulate sinapoyl esters that are sequestered into the vacuole (Figure 1.1), so increased flux to the phenylpropanoid and monolignol biosynthetic pathways could be safely diverted to sinapoyl esters (Bonawitz et al., 2012). These results suggest that generating high-lignin plants without growth penalty should be considered in the context of each cell 35 type’s typical phenylpropanoid metabolism, with lignifying cell types already equipped to cope with increased monolignol production. Non-lignifying cell types are more sensitive to MYB63 overexpression. Plant lines with the highest MYB63 expression in non-lignifying xylem parenchyma (line #2, #3) and phloem companion cells (line #4) displayed delayed stem development (Figure 2.3, Figure 2.4, Figure 2.5) and less lignin than wild type (Figure 2.7). This agrees with the hypothesis that non-lignifying cell types are not equipped for lignification and the associated oxidative stress, adversely impacting plant growth and development. Surprisingly, the severely dwarf pro35S::MYB63 plants also had less lignin than wild type at 6 weeks (Figure 2.4, Figure 2.7), contrary to the increased lignin at 8-10 weeks reported by Perkins et al. (2020). It is possible that in 6-week-old plants, constitutive MYB63 overexpression in non-lignifying cells severely delayed stem development, resulting in less lignin in the stem compared to a tall, normally developing stem. To summarize, lignifying cell types are ideal targets for enhancing lignin without growth defects. A better understanding of the genetic regulation of monolignol biosynthesis, cell type-specific effects of increased flux through the phenylpropanoid and monolignol pathways, and mechanisms of lignin modification-induced dwarfism will enable lignin modification for biofuel and bioproducts. 2.4 Methods 2.4.1 Plant growth conditions Arabidopsis seeds were sterilized with chlorine gas, generated by mixing 100 mL bleach and 10 mL concentrated hydrochloric acid, in a sealed box for 2-4 hours. Sterilized seeds were plated on Germination Media (1× Murashige-Skoog basal salts, 1× Gamborg’s vitamin mix, 10 g L sucrose, 0.5 g L M S, 0.75 g L agar, pH 5.8 with KOH), and stratified at 4°C for 3 days. Seeds were germinated at 21°C under 24 hours of light. Seedlings were transferred to soil and grown at 21°C under 16 hours of light and 8 hours of darkness. Plants were supplemented with a 20-20-20 inorganic fertilizer once a week. 36 2.4.2 Cloning cell type-specific promoter::MYB63 constructs To drive cell type-specific MYB63 overexpression in Arabidopsis, cell type-specific promoters were paired with the MYB63 gene using Multisite Gateway™ cloning (Invitrogen) (Figure 2.9). To generate promoter entry clones, proVND7 (2000 bp upstream of ATG; Kubo et al., 2005), proPRX47 (1593 bp upstream of ATG; Smith et al., 2017; Tokunaga et al., 2009), proPRX64 (900 bp upstream of ATG; Smith et al., 2017), proCER6 (1209 bp upstream of ATG; Hooker et al., 2002), proSCR (2468 bp upstream of ATG; Malamy & Benfey, 1997), and proSUC2 (945 bp upstream of ATG; Imlau et al., 1999) were PCR-amplified from wild-type Arabidopsis (Columbia ecotype, Col-0) genomic DNA and flanked with N-terminal attB4 and C-terminal attB1R sites. Each amplified product was gel-purified with the Monarch® DNA Gel Extraction Kit (New England Biolabs) and incorporated into the pDONR-P4-P1R-Gent attP4-ccdB-CmR-attP1R vector via the BP reaction. The resultant pDONR attL4-promoter-attR1 entry clones, and the pDONR221 attL1-MYB63-attL2 entry clone (Perkins et al., 2020), were incorporated into the R4pGWB501 attR4-CmR-ccdB-attR2 destination vector (Nakagawa et al., 2008) via the LR reaction. All cell type-specific promoter::MYB63 plant expression clones were transformed into chemically competent Subcloning Efficiency™ DH5α E. coli (Invitrogen) and miniprepped with the Presto™ Mini Plasmid Kit (Geneaid Biotech). Sequences of expression clones were verified (Azenta Life Sciences) before transformation into electrocompetent Agrobacterium tumefaciens GV3101 strain (GoldBio). All primers and PCR conditions are listed in Supplementary Tables 1 and 2. 37 Figure 2.9. (A) Construction of MYB63 plant expression clones driven by cell type-specific promoters using nvitrogen MultiSite Gateway™ BP and LR recombination reactions. The MYB63 gene entry clone and the R4pGWB501 destination vector were described in Perkins et al. (2020) and Nakagawa et al. (2008), respectively. attB attP attL attR = Gateway sites; m = chloramphenicol resistance marker for bacterial selection; ccdB = toxin-encoding marker for negative bacterial selection; Tnos = OS terminator; Hyg = hygromycin resistance marker for plant selection; LB RB = Agrobacterium T-DNA left and right borders. ( ) Cell type-specific promoter::MYB63 expression clones created in this thesis work. ach cell type-specific promoter is paired with the MYB63 genomic sequence. Black boxes represent exons, solid horizontal lines attP4 attP1RccdB m attB4 attB1RattP1 attP2ccdB m attB1 attB2Genomic MYB63attL4 attR1attL1 attL2attR4 attR2 m ccdBattB4 attB2attB1 Hyg Hyg RBRBLBLBTnosTnospro attB4 attB1 attB2pro attB4 attB1 attB2pro 6 attB4 attB1 attB2pro 6attB4 attB1 attB2proS attB4 attB1 attB2proS attB4 attB1 attB2Genomic MYB63A BPBPLRPromoter (genomic region upstream of ATG)PromoterPromoterPromoter38 represent introns, and dashed horizontal lines represent potentially retained introns. xon-intron structures are taken from The Arabidopsis nformation Resource (TA R) database (https: www.arabidopsis.org ). 2.4.3 Arabidopsis transformation with Agrobacterium floral dip Wild-type Arabidopsis Col-0 plants were transformed with cell type-specific promoter::MYB63 constructs using the Agrobacterium floral dip method (Zhang et al., 2006) with modifications. 1 mL overnight culture of Agrobacterium tumefaciens containing cell type-specific promoter::MYB63 constructs was inoculated into 50 mL liquid YEB media (5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/L MgCl2) supplemented with 50 µg/mL gentamycin and 100 µg/mL spectinomycin. The culture was shaken at 30°C for 16 hours. The culture was centrifuged at 4000 × g for 10 minutes at room temperature and the supernatant was removed. Cells were resuspended in liquid YEB media without antibiotics, centrifuged at 4000 × g for 10 minutes at room temperature, and the supernatant was removed. Cells were resuspended in 50 mL liquid YEB media without antibiotics, and 10 µL Silwet L-77 (Phyto Tech Lab) was added to a final concentration of 0.02% (v/v). The Agrobacterium media was poured into a shallow dish and Arabidopsis floral buds were dipped for 1 minute. The media was slightly agitated with a transfer pipet while dipping. Dipped plants were placed in a dark, humid chamber for 24 hours before being transferred to the growth chamber for recovery (21°C, 16 hours of light and 8 hours of darkness). The dipping was repeated after one week. 2.4.4 Selection of plant transformants T1 seeds were collected and screened for hygromycin resistance. Chlorine gas-sterilized seeds were plated on Germination Media supplemented with 25 μg mL hygromycin, stratified at 4°C for 3 days, germinated at 21°C in the dark for 4 days, and grown at 21°C under 24 hours of light. Only seedlings showing elongated hypocotyls, green true leaves, and root growth were transferred to soil. Refer to section 2.4.1 for general plant growth conditions. 39 Genomic DNA was extracted from the rosette leaves of T1 plants with Edwards buffer (Edwards et al., 1991). DNA extracts were used as PCR templates to confirm the presence of cell type-specific promoter::MYB63 constructs. Confirmed T1 plants were taken to the T2 generation. Segregating T2 plants selected on hygromycin were used for experiments. All primers and PCR conditions are listed in Supplementary Tables 1 and 2. 2.4.5 RNA extraction, cDNA synthesis, and quantitative real-time PCR Rosette leaves of 4-week-old plants or top 20 cm primary inflorescence stems (up to the uppermost leaf node) of 6-week-old plants were frozen in liquid nitrogen and ground into powder with a mortar and pestle. Total RNA was extracted with the TRIzol™ reagent (Invitrogen), treated with DNAse I, RNAse-free (Thermo Scientific) to remove genomic DNA contamination, and converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), per the manufacturers’ instructions. Quantitative PCRs were prepared with the SensiFAST™ SYBR® No-ROX Kit (Bioline) per the manufacturer’s instructions, and amplification was detected with the CFX96 Touch Real-Time PCR Detection System (BioRad). Three technical replicates were performed for each cDNA sample and primer pair. EF1-α (AT1G07940) was used as a reference gene. Relative gene expression was calculated with the 2-ΔΔCt method (Livak & Schmittgen, 2001). All primers and PCR conditions are listed in Supplementary Tables 1 and 2. 2.4.6 Liquid chromatography-mass spectrometry (LC-MS) Phenolic extracts of Arabidopsis tissues were prepared by adding 1 mL extraction solvent (80% MeOH (v/v) and 1% glacial acetic acid (v/v) in H2O) per 150 mg fresh tissue. For the initial extraction, the extraction solvent contained 0.1 mg/mL morin hydrate (Sigma-Aldrich, M4008) as an internal standard to account for the loss of soluble phenolics in the extraction process. Tissues were incubated at 70°C for 15 minutes with shaking at 200 rpm, centrifuged at 18,000 × g for 5 minutes, and the supernatant was transferred to a new tube. The extraction was repeated two more times on the same tissue using fresh extraction solvent without an internal standard, and all 40 supernatants were pooled. The solvent was removed by vacuum centrifugation at room temperature for 6-8 hours. Soluble phenolic compounds were then resuspended in 50% acetonitrile:50% H2O (v/v), sonicated in a water bath for 10 minutes, and heated at 45°C for an hour. Phenolic extracts were filtered through 0.45 µm filters (Chromatographic Specialities, CSUV04N451) into glass vials with plastic inserts (Chromatographic Specialities, C226310), closed with lids with PTFE/red silicone septa (Agilent Technologies, 5182-0717). Phenolic extracts were stored at 4°C until UHPLC analysis or frozen at -20°C long-term. Phenolic extracts were analyzed on the Agilent Infinity II 1290 UHPLC system with an Agilent Zorbax Eclipse Plus C18 column (2.1 × 50 mm, particle size = 1.8 μm) and a diode array detector (DAD) (Agilent Technologies). To separate phenolic compounds, 1 µL of phenolic extract was injected into the system with a solvent gradient from 90% solvent A (0.1% trifluoroacetic acid in H2O, v/v) and 10% solvent B (0.1% trifluoroacetic acid in acetonitrile, v/v) to 50% A:50% B over 9 minutes, at a flow rate of 0.3 mL/min. DAD signals were captured at 260 nm and 330 nm. External standards of coniferin, syringin (gifted by Dr. Sally Ralph, USDA Forest Products Laboratory, Madison, Wisconsin), and sinapic acid (Sigma-Aldrich, D7927) were dissolved in 50% acetonitrile:50% H2O and used to create calibration curves for quantification. To confirm compound identities, phenolic extracts were further analyzed on the Agilent Infinity II 1290 UHPLC system (Agilent Technologies) with a Waters Acquity UPLC BEH C18 column (1 × 100 mm, particle size = 1.7 µm) (Waters Corporation) and an Impact II Ultra-High Resolution Qq-Time-Of-Flight (UHR-QqTOF) mass spectrometer (Bruker Daltonics). 3 µL of phenolic extract was injected into the system with a solvent gradient from 95% solvent A (0.1% formic acid in H2O, v/v) and 5% solvent B (0.1% formic acid in acetonitrile, v/v) to 75% A:25% B over 8 minutes, then to 30% A:70% B over 6 minutes, at a flow rate of 0.15 mL/min. Compound identities were matched to in-house MS-DIAL (Version 15) (Tsugawa et al., 2015) and NIST20 databases (National Institute of Standards and Technology, US Department of Commerce), curated by Dr. Tao Huan’s Lab (University of British Columbia). 41 2.4.7 Acetyl bromide soluble lignin (ABSL) quantification To quantify total lignin content, a microscale acetyl bromide soluble lignin assay (Chang et al., 2008) was performed with modifications. After phenolic extraction (section 2.4.6), the remaining tissues were dried in an oven at 70°C. Soluble phenolics in dried tissues were removed by Soxhlet extraction with hot acetone for 16 hours (approximately 5 cycles per hour). The acetone was evaporated in the fume hood overnight. 2.5 mg or 5 mg of dried tissues were incubated in 250 µL freshly prepared 25% acetyl bromide in glacial acetic acid (v/v) at 50°C for 2 hours without shaking, then for 1 hour with vortexing every 15 minutes. Samples were cooled on ice and centrifuged for 15 minutes at 9,500 × g. To quench the reaction, 100 µL supernatant was added to 400 µL 2 M NaOH and 75 µL freshly prepared 0.5 M hydroxylamine hydrochloride. The mixture was vortexed briefly and diluted with 1.425 mL glacial acetic acid to a final volume of 2 mL. A blank acetyl bromide reaction was created with the same procedure, but without adding dried stem tissues. Within 30 minutes after quenching, each reaction mixture was transferred to a quartz cuvette (Mandel Scientific), and absorbance at 280 nm (Abs280) was measured three times with a BioMate 3S UV-Visible Spectrophotometer (Thermo Scientific). Average Abs280 values were converted to ABSL concentration (CABSL, in g/L) with the Lambert-Beer equation CABSL = Abs280 / εLD, where the extinction coefficient ε = 23.35 g-1 L cm-1 for Arabidopsis stems (Chang et al., 2008), path length L = 1 cm, and dilution factor D = 0.05 (100 µL reaction was diluted to 2 mL for measurement). CABSL (in g/L) was converted to the proportion of lignin in the cell wall = CABSL × Vi / m, where the initial volume of acetyl bromide reaction Vi = 2.50 × 10-4 L, and m = mass of dried, acetone-insoluble stem tissues (in g). 2.4.8 Stem cross-sectioning, staining, and imaging 1 cm-long sections of primary inflorescence stems were collected 20 cm from the shoot apical meristem of 6-week-old plants. Sections were cleared with 50% ethanol (in H2O, v/v) for at least 3 days and embedded in 4% low melting point agarose (in H2O, w/v). 80 µm-thick stem cross-sections were cut with a Leica VT1000S vibratome and stored in H2O. Unstained or stained cross-sections were imaged with a Leica DMR 42 microscope, a 20× objective, and a Canon EOS Rebel T5 camera (0.1 s exposure, ISO 400, auto white balance). Unstained cross-sections were imaged under UV illumination (340-380 nm excitation wavelengths) with a DAPI filter (450-500 nm emission wavelengths), while stained cross-sections were imaged with Kohler illumination. To visualize lignifying and non-lignifying cell walls, cross-sections were stained with 0.02% toluidine blue O (in H2O, w/v) for 2 minutes, rinsed with H2O twice, and mounted in 75% glycerol (in H2O, v/v) for imaging. For Wiesner staining, cross-sections were stained in two parts of 3% phloroglucinol (in ethanol, w/v) and one part of concentrated hydrochloric acid for 3 minutes, mounted in 75% glycerol, and imaged within 10 minutes. For Mäule staining, cross-sections were stained in 0.5% potassium permanganate (in H2O, w/v) for 5 minutes, rinsed with H2O twice, decolourized in 1 M hydrochloric acid for 3 minutes, treated with 1 M sodium carbonate for 30 seconds, mounted in 75% glycerol, and imaged within 10 minutes. Stains were freshly prepared on the day of the experiment. 2.4.9 Statistical analysis All statistical analyses were performed with R 4.1.2 (R Core Team, 2021) with the following packages: rcompanion (Mangiafico, 2023) and FSA (Ogle et al., 2023). Plots were produced in R with ggplot2 (Wickham, 2016) and ggpubr (Kassambara, 2023), and in Microsoft Excel. 43 3 Targeted lignin removal in the secondary cell wall leads to increased vessel irregularity without additional growth penalty 3.1 Introduction 3.1.1 Laccases and peroxidases control lignin quantity and composition in distinct cell wall domains The quantity and composition of lignin in different cell wall domains are tightly controlled. Lignin deposition typically begins in the pectin-rich cell corners and the middle lamella, followed by the cellulose- and hemicellulose-rich secondary cell wall (Donaldson, 2001; Terashima & Fukushima, 1988). When stem cross-sections are illuminated with UV light, lignin autofluorescence is often the strongest in the middle lamella and cell corners where lignin is the most concentrated. In general, lignin deposited in the middle lamella and the cell corners is enriched in p-hydroxyphenyl (H) units, while lignin deposited in the secondary cell wall is primarily made up of guaiacyl (G) and syringyl (S) units; fibre cell walls are rich in S units, while vessel cell walls are rich in G units (Donaldson, 2001; Terashima & Fukushima, 1988). What mechanisms control lignin quantity and composition in each cell wall domain and cell type? In addition to controlling monolignol composition, LACs and PRXs have distinct localization to different cell wall domains and cell types (Chou et al., 2018; Hoffmann et al., 2020; Schuetz et al., 2014). Several lines of evidence suggest that some LACs and PRXs have monolignol substrate specificities, and that complex regulation of substrate preferences exists. In Arabidopsis, single or double mutants of lac4-2 and lac17 (Berthet et al., 2011), and single mutants of prx2, prx25, and prx71 (Shigeto et al., 2013) had increased syringyl-to-guaiacyl (S/G) ratio; while single mutants of prx4, prx52, and prx72 had decreased S/G ratio (Fernández-Pérez et al., 2015a, 2015b, 2015c; Herrero et al., 2013). Altered lignin composition suggested that these oxidative enzymes may preferentially activate coniferyl alcohol or sinapyl alcohol into G or S lignin units, respectively. However, in vitro assessments of Arabidopsis PRX substrate specificities with oxidative assays showed that while recombinant PRX2, PRX25, and PRX71 had high specificities for syringyl compounds, they could also 44 oxidize guaiacyl compounds and ferrocytochrome c, a large macrocyclic molecule, suggesting a broad substrate specificity (Shigeto et al., 2014). Another system for assessing substrate specificities of oxidative enzymes involves Cleome hassleriana seed coats where G-rich lignin first accumulates, followed by the caffeyl alcohol-rich catechyl (C) lignin (Zhuo et al., 2022). In cell wall extracts of the developing seed coats, there was similar LAC activity against either coniferyl or caffeyl alcohols; however, LAC activity was non-competitively inhibited by caffeyl alcohol, leading to less oxidation of coniferyl alcohol and preferential incorporation of caffeyl alcohol into the seed coat, despite the continual production of both monolignols (Zhuo et al., 2022). Taken together, these results suggest a complex regulation of oxidative enzyme substrate specificities, requiring the identification of oxidative enzymes involved in lignification for biochemical assays and mutant studies. 3.1.2 Biomechanical roles of lignin in distinct cell wall domains Distinct phenolic and polysaccharide profiles in each cell wall domain likely play unique biomechanical roles in supporting plant growth. Studies of biomechanical contributions of lignin in the cell wall mostly focus on lignin variation among woody species. Comparing wood samples from three pine variants showed that decreased lignin between the S1 and S2 secondary cell wall layers led to more fracture events between these layers, rather than between the middle lamella and the S1 layer (Donaldson, 1995). Eastern leatherwood has no lignin in the cell corners and the middle lamella, resulting in flexible stems (Mottiar et al., 2020). To understand the biomechanical contributions of lignin in each cell wall domain within the plant, a study compared mechanical properties of the compound middle lamella and the secondary cell wall in larch sapwood, showing that the middle lamella had a lower elastic modulus but similar hardness compared to the secondary cell wall (Qin et al., 2018). Recently, mutant studies in Arabidopsis and Populus showed that cad mutants deposited more aldehydes than monolignols into cell walls, resulting in reduced stiffness (Ménard et al., 2022; Özparpucu et al., 2017). Although cad mutations likely have a larger impact on the lignin composition of the cell corners and the middle lamella 45 where aldehyde content is normally low, the lignin composition of the secondary cell wall is also altered (Liu et al., 2015; Özparpucu et al., 2017). Probing the biomechanical contributions of lignin composition in distinct cell wall domains will require experimental tools where only lignin in each domain is altered, without global changes to lignin compositions. Since oxidative enzymes localized to each cell wall domain have been documented (Hoffmann et al., 2020), mutating oxidative enzymes may enable targeted alteration in lignin quantities and compositions in each domain. To achieve this, two higher order oxidative enzyme mutants were created: lac4 lac17 prx72, representing oxidative enzymes localized to the thick secondary cell wall; and lac4 prx64 prx71, representing oxidative enzymes localized to the cell corners and the middle lamella (Hoffmann et al., 2020). Interestingly, LAC4 was expressed strongly in the secondary cell wall throughout development, but it also appeared transiently in the cell corners and middle lamella during early fibre development (Hoffmann et al., 2020), so a LAC4 mutation had to be engineered into both higher order mutants. While most mutant studies focussed on creating higher order mutants in either LACs or PRXs, this study is the first to create mutants in both oxidative enzyme families based on their co-occurrence in distinct cell wall domains. 3.1.3 Genetic strategies for creating higher order oxidative enzyme mutants To create higher order oxidative enzyme mutants, a common strategy is to cross existing T-DNA insertional mutant lines, and screen for homozygous mutants in the F2 generation. However, only 1 out of 64 individuals in the F2 generation would be a homozygous triple mutant. Potential genetic linkage between LAC17, PRX64, PRX71, and PRX72, all located on chromosome 5, further decreases the likelihood of generating homozygous triple mutants. An alternative to crossing mutant lines is to use CRISPR/Cas9 to mutate additional oxidative enzyme genes in existing oxidative enzyme T-DNA mutant lines. In this system, genomic sequences immediately upstream of an NGG motif in the target gene are fused with a palindromic sequence; when transcribed, the resulting guide RNA 46 (gRNA) will bind the target gene and recruit the heterologously expressed Streptococcus pyogenes Cas9 endonuclease to make double-stranded cuts in the genomic DNA adjacent to the NGG motif (Hsu et al., 2014). As the cell repairs the damaged DNA, indel mutations are often introduced (Hsu et al., 2014). In Arabidopsis, CRISPR/Cas9 reliably generates heritable indel mutations by expressing gRNAs and the Cas9 gene with egg cell-specific promoters (Wang et al., 2015). The ability to target multiple genes with a single Cas9 construct will generate higher order mutants within a shorter timeframe. The efficiency and specificity of the CRISPR/Cas9 system are highly dependent on the chosen target sequences. When choosing target sequences, the following should be considered: (1) target sequences must be immediately upstream of an NGG motif recognized by Cas9; (2) certain target sequences have been shown to decrease Cas9 efficiency (Graf et al., 2019) and should be avoided; (3) target sequences should be unique to the target gene to minimize off-target edits. Therefore, in silico efficiency prediction and off-target searches within the organism’s genome (Concordet & Haeussler, 2018; Doench et al., 2016) can be used to predict the outcomes of CRISPR/Cas9 mutagenesis. The objective of this study was to use CRISPR/Cas9 to generate higher order oxidative enzyme mutants to deplete lignin, either in the thick secondary cell wall (lac4 lac17 prx72) or in cell corners and middle lamella (lac4 prx64 prx71). I hypothesize that if lignin is reduced in each cell wall domain independently, I will observe phenotypes that reflect altered biophysical properties of each domain. 3.2 Results 3.2.1 CRISPR/Cas9 generates indel mutations in PRX64, PRX71, and PRX72 To generate higher order oxidative enzyme mutants, oxidative enzymes with known localization to specific cell wall domains were mutated with CRISPR/Cas9 (Wang et al., 2015). To mutate LAC4, PRX64, and PRX71 found in the middle lamella and cell corners, gRNAs were designed to target genomic sequences of PRX64 and PRX71 in 47 the lac4-2 mutant background (Berthet et al., 2011). To mutate LAC4, LAC17, and PRX72 oxidative enzymes found in the secondary cell wall, gRNAs were designed to target the genomic sequence of PRX72 in the lac4-2 lac17 mutant background (Berthet et al., 2011). 3-10% of T1 plants transformed with the Cas9 construct containing gRNA sequences had monoallelic indel mutations that could be detected by PCR and gel electrophoresis. If no major indel mutation was detected in T1 plants, some T1 plants were taken to the T2 generation to be screened for mutations. Sequencing confirmed the creation of one mutant allele of prx64, two mutant alleles of prx71 with the same mutation, and three mutant alleles of prx72, all of which were indel frameshift mutations predicted to generate premature stop codons (Figure 3.1B, C, D). Figure 3.1. Arabidopsis laccase and peroxidase mutants and predicted amino acid sequences of mutant proteins. (A) lac and lac were T-DNA insertional mutations characterized by Berthet et al. (2011). ( ) prx6 , ( ) prx , and ( ) prx were Wild type prx lines 15, 53 (1 bp insertion) A (AT 3 ) lac (GK-720G02)P A (AT5 6 )P lac (SALK 016748) (AT5 6 )P Wild type prx6 line 26 (91 bp deletion) P 6 (AT5 ) Wild type prx line 19 (244 bp deletion) A prx line 3 (1 bp deletion + 101 bp insertion + 1 bp insertion) prx line 49 (1 bp insertion + 50 bp deletion) Wild type (AT5 663 )P48 CR SPR Cas9-induced indel mutations (marked red), confirmed by sequencing. Arrows and brackets indicate the position of the mutations in each gene. Asterisks indicate premature stop codons. Grey boxes represent promoters, white boxes represent untranslated regions, black boxes represent exons, and horizontal lines represent introns. xon-intron structures are based on representative gene models in The Arabidopsis nformation Resource (TA R) database (https: www.arabidopsis.org ). After generating lac4-2 lac17 prx72 triple mutants, qRT-PCR was used to quantify LAC4, LAC17, and PRX72 expression levels in whole seedlings. Preliminary analysis on one biological replicate revealed that the lac4-2 lac17 double mutant and all three triple mutant lines consistently showed a knockdown of LAC4 and LAC17 expression levels compared to wild type, validating previous results by Berthet et al. (2011). Compared to wild-type seedlings, PRX72 expression increased 1.4-fold in the lac4-2 lac17 double mutant, while triple mutant lines #3 (0.05-fold) and #49 (0.09-fold) showed PRX72 knockdown (Figure 3.2A). Interestingly, triple mutant line #19 showed the same PRX72 expression (1.4-fold) as the lac4-2 lac17 double mutant (Figure 3.2A), despite a 244 bp deletion where the start codon and the entire exon 1 of PRX72 would be missing (Figure 3.1D). These data revealed compensatory gene regulation of PRX72 in lac4-2 lac17 seedlings, as well as variability in PRX72 transcript levels among CRISPR/Cas9 mutant lines. Although the transcript is upregulated in line 19, I predict that the protein will not be made due to the lack of a start codon. In addition to seedlings, gene expression in the top 20 cm of primary inflorescence stems was also tested. Compared to wild-type stems, and in contrast to the seedling data, PRX72 transcript levels decreased in the lac4-2 lac17 double mutant (0.4-fold), while triple mutant line #3 (0.11-fold) and #49 (0.07-fold) showed further PRX72 knockdown (Figure 3.2B). Triple mutant line #19 showed a similar PRX72 expression (0.3-fold) as the lac4-2 lac17 double mutant (Figure 3.2B). Therefore, stems of double and triple mutants showing PRX72 knockdown were used in further analyses below. 49 Figure 3.2. Preliminary quantification of A , A , and expression levels in lac lac prx plants. qRT-PCR quantified gene expression in (A) 7-day-old whole seedlings, and ( ) the top 20 cm primary inflorescence stems of 6-week-old T3 lac lac prx plants, relative to wild-type Col-0 plants and lac lac plants. Wild-type gene expression was normalized to 1. Three independent mutant lines of lac lac prx plants were analyzed. 4-5 plants were pooled per line, and three technical replicates were performed. One biological replicate was analyzed. 3.2.2 lac4-2 lac17 prx72 triple mutants are not phenotypically different from the lac4-2 lac17 double mutant, while the lac4-2 prx64 double mutant showed delayed stem growth Previous studies showed that mutations in oxidative enzymes resulted in dwarfism, including the lac4-2 lac17 double mutant which showed a semi-dwarf phenotype when grown under continuous light conditions, but not under long-day conditions (Berthet et al., 2011). I hypothesized that additional PRX72 mutations in the lac4-2 lac17 background would lead to further growth penalty, even when grown under long-day conditions. At four and six weeks old, lac4-2 lac17 had slightly shorter stems than wild type, and all three lac4-2 lac17 prx72 triple mutant lines did not show significantly shorter stems than lac4-2 lac17 (Figure 3.3). This suggests that additional PRX72 mutations did not worsen the phenotype of lac4-2 lac17. 00.20.40.60.81 A A 00.20.40.60.811.21.41.6 A A Relative expression (fold change) Seedling StemA 50 Figure 3.3. (A) Phenotypes and ( ) stem heights (in cm) of 4- and 6-week-old T3 lac lac prx plants. Stem height data were from three independent experiments (8-10 plants per line in each independent experiment), grown under long-day conditions. Scale bar = 10 cm. Kruskal-Wallis χ2(Week 4) = 32.1, p(Week 4) = 1.8e-6; χ2(Week 6) = 18.3, p(Week 6) = 1.1e-3. Different letters denote significant statistical difference calculated with post-hoc Dunn’s test and Benjamini-Hochberg adjustments. A previous study showed that the prx64 single mutant had reduced lignification in the Casparian strip (Lee et al., 2013), but no qualitative mutant phenotype was observed in the stem (Hoffmann, 2019). Other studies found that the prx71 single mutant had increased growth (Raggi et al., 2015) and altered β-O-4 lignin linkages, but no change in total lignin (Shigeto et al., 2013). Given that LAC4, PRX64, and PRX71 localized to the cell corners and the middle lamella of interfascicular fibres (Hoffmann et al., 2020), functional redundancy of oxidative enzymes occupying the same cell wall domain likely compensated for the loss of PRX64 activity to maintain proper growth. I hypothesized that additional prx64 and prx71 mutations in the lac4-2 background would 0102030405060a b b b ba b b ab bStem height (cm)10 cmWeek 4 Week 6A 51 lead to delayed stem development similar to the lac4-2 lac17 double mutant (Berthet et al., 2011) (Figure 3.3). While lac4-2 prx64 prx71 triple mutants were still segregating, homozygous lac4-2 prx64 double mutants were isolated for preliminary analysis. At 4 and 6 weeks, lac4-2 prx64 had shorter stems than the wild type and the lac4-2 single mutant (Figure 3.4). Delayed stem growth of lac4-2 prx64 suggests that disrupting oxidative enzyme activity in the cell corners and middle lamella of interfascicular fibres negatively impacts plant growth. Figure 3.4. (A) Phenotypes and ( ) stem heights (in cm) of 4- and 6-week-old T3 lac prx6 plants. Stem height data were from three biological replicates of lac and lac prx6 (8-10 plants per line per biological replicate), and one biological replicate of wild-type Col-0 (8-10 plants), grown under long-day conditions. Scale bar = 10 cm. Kruskal-Wallis χ2(Week 4) = 20.8, p(Week 4) = 3.1e-5; χ2(Week 6) = 16.2, p(Week 6) = 3.1e-4. Different letters denote significant statistical difference calculated with post-hoc Dunn’s test and Benjamini-Hochberg adjustments. 0102030405060a a ba a bWeek 4 Week 6Stem height (cm)10 cmA 52 3.2.3 lac4-2 lac17 prx72 triple mutants showed a slight decrease in stem lignin compared to the lac4-2 lac17 double mutant Since the lac4-2 lac17 double mutant had decreased lignin in the stem (Berthet et al., 2011), and that prx72 single mutant had altered lignin composition and thinner fibre cell wall (Fernández-Pérez et al., 2015a; Herrero et al., 2013), I hypothesized that lac4-2 lac17 prx72 triple mutants would show a further decrease in lignin. Acetone-insoluble stem tissues of lac4-2 lac17 prx72 were treated with acetyl bromide, and the amount of acetyl bromide soluble lignin (ABSL) was quantified with UV-Visible spectrophotometry. All three lac4-2 lac17 prx72 triple mutant lines showed a slight decrease in ABSL relative to the lac4-2 lac17 double mutant (Figure 3.5A, Table 3.1). To account for the variation observed between independent experiments, quantities of ABSL relative to wild-type plants in each independent experiment were calculated, confirming that prx72 mutations further decreased lignin content (Figure 3.5B, Table 3.1). In particular, lac4-2 lac17 prx72 line #19, which was predicted to have a loss-of-function PRX72 mutation (Figure 3.1), had the least ABSL (Figure 3.5, Table 3.1). These data suggest that PRX72 is involved in stem lignification despite PRX72 knockdown in lac4-2 lac17 stems (Figure 3.2), and that lignification can be further decreased by knocking out additional secondary cell wall-bound oxidative enzymes. 53 Figure 3.5. Quantification of acetyl bromide soluble lignin (ABSL) in the top 20 cm primary inflorescence stems of 6-week-old T3 lac lac prx plants. (A) Quantity of ABSL (in grams) per gram of dried, acetone-insoluble stem tissues. ( ) Quantity of ABSL relative to wild-type plants (wild-type quantity was normalized to 1). lac lac (Berthet et al., 2011) was used as a control. Three independent mutant lines were analyzed. 4-5 plants were pooled per line in each independent experiment. Bars and error bars represent mean quantity from three independent experiments ± SD, with three technical replicates per sample in each independent experiment. Table 3.1. Quantification of acetyl bromide soluble lignin (ABSL) in dried, acetone-insoluble, top 20 cm primary inflorescence stems of 6-week-old T3 lac lac prx plants. Three independent mutant lines were analyzed. 4-5 plants were pooled per line in each independent experiment. Mean values are from three independent experiments ± S of the mean, with three technical replicates per sample in each independent experiment. Genotype ABSL (g / g stem) (± SEM) ABSL Fold change (± SEM) Col-0 0.127 ± 0.007 1.00 ± 0.03 lac4-2 lac17 0.075 ± 0.006 0.60 ± 0.03 lac4-2 lac17 prx72 #3 0.065 ± 0.008 0.54 ± 0.02 lac4-2 lac17 prx72 #19 0.063 ± 0.007 0.52 ± 0.01 lac4-2 lac17 prx72 #49 0.067 ± 0.009 0.56 ± 0.04 0.000.050.100.150.00.30.60.9Relative ABSL (Fold change)g ABSL g stemA 54 3.2.4 Histochemical staining of lac4-2 prx64 and lac4-2 lac17 prx72 stem cross-sections showed altered lignin composition The S/G ratio of the lac4-2 single mutant was similar to wild-type plants, while the lac4-2 lac17 double mutant was shown to have a higher S/G ratio than wild-type plants (Berthet et al., 2011). I hypothesized that lignin composition of the lac4-2 single mutant would be altered by an additional prx64 mutation. To visualize lignin composition in stem cross-sections, UV autofluorescence was used to detect total lignin, Wiesner staining was used to detect coniferaldehyde as a proxy for G-rich lignin (Blaschek et al., 2020), and Mäule staining was used to detect S-rich lignin. Stem cross-sections of Col-0 and lac4-2 showed similar UV autofluorescence (Figure 3.6A, D), a strong red colour in G-rich vessels after Wiesner staining (Figure 3.6B, E), and a strong red colour in S-rich fibres after Mäule staining (Figure 3.6C, F), consistent with unaltered lignin composition in lac4-2. An additional prx64 mutation in the lac4-2 background led to weaker lignin autofluorescence (Figure 3.6D, G) and weaker Mäule staining in S-rich fibres (Figure 3.6F, I), while Wiesner staining patterns were similar (Figure 3.6E, H). This suggests that lac4-2 prx64 plants could have a decreased S/G ratio compared to lac4-2. However, the cell corners and the middle lamella remained lignified (Figure 3.6G). Since lac4-2 lac17 prx72 plants had less stem lignin than lac4-2 lac17 plants (Figure 3.5), and previous thioacidolysis data showed a decrease in S-units in prx72 T-DNA single mutants (Fernández-Pérez et al., 2015a), I hypothesized that additional prx72 mutations would alter the lignin composition of lac4-2 lac17. Stem cross-sections of lac4-2 lac17 showed much weaker UV autofluorescence, Wiesner staining, and Mäule staining in fibres compared to Col-0 (Figure 3.6A-C, J-L). Additional prx72 mutations in the lac4-2 lac17 background led to even weaker Mäule staining in fibres and vessels (Figure 3.6L, O), while autofluorescence (Figure 3.6J, M) and Wiesner staining patterns (Figure 3.6K, N) remained unchanged. The weaker Mäule staining in lac4-2 lac17 prx72 suggested that additional prx72 mutations in the lac4-2 lac17 background led to a global decrease in S-rich lignin. The cell corners and the middle lamella remained lignified in lac4-2 lac17 prx72 (Figure 3.6M), confirming that these mutations only reduced lignification in the secondary cell wall. 55 UV Wiesner MäuleCol-0lac lac lac lac prx6 lac lac prx A 56 Figure 3.6. UV lignin autofluorescence and histological staining of lac prx6 and lac lac prx stem cross sections. Serial cross-sections were taken 20 cm from the shoot apical meristem of the primary inflorescence stem. Cross-sections were stained with the Wiesner method (phloroglucinol-HCl) to detect coniferaldehyde or the Mäule method (KMnO4, HCl, Na2CO3) to detect syringyl lignin units. Wild-type Col-0, lac , and lac lac stem cross-sections were imaged as controls. Scale bar = 50 µm. 3.2.5 Xylem vessels in lac4-2 lac17 prx72 triple mutants were more irregularly shaped than in the lac4-2 lac17 double mutant Irregular xylem vessels were observed in the stem cross-sections of the lac4-2 lac17 double mutant (Berthet et al., 2011). To assess if additional mutations in PRX72 in the lac4-2 lac17 mutant background would result in further vessel irregularity, stem cross-sections were stained with safranin and imaged with confocal microscopy to visualize vessel cell walls. Curvature values were mapped at each point of the cell wall for shape analysis (Figure 3.7A-C). More extreme curvature values were observed in the lac4-2 lac17 prx72 triple mutant compared to the lac4-2 lac17 double mutant (Figure 3.7A-D), confirmed by increased standard deviation of curvature values within individual vessels (Figure 3.7E). Vessel irregularity was further characterized by two shape descriptors: circularity, which describes deviations from a perfect circle, and solidity, which describes deviations from an entirely convex shape. Vessels in lac4-2 lac17 prx72 deviated more from a perfect circle (Figure 3.7F), but were not less convex (Figure 3.7G) than vessels in lac4-2 lac17. This suggests that vessel irregularity in lac4-2 lac17 prx72 plants is dominated by extreme convexity, rather than extreme concavity. 57 Figure 3.7. Measures of xylem vessel irregularity from stem cross-sections of lac4-2 lac17 prx72 triple mutants. (A-C) Representative vessel cell wall outlines from Col-0, lac4-2 lac17 double mutant, and lac4-2 lac17 prx72 triple mutant stem cross-sections. Curvature values (κ, μm−1) were calculated and mapped at each pixel. Curvature > 0 (red) represents convex vessel cell walls, while curvature < 0 (blue) represents concave vessel cell walls. Scale bars = 10 μm. (D) Distribution of curvature values along vessel -1012Curvature-1012Curvature-7.5-5.0-2.50.02.55.00.000.250.500.751.000.60.81.00.800.850.900.951.00Curvature (µm-1) SD(Curvature ) (µm-1) Circularity Solidity abbccddab bccddabbbbA Col-0 lac lac lac lac prx -1012Curvature10 µm 10 µm 10 µm58 cell walls. Black dots indicate mean curvature values. (E) Standard deviation of cell wall curvature values within individual vessels. Kruskal-Wallis χ2 = 117.1, p < 2.2e-16. (F) Circularity (4π × area perimeter2) of vessel cell walls. Circularity = 1 represents a perfect circle. Kruskal-Wallis χ2 = 96.1, p < 2.2e-16. (G) Solidity of vessel cell walls (area / area of the convex hull). Solidity = 1 represents an entirely convex shape. Kruskal-Wallis χ2 = 111.3, p < 2.2e-16. Two serial cross-sections from each of 2-3 stems were sampled. 2-3 vascular bundles were imaged per cross-section, and 3-4 vessels were analyzed per vascular bundle. Different letters denote significant statistical difference calculated with post-hoc Dunn’s test and Benjamini-Hochberg adjustments. 3.3 Discussion 3.3.1 Targeted lignin removal from distinct cell wall domains has unique phenotypic outcomes In this study, higher order oxidative enzyme mutants were constructed to reflect the subsets of LACs and PRXs found in different cell wall domains. The lac4-2 lac17 prx72 triple mutants, where oxidative enzymes are depleted in the thick secondary cell wall, showed lower ABSL (Figure 3.5) and more irregular xylem (Figure 3.7) compared to the lac4-2 lac17 double mutant. Mäule staining of lac4-2 lac17 and lac4-2 lac17 prx72 stem cross-sections suggested that prx72 mutations led to a decrease in S lignin units (Figure 3.6), agreeing with the decreased S/G ratio in the prx72 T-DNA single mutant (Fernández-Pérez et al., 2015a; Herrero et al., 2013). This indicates that the PRX72 works non-redundantly with LAC4 and LAC17 in the developmental lignification of Arabidopsis stems, and is functionally important in making xylem vessels that can resist negative pressure. Based on the observation that LAC4, PRX64, and PRX71 co-localize to the cell corners and the middle lamella (Hoffmann et al., 2020), the lac4-2 prx64 double mutant and the lac4-2 prx64 prx71 triple mutant were generated (Figure 3.1) to test if targeted lignin removal from cell corners and the middle lamella is possible. This study showed that the lac4-2 prx64 double mutant had shorter stems (Figure 3.4) and less lignin in interfascicular fibres (Figure 3.6D, F, G, I) than the lac4-2 single mutant. However, lac4-2 prx64 still had lignified cell corners and middle lamella (Figure 3.6G), suggesting that additional oxidative enzymes at this cell wall domain compensated for the loss of 59 PRX64. Future gene expression, chemical, and biomechanical analyses of lac4-2 prx64 and lac4-2 prx64 prx71 mutants will determine if targeted lignin removal from the cell corners and the middle lamella is possible, and establish the biomechanical role of lignin in this cell wall domain. 3.3.2 PRX72 compensates for lac4 lac17 mutations in seedlings but not stems Given the co-localization of LAC4, LAC17, and PRX72 in the secondary cell wall of lignifying cells (Hoffmann et al., 2020), it can be hypothesized that the expression of LAC4, LAC17, and PRX72 are co-regulated. RNA-seq analysis showed that PRX72 had higher magnitudes of upregulation or downregulation than LAC4 or LAC17 in the stems of phenylpropanoid and monolignol pathway mutants (Wang et al., 2022), but changes in PRX72 expression were not previously studied in oxidative enzyme mutants. Preliminary gene expression data showed that in seedlings, where tracheary elements in roots, hypocotyls, and cotyledons are actively lignifying, PRX72 is overexpressed in the lac4-2 lac17 double mutant relative to wild type (Figure 3.2). This suggests a feedback mechanism that upregulates PRX72 expression to compensate for reduced LAC4 and LAC17 expression levels during the lignification of tracheary elements. In contrast, preliminary gene expression data showed that in stems, where more lignifying cell types are present, PRX72 is downregulated in the lac4-2 lac17 double mutant relative to wild type (Figure 3.2). This suggests that PRX72 does not compensate for reduced LAC4 and LAC17 expression levels in stems. 3.3.3 Increased vessel irregularity and reduced lignin do not contribute directly to additional growth penalty The relationship between lignin deficiency and poor plant growth, which includes extreme cases of lignin modification-induced dwarfism (LMID), remains unclear. Previous studies showed that oxidative enzyme mutants lac4-2 lac17 (Berthet et al., 2011), lac4-2 lac5-1 lac10-1 lac12-2 lac17-1 (Blaschek et al., 2023), and prx72 (Fernández-Pérez et al., 2015a; Herrero et al., 2013) showed irregular xylem, reduced lignin, and reduced growth. It has been proposed that lignin perturbation in the vasculature, but not interfascicular fibres, will lead to dwarfism. This proposition seemed 60 to be supported by studies that produced non-dwarf plants by restoring CCR1 expression with a vessel-specific synthetic promoter in the lignin-deficient ccr1 mutant (De Meester et al., 2018), or by selectively knocking down CCR1 expression with the interfascicular fibre-specific proPRX64 promoter (Smith et al., 2017). However, these studies did not directly correlate vessel irregularity with dwarfism. My study showed that lac4-2 lac17 prx72 mutants had more irregular vessels (Figure 3.7) but not shorter stems than lac4-2 lac17 (Figure 3.3), suggesting that increased xylem irregularity does not necessarily lead to more growth penalty. A study of lignin-deficient C3'H knockdown poplar plants suggested that despite collapsed vessels and less efficient water transport, photosynthesis is not limited because sugar accumulation increased in the leaves compared to wild type (Coleman et al., 2008b). They concluded that stunted plant growth is not due to vessel collapse or water usage, but instead due to the blockage of a rate-limiting step in the monolignol biosynthetic pathway, which reduced carbon sink strength and carbon flow from the leaves to the rest of the plant (Coleman et al., 2008b). Therefore, lignin modification-induced dwarfism may be a systemic response to altered metabolism in lignifying cells, a significant carbon sink in vascular plants. 3.3.4 Perturbing lignification in the interfascicular fibres may hinder plant growth and development The ability to remove lignin in the cell corners and the middle lamella is valuable for creating low-lignin plants for biofuels and flexible wood similar to Eastern leatherwood (Mottiar et al., 2020) for bioproducts. Reduced lignin in the secondary cell wall is observed in numerous phenylpropanoid pathway mutants and oxidative enzyme mutants, but no study has been able to remove lignin specifically from the cell corners and the middle lamella, without affecting the secondary cell wall. A recent study showed that the Arabidopsis lac4-2 lac5-1 lac10-1 lac12-2 lac17-1 quintuple mutant had weak lignin autofluorescence in the cell corners and the secondary cell wall, but lignin was not reduced in the middle lamella of interfascicular fibres (Blaschek et al., 2023). Restoring LAC12 expression in the quintuple mutant reverted cell corner autofluorescence to wild-61 type levels, but secondary cell wall autofluorescence remained weak (Blaschek et al., 2023). Arabidopsis PRX64 and PRX71 localize to the cell corners and the middle lamella of interfascicular fibres (Hoffmann et al., 2020), making them desirable targets of mutagenesis. The prx64 single mutant had less lignin in the Casparian strip (Lee et al., 2013) and the prx71 single mutant had increased plant growth (Raggi et al., 2015) and altered lignin linkage (Shigeto et al., 2013), but no major lignification defects were observed in the stem (Hoffmann, 2019; Shigeto et al., 2013). In the lac4-2 prx64 double mutant, it is observed that lignification was reduced in the secondary cell wall of interfascicular fibres, rather than in the cell corners or the middle lamella (Figure 3.6D-I). To explain why lignification in the secondary cell wall is affected by an additional prx64 mutation, it can be speculated that while PRX71 is compensating for lignification at the cell corners and the middle lamella, the loss of LAC4 and PRX64 weakens the concentration gradient driving monolignol diffusion to the cell wall, leading to less lignified secondary cell walls. This suggests that it may not be possible to remove lignin from the cell corners or the middle lamella without affecting the secondary cell wall. Characterizing the lac4-2 prx64 prx71 triple mutant will determine if these three oxidative enzymes are responsible for lignification in the cell corners and the middle lamella, or if lignification in the secondary cell wall will be further reduced. Compared to tracheary elements, interfascicular fibres are more amenable to lignin modification. Multiple mutants with hypolignified interfascicular fibres, such as snd1 nst1 (Zhong et al., 2010a), and CCR1 RNA interference knockdown in interfascicular fibres (Smith et al., 2017), showed floppy stems but not dwarfism. In this study, hypolignified interfascicular fibres of lac4-2 prx64 (Figure 3.6D-I) led to delayed stem growth compared to lac4-2 (Figure 3.4), without further mutations in oxidative enzymes localized to tracheary elements. This suggests that in addition to vessel integrity, fibre integrity also contributes to proper plant growth and development. To conclude, LACs and PRXs are essential to lignification in distinct cell wall domains, and their activity is controlled by substrate specificity or more complicated 62 biochemical mechanisms. Targeted lignin removal from each cell wall domain by mutating LACs and PRXs leads to different phenotypic outcomes, suggesting the unique contribution of lignin in each cell wall domain to plant growth and development. A better understanding of oxidative enzyme activity, lignin deposition in the cell wall, and lignin modification-induced dwarfism will inform strategies for lignin modification for biofuels and bioproducts. 3.4 Methods 3.4.1 Plant growth condition Refer to section 2.4.1. 3.4.2 Designing and cloning CRISPR/Cas9 constructs Mutants of peroxidases in Arabidopsis were created with CRISPR/Cas9 for dicots (Wang et al., 2015). pCBC-DT1T2, pCBC-DT2T3, pCBC-DT3T4, and pKEE401 constructs were gifted by Qi-Jun Chen (China Agricultural University) (Wang et al., 2015) and obtained from Addgene (plasmids #50590, 50591, 50592, 91715). The pBEE401 construct was modified from pKEE401 (Wang et al., 2015) by replacing the kanamycin resistance marker with a BASTA resistance marker, gifted by Yuelin Zhang (University of British Columbia). Two 20 bp exonic sequences within each of Arabidopsis PRX64 (AT5G42180), PRX71 (AT5G64120), and PRX72 (AT5G66390) were identified as CRISPR/Cas9 targets with CRISPOR (http://crispor.tefor.net/) based on efficiency and specificity (Concordet & Haeussler, 2018). To incorporate PRX64 and PRX71 targets into gRNA sequences, pCBC-DT1T2, pCBC-DT2T3, and pCBC-DT3T4 were PCR amplified with primers containing exonic target sequences; to incorporate PRX72 targets into gRNA sequences, only pCBC-DT1T2 was PCR amplified with primers containing exonic target sequences. Detailed primer design and PCR procedures were described by Wang et al. (2015). PCR products were incorporated into the pBEE401 Cas9 destination vector through Golden Gate Assembly (BsaI and T4 ligase, New England Biolabs) with a 37°C/16°C thermal cycle (Supplementary Table 2). All Cas9 plant expression clones 63 containing gRNA sequences were transformed into chemically competent Subcloning Efficiency™ DH5α E. coli (Invitrogen) and miniprepped with the Presto™ Mini Plasmid Kit (Geneaid Biotech). Sequences of expression clones were verified (Azenta Life Sciences) before transformation into electrocompetent Agrobacterium tumefaciens GV3101 strain (GoldBio). All primers and PCR conditions are listed in Supplementary Tables 1 and 2. 3.4.3 Arabidopsis transformation with Agrobacterium floral dip CRISPR/Cas9 constructs containing gRNA sequences for PRX64 and PRX71 were transformed into lac4-2 mutants (Berthet et al., 2011), and constructs containing guide RNA sequences for PRX72 were transformed into lac4-2 lac17 mutants (Berthet et al., 2011). 1 mL overnight culture of Agrobacterium tumefaciens containing CRISPR/Cas9 constructs was inoculated into 50 mL liquid YEB media (5 g/L beef extract, 1 g/L yeast extract, 5 g/L peptone, 5 g/L sucrose, 0.5 g/L MgCl2) supplemented with 50 µg/mL gentamycin and 50 µg/mL kanamycin. Refer to section 2.4.3 for the remaining transformation procedure. 3.4.4 Selection of plant transformants and screening for gene mutations T1 seeds were collected and screened for BASTA resistance. Chlorine gas-sterilized seeds were plated on Germination Media supplemented with 25 μg mL DL-phosphinothricin (BASTA, Phytotech Labs, P679), stratified at 4°C for 3 days, and germinated at 21°C under 24 hours of light. Only seedlings showing green true leaves and root growth were transferred to soil. Refer to section 2.4.1 for general plant growth conditions. Genomic DNA was extracted from the rosette leaves of T1 plants with Edwards buffer (Edwards et al., 1991). DNA extracts were used as PCR templates to screen for gene mutations. PCR products were gel purified with the Monarch® DNA Gel Extraction Kit (New England Biolabs) and sequenced (Azenta Life Sciences) to confirm the mutations. T1 plants heterozygous for the mutations were taken to the T2 generation to be screened for plants homozygous for the mutations. T3 homozygotes were used for 64 experiments. All primers and PCR conditions are listed in Supplementary Tables 1 and 2. 3.4.5 RNA extraction, cDNA synthesis, and quantitative real-time PCR The top 20 cm primary inflorescence stems (up to the uppermost leaf node) of 6-week-old plants and 7-day-old seedlings germinated on Germination Media (Section 2.4.1) were frozen in liquid nitrogen and ground into a powder with a mortar and pestle. Refer to section 2.4.5 for the remaining procedure. 3.4.6 Acetyl bromide soluble lignin (ABSL) quantification Frozen stem tissues were first incubated in 80% methanol (in H2O, v/v) for 15 minutes at 70°C, centrifuged at 18,000 × g for 5 minutes, and the supernatant was removed. The remaining tissues were subject to acetone extraction and ABSL quantification. Refer to section 2.4.7 for the remaining procedure. 3.4.7 Stem cross-sectioning, staining, and microscopy 1 cm-long sections of primary inflorescence stems were taken 20 cm from the shoot apical meristem of 6-week-old plants. Sections were cleared with 50% ethanol (in H2O, v/v) for at least 3 days and embedded in 4% low melting point agarose. 80 µm-thick stem cross-sections were cut with a Leica VT1000S vibratome, stored in H2O, and stained with 0.2% safranin O (w/v) (Sigma-Aldrich, S2255) in 50% ethanol as described by Baldacci-Cresp et al. (2020b). Images of safranin O-stained cross-sections were acquired with the Olympus FV1000 Laser Scanning Microscope, a Spectra-Physics Mai Tai DeepSee pulsed laser, and the Olympus UPLSAPO60×W 60× water immersion objective (NA = 1.2) (Bioimaging Facility, University of British Columbia). Images were acquired with a 473 nm laser and 530-560 nm emission wavelengths (green channel), and with a 559 nm laser and 570-600 nm emission wavelengths (red channel). Refer to section 2.4.8 for UV illumination, Wiesner staining, and Mäule staining protocols. 65 3.4.8 Cell wall thickness and curvature analysis To analyze irregularity in vessel cell walls, outlines of vessel cell walls were traced counterclockwise with the Kappa tool (Mary & Brouhard, 2019) in ImageJ (Schindelin et al., 2012) to generate a closed B-spline for each outline. X- and Y-coordinates of all pixels defining the B-splines and curvature values at each pixel were exported and analyzed with R 4.1.2 (R Core Team, 2021). The following functions or packages were used: chull, to extract x- and y-coordinates that make up the convex hull from each B-spline; pracma (Borchers, 2022), to calculate the perimeter of and the area enclosed by each B-spline, and the area of the convex hull; and plyr (Wickham, 2011) and dplyr (Wickham et al., 2023), to organize the data. Circularity is calculated as (4π × area / perimeter2) of the shape enclosed by the B-spline, and solidity is calculated as the area enclosed by the B-spline divided by the area of the convex hull. 3.4.9 Statistical analyses All statistical analyses were performed with R 4.1.2 (R Core Team, 2021) with the following packages: rcompanion (Mangiafico, 2023) and FSA (Ogle et al., 2023). Plots were produced in R with ggplot2 (Wickham, 2016) and ggpubr (Kassambara, 2023), and in Microsoft Excel. 66 4 Conclusion 4.1 Major findings 4.1.1 Dwarfism due to MYB63 overexpression is triggered in non-lignifying cells Chapter 2 of this thesis aims to address two objectives: to assess the capacity of lignification in lignifying cell walls; and to test the consequences of additional or ectopic monolignol production in lignifying and non-lignifying cell types in Arabidopsis stems. Arabidopsis MYB63 is a transcriptional activator of the shikimate and the phenylpropanoid pathways, leading to monolignol production (Figure 1.1, Table 2.1) (Perkins et al., 2020; Zhou et al., 2009). MYB63 overexpression in all cell types resulted in dwarf plants with high monolignol levels and a stress-associated transcriptome (Perkins et al., 2020; Zhou et al., 2009). Given monolignols are cytotoxic and lignification involves genes and reactive oxygen species that can signal stress (section 2.1.2, Table 2.1), do high monolignol levels in specific cell types trigger dwarfism? To address this, MYB63 was overexpressed in wild-type Arabidopsis plants with cell type-specific promoters targeting lignifying cell types (vessels, interfascicular fibres) and non-lignifying cell types (xylem parenchyma, epidermis, starch sheath, phloem companion cells) (Figure 2.1, Table 2.2, Figure 2.9). After confirming MYB63 overexpression in rosette leaves and stems with qRT-PCR (Figure 2.2, Figure 2.3), stems were used for phenotypic, chemical, and histochemical analysis. MYB63 overexpression in vessels, interfascicular fibres, and epidermal cells did not affect plant growth (Figure 2.4), while high MYB63 expression in xylem parenchyma, starch sheath, and phloem companion cells led to delayed stem development (Figure 2.4, Figure 2.5). UHPLC-MS analysis revealed hyperaccumulation of sinapoyl esters when MYB63 was overexpressed in each cell type except the starch sheath (Figure 2.6). Quantities of acetyl bromide soluble lignin (ABSL) increased when MYB63 was overexpressed in lignifying vessels and interfascicular fibres (Figure 2.7), without qualitative changes in lignin composition based on histochemical staining of stem cross-sections (Figure 2.8). Taken together, the data suggest that vessel and interfascicular fibre cell walls have the capacity for further lignification without growth penalty. Ectopic lignification is 67 not observed in non-lignifying cell types despite altered soluble phenolic profiles, suggesting lignification cannot be triggered by increased monolignol availability alone, but also requires oxidative enzyme activity in the cell wall. High MYB63 overexpression in non-lignifying cell types adversely affects plant growth, suggesting the possibility of stress sensing in these cell types; epidermal cells are the exception, since they naturally accumulate sinapoyl esters and may be able divert phenylpropanoid compounds into hydroxycinnamate esters for vacuolar sequestration. The ability to increase lignin without growth penalty is desirable for creating high-lignin plants for renewable bioproducts. 4.1.2 Dwarfism associated with lignin deficiency is not due to vessel irregularity Chapter 3 of this thesis aims to address the following objective: to assess the role of lignin in plant growth and development through targeted lignin removal from specific cell wall domains in lignifying cell types. Lignification typically begins in the middle lamella and cell corners, followed by the secondary cell wall (Donaldson, 2001; Terashima & Fukushima, 1988). How does lignification in different cell wall domains contribute to plant growth and development? Is it possible to remove lignin from specific cell wall domains by knocking out oxidative enzymes with known localization? In Arabidopsis, LACs and PRXs co-localize to distinct cell wall domains, including cell corner- and middle lamella-bound LAC4, PRX64, and PRX71, and secondary cell wall-bound LAC4, LAC17, and PRX72 (Hoffmann et al., 2020). CRISPR/Cas9 mutagenesis generated higher order oxidative enzyme mutants, including prx64 prx71 mutations in the lac4-2 background, and prx72 mutations in the lac4-2 lac17 background (Figure 3.1). Preliminary qRT-PCR data confirmed PRX72 knockdown in lac4-2 lac17 prx72 mutants, and showed PRX72 compensation for lac4-2 lac17 mutations in seedlings, but not in the stem (Figure 3.2). Additional prx72 mutations in the lac4-2 lac17 background did not lead to further growth penalty (Figure 3.3) despite a slight decrease in ABSL (Figure 3.5), which was reflected by weaker Mäule staining in interfascicular fibres (Figure 3.6) and more irregular vessels (Figure 3.7). This provides further evidence that lignin deficiency and vessel irregularity do not lead to dwarfism directly. Additional prx64 mutations in the lac4-2 background resulted in a qualitative reduction of lignin in the 68 secondary cell walls of interfascicular fibres and delayed stem development (Figure 3.4), suggesting that lignification defects in the interfascicular fibres can also hinder plant growth and development. Cell corners and the middle lamella remained lignified in lac4-2 prx64 (Figure 3.6), so it can be hypothesized that mutating oxidative enzymes in the cell corners and the middle lamella weakens the concentration gradient driving monolignol diffusion to the cell wall, resulting in less lignified secondary cell walls. 4.2 Future directions 4.2.1 Establishing a system in woody gymnosperms and angiosperms to study and manipulate lignification This study describes a system where upregulation of phenylpropanoid and monolignol biosynthetic pathways in lignifying cell types can increase lignification without growth penalty. A previous study in Arabidopsis showed that expressing VND6/7 with an interfascicular fibre- and xylary fibre-specific promoter (proSND1) rescued the floppy stem phenotype and restored lignification in the snd1 nst1 mutant with hypolignified fibres (Zhong et al., 2010a), suggesting that vessel-specific lignification system is compatible with fibres. Can these systems be established in woody gymnosperms and angiosperms to increase lignification? Our increased understanding of transcriptional switches involved in the lignification of pine (Akiyoshi et al., 2020) and poplar (Ohtani et al., 2011; Zhong et al., 2010b) could help us establish genetic strategies to modify lignin for biofuel and bioproducts. 4.2.2 Connecting MYB63 overexpression in non-lignifying cell types and lignin modification-induced dwarfism Mechanisms underlying delayed stem development when MYB63 is overexpressed in non-lignifying cells remain unclear. Xylem parenchyma cells are known to supply monolignols to neighbouring vessels (Smith et al., 2013, 2017), but MYB63 overexpression in this cell type somehow impacted plant growth and development. Given that MYB63 is not typically expressed in the xylem parenchyma (Zhou et al., 2009), are downstream targets of MYB63 incompatible with existing 69 genetic regulations in the cell type? Given that MYB58 is strongly expressed in tracheary elements (Zhou et al., 2009), is MYB58 overexpression more compatible with xylem parenchyma cells? To understand monolignol production in xylem parenchyma cells, single-cell transcriptomes of xylem parenchyma cells, which are defined by the expression of PRX47 (Smith et al., 2017; Tokunaga et al., 2009), can be analyzed to identify transcription factors that activate monolignol production. A recent study used this strategy to characterize single-cell transcriptomes in the differentiating xylem of woody angiosperms, revealing cell type-specific features that are conserved across taxa (Tung et al., 2023). Alternatively, a proPRX47::MYB58 line can be created to test if MYB58 overexpression in the xylem parenchyma can lead to additional monolignol production and vessel lignification. The phloem typically transports organic nutrients such as sucrose, but the woody angiosperm Syringa vulgaris is known to have high amounts of syringin in the phloem (Aoki et al., 2019). While the presence of monolignol glucosides in the phloem can be explained by the transport of lignification intermediates to lignifying cells, it is unclear if plant growth will be affected if monolignol glucoside content increases in the phloem. In Arabidopsis plants where monolignol glucosides are not typically transported or accumulated, high MYB63 expression in Arabidopsis phloem companion cells resulted in sinapoyl malate accumulation and delayed stem growth (Figure 2.3, Figure 2.4, Figure 2.5, Figure 2.6). Are sinapoyl malate or other phenolic compounds stored within the vacuoles of companion cells or transported in the phloem throughout the plant? Analyzing the phloem sap of proSUC2::MYB63 collected by aphids will establish a connection between phenylpropanoid metabolism and plant growth. A potential mechanism of LMID is the imbalance between growth-stimulating and grow-inhibiting compounds as a result of altered phenylpropanoid metabolism, as suggested by the growth-stimulating effects of dehydrodiconiferyl glucosides (Binns et al., 1987; Teutonico et al., 1991). Recently, pinoresinol, a dimer of coniferyl alcohol, was found to rescue the dwarfism in Arabidopsis plants overexpressing FERULATE 5-HYDROXYLASE (F5H) in the cinnamyl alcohol dehydrogenase c and d (cadc cadd) mutant background (Muro-Villanueva et al., 2023), further establishing growth-70 stimulating effects of certain phenylpropanoid compounds. Similarly, it will be interesting to test if pinoresinol will restore the dwarfism of MYB58/63 overexpression plants. 4.2.3 Probing biomechanical contribution of lignin in different cell wall domains Plant cell walls are a matrix of polysaccharides, lignin, and proteins, all of which contribute to unique biomechanical properties of plant tissues. Distinct biomechanical roles of polysaccharides and lignin are seen in reaction wood where increased cellulose and xylans contribute to tensile strength in tension wood, while increased lignin contributes to compressive forces in compression wood (Donaldson & Singh, 2016). Glycoproteins such as arabinogalactan proteins also contribute to tensile strength (MacMillan et al., 2010). Stem biomechanical properties such as stiffness and elasticity can be measured with flexural tests and axial loading tests (Shah et al., 2017). Given that lignification occurs in distinct cell wall domains, how will biomechanical properties change when lignin is removed from each cell wall domain? Based on the differential phenotypic outcomes of lac4-2 lac17 prx72 (depleted oxidative enzymes in the secondary cell wall) and lac4-2 prx64 (depleted oxidative enzymes in the cell corners and the middle lamella) (Figure 3.3, Figure 3.4), it is likely that they have differential biomechanical alterations. The lignified secondary cell wall has a higher Young’s modulus, meaning higher tensile or compressive stiffness (Niklas, 1992); and the lignified middle lamella has a higher shear modulus, meaning higher rigidity (Mark (1967) as cited by Niklas (1992)). If lignin removal from distinct cell wall domains is achieved, lac4-2 lac17 prx72 might have reduced stiffness, while lac4-2 prx64 might have reduced rigidity. Similarly, given the increased lignin deposition in vessel and fibre cell walls (Figure 2.7), stems might gain rigidity or stiffness depending on the amount of lignin additionally deposited into each cell wall domain. This presents an exciting opportunity to study lignin’s biomechanical contribution to plant structures and modify wood properties for bioproducts. 71 References Akiyoshi, N., Nakano, Y., Sano, R., Kunigita, Y., Ohtani, M., & Demura, T. (2020). Involvement of VNS NAC-domain transcription factors in tracheid formation in Pinus taeda. Tree Physiology, 40(6), 704–716. https://doi.org/10.1093/treephys/tpz106 Aoki, D., Okumura, W., Akita, T., Matsushita, Y., Yoshida, M., Sano, Y., & Fukushima, K. (2019). Microscopic distribution of syringin in freeze-fixed Syringa vulgaris stems. Plant Direct, 3(8), e00155. https://doi.org/10.1002/pld3.155 Balakshin, M. Y., Capanema, E. A., Sulaeva, I., Schlee, P., Huang, Z., Feng, M., Borghei, M., Rojas, O. J., Potthast, A., & Rosenau, T. (2021). New Opportunities in the Valorization of Technical Lignins. ChemSusChem, 14(4), 1016–1036. https://doi.org/10.1002/cssc.202002553 Baldacci-Cresp, F., Le Roy, J., Huss, B., Lion, C., Créach, A., Spriet, C., Duponchel, L., Biot, C., Baucher, M., Hawkins, S., & Neutelings, G. (2020a). UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis. International Journal of Molecular Sciences, 21(17), 6094. https://doi.org/10.3390/ijms21176094 Baldacci-Cresp, F., Spriet, C., Twyffels, L., Blervacq, A. S., Neutelings, G., Baucher, M., & Hawkins, S. (2020b). A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification. Plant Journal, 102(5), 1074–1089. https://doi.org/10.1111/tpj.14675 Barros, J., Escamilla-Trevino, L., Song, L., Rao, X., Serrani-Yarce, J. C., Palacios, M. D., Engle, N., Choudhury, F. K., Tschaplinski, T. J., Venables, B. J., Mittler, R., & Dixon, R. A. (2019). 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. Nature Communications, 10(1), 1994. https://doi.org/10.1038/s41467-019-10082-7 Berthet, S., Demont-Caulet, N., Pollet, B., Bidzinski, P., Cézard, L., Le Bris, P., Borrega, N., Hervé, J., Blondet, E., Balzergue, S., Lapierre, C., & Jouanin, L. (2011). Disruption of LACCASE4 and 17 Results in Tissue-Specific Alterations to Lignification of Arabidopsis thaliana Stems. Plant Cell, 23(3), 1124–1137. https://doi.org/10.1105/tpc.110.082792 Binns, A. N., Chen, R. H., Wood, H. N., & Lynn, D. G. (1987). Cell division promoting activity of naturally occurring dehydrodiconiferyl glucosides: do cell wall components control cell division? Proceedings of the National Academy of Sciences, 84(4), 980–984. https://doi.org/10.1073/pnas.84.4.980 Blaschek, L., Champagne, A., Dimotakis, C., Nuoendagula, Decou, R., Hishiyama, S., Kratzer, S., Kajita, S., & Pesquet, E. (2020). Cellular and Genetic Regulation of Coniferaldehyde Incorporation in Lignin of Herbaceous and Woody Plants by Quantitative Wiesner Staining. Frontiers in Plant Science, 11, 109. https://doi.org/10.3389/fpls.2020.00109 Blaschek, L., Murozuka, E., Serk, H., Ménard, D., & Pesquet, E. (2023). Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis. Plant Cell, 35(2), 889–909. https://doi.org/10.1093/plcell/koac344 Blokhina, O., Laitinen, T., Hatakeyama, Y., Delhomme, N., Paasela, T., Zhao, L., Street, N. R., Wada, H., Kärkönen, A., & Fagerstedt, K. (2019). Ray Parenchymal Cells Contribute to 72 Lignification of Tracheids in Developing Xylem of Norway Spruce. Plant Physiology, 181(4), 1552–1572. https://doi.org/10.1104/pp.19.00743 Bonawitz, N. D., & Chapple, C. (2010). The genetics of lignin biosynthesis: Connecting genotype to phenotype. Annual Review of Genetics, 44, 337–363. https://doi.org/10.1146/annurev-genet-102209-163508 Bonawitz, N. D., Kim, J. I., Tobimatsu, Y., Ciesielski, P. N., Anderson, N. A., Ximenes, E., Maeda, J., Ralph, J., Donohoe, B. S., Ladisch, M., & Chapple, C. (2014). Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant. Nature, 509(7500), 376–380. https://doi.org/10.1038/nature13084 Bonawitz, N. D., Soltau, W. L., Blatchley, M. R., Powers, B. L., Hurlock, A. K., Seals, L. A., Weng, J.-K., Stout, J., & Chapple, C. (2012). REF4 and RFR1, Subunits of the Transcriptional Coregulatory Complex Mediator, Are Required for Phenylpropanoid Homeostasis in Arabidopsis. Journal of Biological Chemistry, 287(8), 5434–5445. https://doi.org/10.1074/jbc.M111.312298 Borchers, H. W. (2022). pracma: Practical Numerical Math Functions. https://CRAN.R-project.org/package=pracma Brown, D. M., Zeef, L. A. H., Ellis, J., Goodacre, R., & Turner, S. R. (2005). Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics. Plant Cell, 17(8), 2281–2295. https://doi.org/10.1105/tpc.105.031542 Chang, X. F., Chandra, R., Berleth, T., & Beatson, R. P. (2008). Rapid, Microscale, Acetyl Bromide-Based Method for High-Throughput Determination of Lignin Content in Arabidopsis thaliana. Journal of Agricultural and Food Chemistry, 56(16), 6825–6834. https://doi.org/10.1021/jf800775f Cho, M.-H., Corea, O. R. A., Yang, H., Bedgar, D. L., Laskar, D. D., Anterola, A. M., Moog-Anterola, F. A., Hood, R. L., Kohalmi, S. E., Bernards, M. A., Kang, C., Davin, L. B., & Lewis, N. G. (2007). Phenylalanine Biosynthesis in Arabidopsis thaliana. Journal of Biological Chemistry, 282(42), 30827–30835. https://doi.org/10.1074/jbc.M702662200 Chou, E. Y., Schuetz, M., Hoffmann, N., Watanabe, Y., Sibout, R., & Samuels, A. L. (2018). Distribution, mobility, and anchoring of lignin-related oxidative enzymes in Arabidopsis secondary cell walls. Journal of Experimental Botany, 69(8), 1849–1859. https://doi.org/10.1093/jxb/ery067 Chun, H. J., Baek, D., Cho, H. M., Lee, S. H., Jin, B. J., Yun, D.-J., Hong, Y.-S., & Kim, M. C. (2019). Lignin biosynthesis genes play critical roles in the adaptation of Arabidopsis plants to high-salt stress. Plant Signaling & Behavior, 14(8), 1625697. https://doi.org/10.1080/15592324.2019.1625697 Cochrane, F. C., Davin, L. B., & Lewis, N. G. (2004). The Arabidopsis phenylalanine ammonia lyase gene family: Kinetic characterization of the four PAL isoforms. Phytochemistry, 65(11), 1557–1564. https://doi.org/10.1016/j.phytochem.2004.05.006 Coleman, H. D., Park, J.-Y., Nair, R., Chapple, C., & Mansfield, S. D. (2008a). RNAi-mediated suppression of p-coumaroyl-CoA 3'-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism. Proceedings of the National Academy of Sciences, 105(11), 4501–4506. https://doi.org/10.1073/pnas.0706537105 73 Coleman, H. D., Samuels, A. L., Guy, R. D., & Mansfield, S. D. (2008b). Perturbed Lignification Impacts Tree Growth in Hybrid Poplar—A Function of Sink Strength, Vascular Integrity, and Photosynthetic Assimilation. Plant Physiology, 148(3), 1229–1237. https://doi.org/10.1104/pp.108.125500 Concordet, J.-P., & Haeussler, M. (2018). CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Research, 46(W1), W242–W245. https://doi.org/10.1093/nar/gky354 Cosgrove, D. J. (2022). Building an extensible cell wall. Plant Physiology, 189(3), 1246–1277. https://doi.org/10.1093/plphys/kiac184 Costa, M. A., Bedgar, D. L., Moinuddin, S. G. A., Kim, K. W., Cardenas, C. L., Cochrane, F. C., Shockey, J. M., Helms, G. L., Amakura, Y., Takahashi, H., Milhollan, J. K., Davin, L. B., Browse, J., & Lewis, N. G. (2005). Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: Syringyl lignin and sinapate/sinapyl alcohol derivative formation. Phytochemistry, 66(17), 2072–2091. https://doi.org/10.1016/j.phytochem.2005.06.022 De Meester, B., de Vries, L., Özparpucu, M., Gierlinger, N., Corneillie, S., Pallidis, A., Goeminne, G., Morreel, K., De Bruyne, M., De Rycke, R., Vanholme, R., & Boerjan, W. (2018). Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass. Plant Physiology, 176(1), 611–633. https://doi.org/10.1104/pp.17.01462 Denness, L., McKenna, J. F., Segonzac, C., Wormit, A., Madhou, P., Bennett, M., Mansfield, J., Zipfel, C., & Hamann, T. (2011). Cell Wall Damage-Induced Lignin Biosynthesis Is Regulated by a Reactive Oxygen Species- and Jasmonic Acid-Dependent Process in Arabidopsis. Plant Physiology, 156(3), 1364–1374. https://doi.org/10.1104/pp.111.175737 Dixon, R. A., & Barros, J. (2019). Lignin biosynthesis: old roads revisited and new roads explored. Open Biology, 9(12), 190215. https://doi.org/10.1098/rsob.190215 Do, C. T., Pollet, B., Thévenin, J., Sibout, R., Denoue, D., Barrière, Y., Lapierre, C., & Jouanin, L. (2007). Both caffeoyl Coenzyme A 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 are involved in redundant functions for lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis. Planta, 226(5), 1117–1129. https://doi.org/10.1007/s00425-007-0558-3 Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., Smith, I., Tothova, Z., Wilen, C., Orchard, R., Virgin, H. W., Listgarten, J., & Root, D. E. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology, 34(2), 184–191. https://doi.org/10.1038/nbt.3437 Donaldson, L. A. (1995). Cell wall fracture properties in relation to lignin distribution and cell dimensions among three genetic groups of radiata pine. Wood Science and Technology, 29, 51–63. https://doi.org/10.1007/BF00196931 Donaldson, L. A. (2001). Lignification and lignin topochemistry — an ultrastructural view. Phytochemistry, 57(6), 859–873. https://doi.org/10.1016/S0031-9422(01)00049-8 Donaldson, L. A., & Singh, A. P. (2016). Reaction Wood. In Secondary Xylem Biology (pp. 93–110). Elsevier. https://doi.org/10.1016/B978-0-12-802185-9.00006-1 Drakakaki, G. (2015). Polysaccharide deposition during cytokinesis: Challenges and future perspectives. Plant Science, 236, 177–184. https://doi.org/10.1016/j.plantsci.2015.03.018 74 Eberhard, J., Ehrler, T. T., Epple, P., Felix, G., Raesecke, H., Amrhein, N., & Schmid, J. (1996). Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: molecular characterization and enzymatic properties. Plant Journal, 10(5), 815–821. https://doi.org/10.1046/j.1365-313X.1996.10050815.x Eberhard, J., Raesecke, H.-R., Schmid, J., & Amrhein, N. (1993). Cloning and expression in yeast of a higher plant chorismate mutase. Molecular cloning, sequencing of the cDNA and characterization of the Arabidopsis thaliana enzyme expressed in yeast. FEBS Letters, 334(2), 233–236. https://doi.org/10.1016/0014-5793(93)81718-F Edwards, K., Johnstone, C., & Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research, 19(6), 1349. https://academic.oup.com/nar/article/19/6/1349/1092879 Ehlting, J., Büttner, D., Wang, Q., Douglas, C. J., Somssich, I. E., & Kombrink, E. (1999). Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms. Plant Journal, 19(1), 9–20. https://doi.org/10.1046/j.1365-313X.1999.00491.x Fernández-Pérez, F., Pomar, F., Pedreño, M. A., & Novo-Uzal, E. (2015a). Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism. Plant Science, 239, 192–199. https://doi.org/10.1016/j.plantsci.2015.08.001 Fernández-Pérez, F., Pomar, F., Pedreño, M. A., & Novo-Uzal, E. (2015b). The suppression of AtPrx52 affects fibers but not xylem lignification in Arabidopsis by altering the proportion of syringyl units. Physiologia Plantarum, 154(3), 395–406. https://doi.org/10.1111/ppl.12310 Fernández-Pérez, F., Vivar, T., Pomar, F., Pedreño, M. A., & Novo-Uzal, E. (2015c). Peroxidase 4 is involved in syringyl lignin formation in Arabidopsis thaliana. Journal of Plant Physiology, 175, 86–94. https://doi.org/10.1016/j.jplph.2014.11.006 Franke, R., Humphreys, J. M., Hemm, M. R., Denault, J. W., Ruegger, M. O., Cusumano, J. C., & Chapple, C. (2002). The Arabidopsis REF8 gene encodes the 3‐hydroxylase of phenylpropanoid metabolism. Plant Journal, 30(1), 33–45. https://doi.org/10.1046/j.1365-313X.2002.01266.x Fraser, C. M., & Chapple, C. (2011). The Phenylpropanoid Pathway in Arabidopsis. The Arabidopsis Book, 9, e0152. https://doi.org/10.1199/tab.0152 Freudenberg, K. (1959). Biosynthesis and constitution of lignin. Nature, 183(4669), 1152-1155. https://doi.org/10.1038/1831152a0 Fucile, G., Falconer, S., & Christendat, D. (2008). Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates. PLoS Genetics, 4(12), e1000292. https://doi.org/10.1371/journal.pgen.1000292 Fucile, G., Garcia, C., Carlsson, J., Sunnerhagen, M., & Christendat, D. (2011). Structural and biochemical investigation of two Arabidopsis shikimate kinases: The heat‐inducible isoform is thermostable. Protein Science, 20(7), 1125–1136. https://doi.org/10.1002/pro.640 Goujon, T., Sibout, R., Pollet, B., Maba, B., Nussaume, L., Bechtold, N., Lu, F., Ralph, J., Mila, I., Barriere, Y., & others. (2003). A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Molecular Biology, 51, 973–989. https://doi.org/10.1023/A:1023022825098 75 Graf, R., Li, X., Chu, V. T., & Rajewsky, K. (2019). sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing. Cell Reports, 26(5), 1098-1103.e3. https://doi.org/10.1016/j.celrep.2019.01.024 Gu, Y., & Rasmussen, C. G. (2022). Cell biology of primary cell wall synthesis in plants. Plant Cell, 34(1), 103–128. https://doi.org/10.1093/plcell/koab249 Halpin, C., Holt, K., Chojecki, J., Oliver, D., Chabbert, B., Monties, B., Edwards, K., Barakate, A., & Foxon, G. A. (1998). Brown-midrib maize (bm1)–a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant Journal, 14(5), 545–553. https://doi.org/10.1046/j.1365-313X.1998.00153.x Hamberger, B., & Hahlbrock, K. (2004). The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proceedings of the National Academy of Sciences, 101(7), 2209–2214. https://doi.org/10.1073/pnas.0307307101 Herrero, J., Fernández-Pérez, F., Yebra, T., Novo-Uzal, E., Pomar, F., Pedreño, M. Á., Cuello, J., Guéra, A., Esteban-Carrasco, A., & Zapata, J. M. (2013). Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis. Planta, 237(6), 1599–1612. https://doi.org/10.1007/s00425-013-1865-5 Hiraga, S., Sasaki, K., Ito, H., Ohashi, Y., & Matsui, H. (2001). A large family of class III plant peroxidases. Plant and Cell Physiology, 42(5), 462–468. https://doi.org/10.1093/pcp/pce061 Hoffmann, L., Besseau, S., Geoffroy, P., Ritzenthaler, C., Meyer, D., Lapierre, C., Pollet, B., & Legrand, M. (2004). Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell, 16(6), 1446–1465. https://doi.org/10.1105/tpc.020297 Hoffmann, N. (2019). Setting the stage for lignin deposition: spatial distribution of enzymes directing lignification in Arabidopsis thaliana [University of British Columbia]. https://doi.org/10.14288/1.0380659 Hoffmann, N., Benske, A., Betz, H., Schuetz, M., & Samuels, A. L. (2020). Laccases and Peroxidases Co-Localize in Lignified Secondary Cell Walls throughout Stem Development. Plant Physiology, 184(2), 806–822. https://doi.org/10.1104/pp.20.00473 Hoffmann, N., Gonzales-Vigil, E., Mansfield, S. D., & Samuels, A. L. (2022). Oxidative enzymes in lignification. In Advances in Botanical Research (Vol. 104, pp. 133–167). Academic Press. https://doi.org/10.1016/bs.abr.2022.02.004 Hooker, T. S., Millar, A. A., & Kunst, L. (2002). Significance of the expression of the CER6 condensing enzyme for cuticular wax production in Arabidopsis. Plant Physiology, 129(4), 1568–1580. https://doi.org/10.1104/pp.003707 Hsu, P. D., Lander, E. S., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010 Huang, J., Gu, M., Lai, Z., Fan, B., Shi, K., Zhou, Y. H., Yu, J. Q., & Chen, Z. (2010). Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress. Plant Physiology, 153(4), 1526–1538. https://doi.org/10.1104/pp.110.157370 76 Iiyama, K., & Pant, R. (1988). The mechanism of the Mäule colour reaction introduction of methylated syringyl nuclei into softwood lignin. Wood Science and Technology, 22(2), 167–175. https://doi.org/10.1007/BF00355852 Imlau, A., Truernit, E., & Sauer, N. (1999). Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell, 11(3), 309–322. https://doi.org/10.1105/tpc.11.3.309 Jones, L., Ennos, A. R., & Turner, S. R. (2001). Cloning and characterization of irregular xylem4 (irx4): A severely lignin-deficient mutant of Arabidopsis. Plant Journal, 26(2), 205–216. https://doi.org/10.1046/j.1365-313X.2001.01021.x Kanaris, M., Poulin, J., Shahinas, D., Johnson, D., Crowley, V. M., Fucile, G., Provart, N., & Christendat, D. (2022). Elevated tyrosine results in the cytosolic retention of 3‐deoxy‐D‐arabino‐heptulosonate 7‐phosphate synthase in Arabidopsis thaliana. Plant Journal, 109(4), 789–803. https://doi.org/10.1111/tpj.15590 Kaneda, M., Rensing, K. H., Wong, J. C. T., Banno, B., Mansfield, S. D., & Samuels, A. L. (2008). Tracking Monolignols during Wood Development in Lodgepole Pine. Plant Physiology, 147(4), 1750–1760. https://doi.org/10.1104/pp.108.121533 Kannan, S., Halter, G., Renner, T., & Waters, E. R. (2018). Patterns of alternative splicing vary between species during heat stress. AoB PLANTS, 10(2), ply013. https://doi.org/10.1093/aobpla/ply013 Kassambara, A. (2023). ggpubr: “ggplot ” Based ublication eady lots. https://CRAN.R-project.org/package=ggpubr Kim, S.-J., Kim, M.-R., Bedgar, D. L., Moinuddin, S. G. A., Cardenas, C. L., Davin, L. B., Kang, C., & Lewis, N. G. (2004). Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proceedings of the National Academy of Sciences, 101(6), 1455–1460. https://doi.org/10.1073/pnas.0307987100 Klee, H. J., Muskopf, Y. M., & Gasser, C. S. (1987). Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Molecular and General Genetics MGG, 210(3), 437–442. https://doi.org/10.1007/BF00327194 Koo, A. J. K., Chung, H. S., Kobayashi, Y., & Howe, G. A. (2006). Identification of a Peroxisomal Acyl-activating Enzyme Involved in the Biosynthesis of Jasmonic Acid in Arabidopsis. Journal of Biological Chemistry, 281(44), 33511–33520. https://doi.org/10.1074/jbc.M607854200 Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi, M., Ito, J., Mimura, T., Fukuda, H., & Demura, T. (2005). Transcription switches for protoxylem and metaxylem vessel formation. Genes and Development, 19(16), 1855–1860. https://doi.org/10.1101/gad.1331305 Landry, L. G., Chapple, CCS., & Last, R. L. (1995). Arabidopsis Mutants Lacking Phenolic Sunscreens Exhibit Enhanced Ultraviolet-B Injury and Oxidative Damage. Plant Physiology, 109(4), 1159–1166. https://doi.org/10.1104/pp.109.4.1159 Lanot, A., Hodge, D., Jackson, R. G., George, G. L., Elias, L., Lim, E.-K., Vaistij, F. E., & Bowles, D. J. (2006). The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana. Plant Journal, 48(2), 286–295. https://doi.org/10.1111/j.1365-313X.2006.02872.x 77 Lauvergeat, V., Lacomme, C., Lacombe, E., Lasserre, E., Roby, D., & Grima-Pettenati, J. (2001). Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry, 57(7), 1187–1195. https://doi.org/10.1016/S0031-9422(01)00053-X Lee, Y., Rubio, M. C., Alassimone, J., & Geldner, N. (2013). A mechanism for localized lignin deposition in the endodermis. Cell, 153(2), 402–412. https://doi.org/10.1016/j.cell.2013.02.045 Lehfeldt, C., Shirley, A. M., Meyer, K., Ruegger, M. O., Cusumano, J. C., Viitanen, P. V., Strack, D., & Chapple, C. (2000). Cloning of the SNG1 Gene of Arabidopsis Reveals a Role for a Serine Carboxypeptidase-like Protein as an Acyltransferase in Secondary Metabolism. Plant Cell, 12(8), 1295–1306. https://doi.org/10.1105/tpc.12.8.1295 Leplé, J.-C., Dauwe, R., Morreel, K., Storme, V., Lapierre, C., Pollet, B., Naumann, A., Kang, K.-Y., Kim, H., Ruel, K., Lefèbvre, A., Joseleau, J.-P., Grima-Pettenati, J., De Rycke, R., Andersson-Gunnerås, S., Erban, A., Fehrle, I., Petit-Conil, M., Kopka, J., … Boerjan, W. (2007). Downregulation of Cinnamoyl-Coenzyme A Reductase in Poplar: Multiple-Level Phenotyping Reveals Effects on Cell Wall Polymer Metabolism and Structure. Plant Cell, 19(11), 3669–3691. https://doi.org/10.1105/tpc.107.054148 Li, W., Zhang, F., Wu, R., Jia, L., Li, G., Guo, Y., Liu, C., & Wang, G. (2017). A Novel N-Methyltransferase in Arabidopsis Appears to Feed a Conserved Pathway for Nicotinate Detoxification among Land Plants and Is Associated with Lignin Biosynthesis. Plant Physiology, 174(3), 1492–1504. https://doi.org/10.1104/pp.17.00259 Li, Y., Kim, J. I., Pysh, L., & Chapple, C. (2015). Four isoforms of Arabidopsis 4-coumarate:CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism. Plant Physiology, 169(4), 2409–2421. https://doi.org/10.1104/pp.15.00838 Lim, E.-K., Jackson, R. G., & Bowles, D. J. (2005). Identification and characterisation of Arabidopsis glycosyltransferases capable of glucosylating coniferyl aldehyde and sinapyl aldehyde. FEBS Letters, 579(13), 2802–2806. https://doi.org/10.1016/j.febslet.2005.04.016 Lin, J.-S., Huang, X.-X., Li, Q., Cao, Y., Bao, Y., Meng, X.-F., Li, Y.-J., Fu, C., & Hou, B.-K. (2016). UDP-glycosyltransferase 72B1 catalyzes the glucose conjugation of monolignols and is essential for the normal cell wall lignification in Arabidopsis thaliana. Plant Journal, 88(1), 26–42. https://doi.org/10.1111/tpj.13229 Liu, B., Wang, P., Kim, J. I., Zhang, D., Xia, Y., Chapple, C., & Cheng, J. X. (2015). Vibrational Fingerprint Mapping Reveals Spatial Distribution of Functional Groups of Lignin in Plant Cell Wall. Analytical Chemistry, 87(18), 9436–9442. https://doi.org/10.1021/acs.analchem.5b02434 Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4), 402–408. https://doi.org/10.1006/meth.2001.1262 MacMillan, C. P., Mansfield, S. D., Stachurski, Z. H., Evans, R., & Southerton, S. G. (2010). Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in Arabidopsis and Eucalyptus. Plant Journal, 62(4), 689–703. https://doi.org/10.1111/j.1365-313X.2010.04181.x 78 Maeda, H., & Dudareva, N. (2012). The Shikimate Pathway and Aromatic Amino Acid Biosynthesis in Plants. Annual Review of Plant Biology, 63(1), 73–105. https://doi.org/10.1146/annurev-arplant-042811-105439 Maeda, H., Yoo, H., & Dudareva, N. (2011). Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate. Nature Chemical Biology, 7(1), 19–21. https://doi.org/10.1038/nchembio.485 Malamy, J. E., & Benfey, P. N. (1997). Analysis of SCARECROW expression using a rapid system for assessing transgene expression in Arabidopsis roots. Plant Journal, 12(4), 957–963. https://doi.org/10.1046/j.1365-313X.1997.12040957.x Mangiafico, S. S. (2023). rcompanion: Functions to Support Extension Education Program Evaluation. https://CRAN.R-project.org/package=rcompanion/ Mark, R. E. (1967). Cell wall mechanics of tracheids. Yale University Press. Mary, H., & Brouhard, G. J. (2019). Kappa (κ): analysis of curvature in biological image data using B-splines. BioRxiv, 852772. https://doi.org/10.1101/852772 Matsuno, M., Compagnon, V., Schoch, G. A., Schmitt, M., Debayle, D., Bassard, J.-E., Pollet, B., Hehn, A., Heintz, D., Ullmann, P., Lapierre, C., Bernier, F., Ehlting, J., & Werck-Reichhart, D. (2009). Evolution of a Novel Phenolic Pathway for Pollen Development. Science, 325(5948), 1688–1692. https://doi.org/10.1126/science.1174095 McCaig, B. C., Meagher, R. B., & Dean, J. F. D. (2005). Gene structure and molecular analysis of the laccase-like multicopper oxidase (LMCO) gene family in Arabidopsis thaliana. Planta, 221(5), 619–636. https://doi.org/10.1007/s00425-004-1472-6 Ménard, D., Blaschek, L., Kriechbaum, K., Lee, C. C., Serk, H., Zhu, C., Lyubartsev, A., Nuoendagula, Bacsik, Z., Bergström, L., Mathew, A., Kajita, S., & Pesquet, E. (2022). Plant biomechanics and resilience to environmental changes are controlled by specific lignin chemistries in each vascular cell type and morphotype. Plant Cell, 34(12), 4877–4896. https://doi.org/10.1093/plcell/koac284 Meyer, K., Cusumano, J. C., Somerville, C., & Chapple, C. C. (1996). Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases. Proceedings of the National Academy of Sciences, 93(14), 6869–6874. https://doi.org/10.1073/pnas.93.14.6869 Mitsuda, N., Seki, M., Shinozaki, K., & Ohme-Takagi, M. (2005). The NAC Transcription Factors NST1 and NST2 of Arabidopsis Regulate Secondary Wall Thickenings and Are Required for Anther Dehiscence. Plant Cell, 17(11), 2993–3006. https://doi.org/10.1105/tpc.105.036004 Mitsuda, N., Iwase, A., Yamamoto, H., Yoshida, M., Seki, M., Shinozaki, K., & Ohme-Takagi, M. (2007). NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell, 19(1), 270–280. https://doi.org/10.1105/tpc.106.047043 Mizutani, M., Ohta, D., & Sato, R. (1997). Isolation of a cDNA and a Genomic Clone Encoding Cinnamate 4-Hydroxylase from Arabidopsis and Its Expression Manner in Planta. Plant Physiology, 113(3), 755–763. https://doi.org/10.1104/pp.113.3.755 Mobley, E. M., Kunkel, B. N., & Keith, B. (1999). Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana. Gene, 240(1), 115–123. https://doi.org/10.1016/S0378-1119(99)00423-0 79 Mottiar, Y., Gierlinger, N., Jeremic, D., Master, E. R., & Mansfield, S. D. (2020). Atypical lignification in eastern leatherwood (Dirca palustris). New Phytologist, 226(3), 704–713. https://doi.org/10.1111/nph.16394 Muro-Villanueva, F., Mao, X., & Chapple, C. (2019). Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition. Current Opinion in Biotechnology, 56, 202–208. https://doi.org/10.1016/j.copbio.2018.12.008 Muro-Villanueva, F., Pysh, L. D., Kim, H., Bouse, T., Ralph, J., Luo, Z., Cooper, B. R., Jannasch, A. S., Zhang, Z., Gu, C., & Chapple, C. (2023). Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE. Proceedings of the National Academy of Sciences, 120(31), e2216543120. https://doi.org/10.1073/pnas.2216543120 Nair, R. B., Bastress, K. L., Ruegger, M. O., Denault, J. W., & Chapple, C. (2004). The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 Gene Encodes an Aldehyde Dehydrogenase Involved in Ferulic Acid and Sinapic Acid Biosynthesis. Plant Cell, 16(2), 544–554. https://doi.org/10.1105/tpc.017509 Nakagawa, T., Nakamura, S., Tanaka, K., Kawamukai, M., Suzuki, T., Nakamura, K., Kimura, T., & Ishiguro, S. (2008). Development of R4 gateway binary vectors (R4pGWB) enabling high-throughput promoter swapping for plant research. Bioscience, Biotechnology and Biochemistry, 72(2), 624–629. https://doi.org/10.1271/bbb.70678 Nakashima, J., Chen, F., Jackson, L., Shadle, G., & Dixon, R. A. (2008). Multi‐site genetic modification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignin composition in specific cell types. New Phytologist, 179(3), 738–750. https://doi.org/10.1111/j.1469-8137.2008.02502.x Niklas, K. J. (1992). Plant biomechanics: an engineering approach to plant form and function. University of Chicago Press. O’Malley, R. C., Huang, S. S. C., Song, L., Lewsey, M. G., Bartlett, A., Nery, J. R., Galli, M., Gallavotti, A., & Ecker, J. R. (2016). Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape. Cell, 165(5), 1280–1292. https://doi.org/10.1016/j.cell.2016.04.038 Ogle, D. H., Doll, J. C., Wheeler, A. P., & Dinno, A. (2023). FSA: Simple Fisheries Stock Assessment Methods. https://CRAN.R-project.org/package=FSA Ohtani, M., Nishikubo, N., Xu, B., Yamaguchi, M., Mitsuda, N., Goué, N., Shi, F., Ohme‐Takagi, M., & Demura, T. (2011). A NAC domain protein family contributing to the regulation of wood formation in poplar. Plant Journal, 67(3), 499–512. https://doi.org/10.1111/j.1365-313X.2011.04614.x Özparpucu, M., Rüggeberg, M., Gierlinger, N., Cesarino, I., Vanholme, R., Boerjan, W., & Burgert, I. (2017). Unravelling the impact of lignin on cell wall mechanics: a comprehensive study on young poplar trees downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD). Plant Journal, 91(3), 480–490. https://doi.org/10.1111/tpj.13584 Pauly, M., & Keegstra, K. (2008). Cell‐wall carbohydrates and their modification as a resource for biofuels. Plant Journal, 54(4), 559–568. https://doi.org/10.1111/j.1365-313X.2008.03463.x 80 Payyavula, R. S., Badmi, R., Jawdy, S. S., Rodriguez, M., Gunter, L., Sykes, R. W., Winkeler, K. A., Collins, C. M., Rottmann, W. H., Chen, J., Yang, X., Tuskan, G. A., & Kalluri, U. C. (2022). Biomass formation and sugar release efficiency of Populus modified by altered expression of a NAC transcription factor. Plant Direct, 6(8), e419. https://doi.org/10.1002/pld3.419 Perkins, M. L., Schuetz, M., Unda, F., Chen, K. T., Bally, M. B., Kulkarni, J. A., Yan, Y., Pico, J., Castellarin, S. D., Mansfield, S. D., & Samuels, A. L. (2022). Monolignol export by diffusion down a polymerization-induced concentration gradient. Plant Cell, 34(5), 2080–2095. https://doi.org/10.1093/plcell/koac051 Perkins, M. L., Schuetz, M., Unda, F., Smith, R. A., Sibout, R., Hoffmann, N. J., Wong, D. C. J., Castellarin, S. D., Mansfield, S. D., & Samuels, L. (2020). Dwarfism of high-monolignol Arabidopsis plants is rescued by ectopic LACCASE overexpression. Plant Direct, 4(9), e00265. https://doi.org/10.1002/pld3.265 Pfalz, M., Mikkelsen, M. D., Bednarek, P., Olsen, C. E., Halkier, B. A., & Kroymann, J. (2011). Metabolic Engineering in Nicotiana benthamiana Reveals Key Enzyme Functions in Arabidopsis Indole Glucosinolate Modification. Plant Cell, 23(2), 716–729. https://doi.org/10.1105/tpc.110.081711 Prabhu, P. R., & Hudson, A. O. (2010). Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) from Arabidopsis thaliana. Biochemistry Research International, 2010, 1–11. https://doi.org/10.1155/2010/549572 Qin, L., Lin, L., Fu, F., & Fan, M. (2018). Micromechanical properties of wood cell wall and interface compound middle lamella using quasi-static nanoindentation and dynamic modulus mapping. Journal of Materials Science, 53(1), 549–558. https://doi.org/10.1007/s10853-017-1185-4 R Core Team. (2021). R: A Language and Environment for Statistical Computing. https://www.R-project.org/ Raes, J., Rohde, A., Christensen, J. H., Van De Peer, Y., & Boerjan, W. (2003). Genome-Wide Characterization of the Lignification Toolbox in Arabidopsis. Plant Physiology, 133(3), 1051–1071. https://doi.org/10.1104/pp.103.026484 Raggi, S., Ferrarini, A., Delledonne, M., Dunand, C., Ranocha, P., De Lorenzo, G., Cervone, F., & Ferrari, S. (2015). The Arabidopsis thaliana Class III Peroxidase AtPRX71 Negatively Regulates Growth under Physiological Conditions and in Response to Cell Wall Damage. Plant Physiology, 169(4), 2513–2525. https://doi.org/10.1104/pp.15.01464 Ralph, J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P. F., Marita, J. M., Hatfield, R. D., Ralph, S. A., Christensen, J. H., & Boerjan, W. (2004). Lignins: Natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochemistry Reviews, 3(1), 29–60. https://doi.org/10.1023/B:PHYT.0000047809.65444.a4 Rezania, S., Oryani, B., Cho, J., Talaiekhozani, A., Sabbagh, F., Hashemi, B., Rupani, P. F., & Mohammadi, A. A. (2020). Different pretreatment technologies of lignocellulosic biomass for bioethanol production: An overview. Energy, 199, 117457. https://doi.org/10.1016/j.energy.2020.117457 Riehl Koch, J., Scherzer, A. J., Eshita, S. M., & Davis, K. R. (1998). Ozone Sensitivity in Hybrid Poplar Is Correlated with a Lack of Defense-Gene Activation. Plant Physiology, 118(4), 1243–1252. https://doi.org/10.1104/pp.118.4.1243 81 Rohde, A., Morreel, K., Ralph, J., Goeminne, G., Hostyn, V., De Rycke, R., Kushnir, S., Van Doorsselaere, J., Joseleau, J. P., Vuylsteke, M., Van Driessche, G., Van Beeumen, J., Messens, E., & Boerjan, W. (2004). Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism. Plant Cell, 16(10), 2749–2771. https://doi.org/10.1105/tpc.104.023705 Rojas-Murcia, N., Hématy, K., Lee, Y., Emonet, A., Ursache, R., Fujita, S., de Bellis, D., & Geldner, N. (2020). High-order mutants reveal an essential requirement for peroxidases but not laccases in Casparian strip lignification. Proceedings of the National Academy of Sciences, 117(46), 29166–29177. https://doi.org/10.1073/pnas.2012728117 Ruegger, M., & Chapple, C. (2001). Mutations That Reduce Sinapoylmalate Accumulation in Arabidopsis thaliana Define Loci With Diverse Roles in Phenylpropanoid Metabolism. Genetics, 159(4), 1741–1749. https://doi.org/10.1093/genetics/159.4.1741 Schilmiller, A. L., Stout, J., Weng, J. K., Humphreys, J., Ruegger, M. O., & Chapple, C. (2009). Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis. Plant Journal, 60(5), 771–782. https://doi.org/10.1111/j.1365-313X.2009.03996.x Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. https://doi.org/10.1038/nmeth.2019 Schoch, G., Goepfert, S., Morant, M., Hehn, A., Meyer, D., Ullmann, P., & Werck-Reichhart, D. (2001). CYP98A3 from Arabidopsis thaliana s a 3′-Hydroxylase of Phenolic Esters, a Missing Link in the Phenylpropanoid Pathway. Journal of Biological Chemistry, 276(39), 36566–36574. https://doi.org/10.1074/jbc.M104047200 Schuetz, M., Benske, A., Smith, R. A., Watanabe, Y., Tobimatsu, Y., Ralph, J., Demura, T., Ellis, B., & Samuels, A. L. (2014). Laccases direct lignification in the discrete secondary cell wall domains of protoxylem. Plant Physiology, 166(2), 798–807. https://doi.org/10.1104/pp.114.245597 Shah, D. U., Reynolds, T. P., & Ramage, M. H. (2017). The strength of plants: theory and experimental methods to measure the mechanical properties of stems. Journal of Experimental Botany, 68(16), 4497–4516. https://doi.org/10.1093/jxb/erx245 Shigeto, J., & Tsutsumi, Y. (2016). Diverse functions and reactions of class III peroxidases. New Phytologist, 209(4), 1395–1402. https://doi.org/10.1111/nph.13738 Shigeto, J., Kiyonaga, Y., Fujita, K., Kondo, R., & Tsutsumi, Y. (2013). Putative cationic cell-wall-bound peroxidase homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignification. Journal of Agricultural and Food Chemistry, 61(16), 3781–3788. https://doi.org/10.1021/jf400426g Shigeto, J., Nagano, M., Fujita, K., & Tsutsumi, Y. (2014). Catalytic profile of Arabidopsis peroxidases, AtPrx-2, 25 and 71, contributing to stem lignification. PLoS ONE, 9(8), e105332. https://doi.org/10.1371/journal.pone.0105332 82 Shockey, J., & Browse, J. (2011). Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants. Plant Journal, 66(1), 143–160. https://doi.org/10.1111/j.1365-313X.2011.04512.x Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., & Séguin, A. (2005). Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell, 17(7), 2059–2076. https://doi.org/10.1105/tpc.105.030767 Singh, S. A., & Christendat, D. (2006). Structure of Arabidopsis dehydroquinate dehydratase-shikimate dehydrogenase and implications for metabolic channeling in the shikimate pathway. Biochemistry, 45(25), 7787–7796. https://doi.org/10.1021/bi060366+ Sinlapadech, T., Stout, J., Ruegger, M. O., Deak, M., & Chapple, C. (2007). The hyper‐fluorescent trichome phenotype of the brt1 mutant of Arabidopsis is the result of a defect in a sinapic acid:UDPG glucosyltransferase. Plant Journal, 49(4), 655–668. https://doi.org/10.1111/j.1365-313X.2006.02984.x Smith, R. A., Schuetz, M., Karlen, S. D., Bird, D., Tokunaga, N., Sato, Y., Mansfield, S. D., Ralph, J., & Samuels, A. L. (2017). Defining the Diverse Cell Populations Contributing to Lignification in Arabidopsis Stems. Plant Physiology, 174(2), 1028–1036. https://doi.org/10.1104/pp.17.00434 Smith, R. A., Schuetz, M., Roach, M., Mansfield, S. D., Ellis, B., & Samuels, L. (2013). Neighboring Parenchyma Cells Contribute to Arabidopsis Xylem Lignification, while Lignification of Interfascicular Fibers Is Cell Autonomous. Plant Cell, 25(10), 3988–3999. https://doi.org/10.1105/tpc.113.117176 Solomon, E. I., Sundaram, U. M., & Machonkin, T. E. (1996). Multicopper oxidases and oxygenases. Chemical Reviews, 96(7), 2563–2606. https://doi.org/10.1021/cr950046o Somssich, I. E., Wernert, P., Kiedrowski, S., & Hahlbrock, K. (1996). Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP+ oxidoreductase. Proceedings of the National Academy of Sciences, 93(24), 14199–14203. https://doi.org/10.1073/pnas.93.24.14199 Sørhagen, K., Laxa, M., Peterhänsel, C., & Reumann, S. (2013). The emerging role of photorespiration and non‐photorespiratory peroxisomal metabolism in pathogen defence. Plant Biology, 15(4), 723–736. https://doi.org/10.1111/j.1438-8677.2012.00723.x Taylor-Teeples, M., Lin, L., de Lucas, M., Turco, G., Toal, T. W., Gaudinier, A., Young, N. F., Trabucco, G. M., Veling, M. T., Lamothe, R., Handakumbura, P. P., Xiong, G., Wang, C., Corwin, J., Tsoukalas, A., Zhang, L., Ware, D., Pauly, M., Kliebenstein, D. J., … Brady, S. M. (2015). An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature, 517(7536), 571–575. https://doi.org/10.1038/nature14099 Terashima, N., & Fukushima, K. (1988). Heterogeneity in formation of lignin-XI: An autoradiographic study of the heterogeneous formation and structure of pine lignin. Wood Science and Technology, 22(3), 259–270. https://doi.org/10.1007/BF00386021 Teutonico, R. A., Dudley, M. W., Orr, J. D., Lynn, D. G., & Binns, A. N. (1991). Activity and Accumulation of Cell Division-Promoting Phenolics in Tobacco Tissue Cultures. Plant Physiology, 97(1), 288–297. https://doi.org/10.1104/pp.97.1.288 83 Tokunaga, N., Kaneta, T., Sato, S., & Sato, Y. (2009). Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana. Physiologia Plantarum, 136(2), 237–249. https://doi.org/10.1111/j.1399-3054.2009.01233.x Truernit, E., & Sauer, N. (1995). The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: Evidence for phloem loading and unloading by SUC2. Planta, 196(3), 564–570. https://doi.org/10.1007/BF00203657 Tsugawa, H., Cajka, T., Kind, T., Ma, Y., Higgins, B., Ikeda, K., Kanazawa, M., VanderGheynst, J., Fiehn, O., & Arita, M. (2015). MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature Methods, 12(6), 523–526. https://doi.org/10.1038/nmeth.3393 Tsuyama, T., Kawai, R., Shitan, N., Matoh, T., Sugiyama, J., Yoshinaga, A., Takabe, K., Fujita, M., & Yazaki, K. (2013). Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants. Plant Physiology, 162(2), 918–926. https://doi.org/10.1104/pp.113.214957 Tung, C.-C., Kuo, S.-C., Yang, C.-L., Yu, J.-H., Huang, C.-E., Liou, P.-C., Sun, Y.-H., Shuai, P., Su, J.-C., Ku, C., & Lin, Y.-C. J. (2023). Single-cell transcriptomics unveils xylem cell development and evolution. Genome Biology, 24(1), 3. https://doi.org/10.1186/s13059-022-02845-1 Turlapati, P. V., Kim, K. W., Davin, L. B., & Lewis, N. G. (2011). The laccase multigene family in Arabidopsis thaliana: Towards addressing the mystery of their gene function(s). Planta, 233(3), 439–470. https://doi.org/10.1007/s00425-010-1298-3 Tzin, V., & Galili, G. (2010). The Biosynthetic Pathways for Shikimate and Aromatic Amino Acids in Arabidopsis thaliana. The Arabidopsis Book, 8, e0132. https://doi.org/10.1199/tab.0132 Vaahtera, L., Schulz, J., & Hamann, T. (2019). Cell wall integrity maintenance during plant development and interaction with the environment. Nature Plants, 5(9), 924–932. https://doi.org/10.1038/s41477-019-0502-0 Väisänen, E. E., Smeds, A. I., Fagerstedt, K. V., Teeri, T. H., Willför, S. M., & Kärkönen, A. (2015). Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings. Planta, 242(3), 747–760. https://doi.org/10.1007/s00425-015-2348-7 Väisänen, E., Takahashi, J., Obudulu, O., Bygdell, J., Karhunen, P., Blokhina, O., Laitinen, T., Teeri, T. H., Wingsle, G., Fagerstedt, K. V, & Kärkönen, A. (2020). Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce. Journal of Experimental Botany, 71(20), 6379–6395. https://doi.org/10.1093/jxb/eraa368 Valério, L., De Meyer, M., Penel, C., & Dunand, C. (2004). Expression analysis of the Arabidopsis peroxidase multigenic family. Phytochemistry, 65(10), 1331–1342. https://doi.org/10.1016/j.phytochem.2004.04.017 Vanholme, R., Cesarino, I., Rataj, K., Xiao, Y., Sundin, L., Goeminne, G., Kim, H., Cross, J., Morreel, K., Araujo, P., Welsh, L., Haustraete, J., McClellan, C., Vanholme, B., Ralph, J., Simpson, G. G., Halpin, C., & Boerjan, W. (2013). Caffeoyl Shikimate Esterase (CSE) Is an 84 Enzyme in the Lignin Biosynthetic Pathway in Arabidopsis. Science, 341(6150), 1103–1106. https://doi.org/10.1126/science.1241602 Vincent, D., Lapierre, C., Pollet, B., Cornic, G., Negroni, L., & Zivy, M. (2005). Water Deficits Affect Caffeate O -Methyltransferase, Lignification, and Related Enzymes in Maize Leaves. A Proteomic Investigation. Plant Physiology, 137(3), 949–960. https://doi.org/10.1104/pp.104.050815 Wadenbäck, J., von Arnold, S., Egertsdotter, U., Walter, M. H., Grima-Pettenati, J., Goffner, D., Gellerstedt, G., Gullion, T., & Clapham, D. (2008). Lignin biosynthesis in transgenic Norway spruce plants harboring an antisense construct for cinnamoyl CoA reductase (CCR). Transgenic Research, 17, 379–392. https://doi.org/10.1007/s11248-007-9113-z Wagner, A., Ralph, J., Akiyama, T., Flint, H., Phillips, L., Torr, K., Nanayakkara, B., & Te Kiri, L. (2007). Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata. Proceedings of the National Academy of Sciences, 104(28), 11856–11861. https://doi.org/10.1073/pnas.0701428104 Wang, H., Avci, U., Nakashima, J., Hahn, M. G., Chen, F., & Dixon, R. A. (2010). Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants. Proceedings of the National Academy of Sciences, 107(51), 22338–22343. https://doi.org/10.1073/pnas.1016436107 Wang, P., Guo, L., Morgan, J., Dudareva, N., & Chapple, C. (2022). Transcript and metabolite network perturbations in lignin biosynthetic mutants of Arabidopsis. Plant Physiology, 190(4), 2828–2846. https://doi.org/10.1093/plphys/kiac344 Wang, Z.-P., Xing, H.-L., Dong, L., Zhang, H.-Y., Han, C.-Y., Wang, X.-C., & Chen, Q.-J. (2015). Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biology, 16(1), 144. https://doi.org/10.1186/s13059-015-0715-0 Weng, J., & Chapple, C. (2010). The origin and evolution of lignin biosynthesis. New Phytologist, 187(2), 273–285. https://doi.org/10.1111/j.1469-8137.2010.03327.x Weng, J.-K., Li, Y., Mo, H., & Chapple, C. (2012). Assembly of an Evolutionarily New Pathway for α-Pyrone Biosynthesis in Arabidopsis. Science, 337(6097), 960–964. https://doi.org/10.1126/science.1221614 Wickham, H. (2011). The Split-Apply-Combine Strategy for Data Analysis. Journal of Statistical Software, 40(1), 1–29. https://www.jstatsoft.org/v40/i01/ Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. https://ggplot2.tidyverse.org Wickham, H., François, R., Henry, L., Müller, K., & Vaughan, D. (2023). dplyr: A Grammar of Data Manipulation. https://CRAN.R-project.org/package=dplyr Wils, C. R., Brandt, W., Manke, K., & Vogt, T. (2013). A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase. FEBS Letters, 587(6), 683–689. https://doi.org/10.1016/j.febslet.2013.01.040 Yang, S. X., Wu, T. T., Ding, C. H., Zhou, P. C., Chen, Z. Z., & Gou, J. Y. (2019). SAHH and SAMS form a methyl donor complex with CCoAOMT7 for methylation of phenolic compounds. Biochemical and Biophysical Research Communications, 520(1), 122–127. https://doi.org/10.1016/j.bbrc.2019.09.101 85 Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W., & Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 1(2), 641–646. https://doi.org/10.1038/nprot.2006.97 Zhao, Q., Nakashima, J., Chen, F., Yin, Y., Fu, C., Yun, J., Shao, H., Wang, X., Wang, Z.-Y., & Dixon, R. A. (2013). LACCASE Is Necessary and Nonredundant with PEROXIDASE for Lignin Polymerization during Vascular Development in Arabidopsis. Plant Cell, 25(10), 3976–3987. https://doi.org/10.1105/tpc.113.117770 Zhong, R., Cui, D., & Ye, Z.-H. (2019). Secondary cell wall biosynthesis. New Phytologist, 221(4), 1703–1723. https://doi.org/10.1111/nph.15537 Zhong, R., Lee, C., & Ye, Z. H. (2010a). Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Molecular Plant, 3(6), 1087–1103. https://doi.org/10.1093/mp/ssq062 Zhong, R., Lee, C., & Ye, Z.-H. (2010b). Functional Characterization of Poplar Wood-Associated NAC Domain Transcription Factors. Plant Physiology, 152(2), 1044–1055. https://doi.org/10.1104/pp.109.148270 Zhong, R., Lee, C., Zhou, J., McCarthy, R. L., & Ye, Z. H. (2008). A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell, 20(10), 2763–2782. https://doi.org/10.1105/tpc.108.061325 Zhong, R., & Ye, Z.-H. (2015). The Arabidopsis NAC transcription factor NST2 functions together with SND1 and NST1 to regulate secondary wall biosynthesis in fibers of inflorescence stems. Plant Signaling & Behavior, 10(2), e989746. https://doi.org/10.4161/15592324.2014.989746 Zhou, J., Lee, C., Zhong, R., & Ye, Z.-H. (2009). MYB58 and MYB63 Are Transcriptional Activators of the Lignin Biosynthetic Pathway during Secondary Cell Wall Formation in Arabidopsis. Plant Cell, 21(1), 248–266. https://doi.org/10.1105/tpc.108.063321 Zhuo, C., Wang, X., Docampo-Palacios, M., Sanders, B. C., Engle, N. L., Tschaplinski, T. J., Hendry, J. I., Maranas, C. D., Chen, F., & Dixon, R. A. (2022). Developmental changes in lignin composition are driven by both monolignol supply and laccase specificity. Science Advances, 8(10), eabm8145. https://doi.org/10.1126/sciadv.abm8145 86 Appendices Supplementary Table 1. List of primers used in this thesis work. Bolded sequences are genomic sequences flanked with Gateway or Golden Gate sequences. Primer name Sequence (5' to 3') Source Generating Gateway promoter entry clones B4-proVND7 fwd GGGGACAACTTTGTATAGAAAAGTTGTTCTGTAGTTCTTCTTCGGGTTTACAAATC This thesis B1r-proVND7 rev GGGGACTGCTTTTTTGTACAAACTTGCCACGATGATCCTATAAACGT This thesis B4-proPRX47 fwd GGGGACAACTTTGTATAGAAAAGTTGGAGAGATGATGTCTTATATCCC This thesis B1r-proPRX47 rev GGGGACTGCTTTTTTGTACAAACTTGGATAAAGATCAGTATAATTAAGATGTTTAATTCA This thesis B4-proPRX64 fwd GGGGACAACTTTGTATAGAAAAGTTGATGTTCATTTAGATTAAATAGATTCAA This thesis B1r-proPRX64 rev GGGGACTGCTTTTTTGTACAAACTTGTTTAACAAACTTTTCGAAATTTTAATT This thesis B4-proCER6 fwd GGGGACAACTTTGTATAGAAAAGTTGCTTCGATATCGGTTGTTG This thesis B1r-proCER6 rev GGGGACTGCTTTTTTGTACAAACTTGCGTCGGAGAGTTTTAATG This thesis B4-proSCR fwd GGGGACAACTTTGTATAGAAAAGTTGCTGCGTTCGAAATTCGAAAA This thesis B1r-proSCR rev GGGGACTGCTTTTTTGTACAAACTTGGGAGATTGAAGGGTTGTTGG This thesis B4-proSUC2 fwd GGGGACAACTTTGTATAGAAAAGTTGCATGCAAAATAGCACACCAT This thesis B1r-proSUC2 rev GGGGACTGCTTTTTTGTACAAACTTGATTTGACAAACCAAGAAAGTAAG This thesis Confirming cell type-specific promoter::MYB63 constructs with sequencing P4P1R fwd CTGGCAGTTCCCTACTCTCG This thesis proVND7 geno F1 ATTTCCAACAAGCCACTCAC This thesis proVND7 geno F2 TGCCGGTAAAGTGGAGAAGATTG This thesis proVND7 geno F3 CAATCATTTGATCCACACCACC This thesis proVND7 geno F4 GATGATACATCGGCGTTTGTG This thesis proVND7 geno F5 GTGTAACACACTCCAACCTCAG This thesis proVND7 geno F6 GGCGAAACAAACCCTTAAACC This thesis proVND7 geno F7 TTGGACCCAAGAGCCATTCG This thesis proPRX47 geno F1 GAGAGATGATGTCTTATATCCC This thesis proPRX47 geno F2 AGCGTATTACTGTGCATAAG This thesis proPRX47 geno F3 GCTAATGGAAGCCAGCTACATG This thesis proPRX47 geno R3 CATGTAGCTGGCTTCCATTAGC This thesis proPRX47 geno F4 CTTGCTGGGTTTGAGACTTC This thesis proPRX47 geno F5 TGATAAGAGTAAGCGGGAAGC This thesis proPRX64 geno F1 GACGAAGAAATAGAAGGCATCC This thesis proPRX64 geno F2 AACTCTACTCCACTGCAACC This thesis 87 Primer name Sequence (5' to 3') Source proPRX64 geno F3 TCTCACTAACAACTCCATCTCC This thesis proCER6 geno F1 CACGTACATGGGTGATAGGTCC This thesis proCER6 geno F2 ACGGGTAAACCAAACCAAAC This thesis proCER6 geno F3 AAAGCTGACACGTCTTGCTG This thesis proCER6 geno F4 CACCTTCCCTCTCTCATCTTC This thesis proSCR geno F1 CGATTGAGAGGAGAGGATTGACC This thesis proSCR geno F2 ATGGATAAGGGATAGAGGAAGAGG This thesis proSCR geno F3 GATATTGTTTAAGTGCATTTGTG This thesis proSCR geno F4 TTGAGCCGTGTGTTTGTGTTGTG This thesis proSCR geno F5 AGCTCTACGTCTTGTCCAATTCC This thesis proSCR geno F6 TCCCATCTCTCATACCAAACC This thesis proSCR geno F7 TTGTGGTTAGTTCGAGAGAGAGG This thesis proSCR geno F8 ATTCATCTCCAACCCACATCC This thesis proSUC2 geno F1 TATCGCGCAGTACAAATAAACAG This thesis proSUC2 geno F2 TATCCGAATTTCTCGCTTCATGG This thesis proSUC2 geno F3 CTTCACTGTCTCCTCTTCG This thesis pDONR221 fwd TTCTGCTTAGTTTGATGCCTGG This thesis MYB63 geno F1 TGTGACAAGACCAAAGTGAAGAG This thesis MYB63 geno F2 GGAGGAAGAAACAATCATTAAG This thesis MYB63 geno F3 TCGTGGGAAAGATGATAAG This thesis MYB63 geno F4 GATGAGGATGAGGTTAAGAAATGG This thesis Confirming transformation of cell type-specific promoter::MYB63 constructs in plants proVND7 plant geno fwd AGCTCCTCCATTGTTGGGTG This thesis proPRX47 plant geno fwd AAGCAAGCGTGTCTCAGTGT This thesis proPRX64 plant geno fwd ACATACGTACGAGAACAAGACGA This thesis proCER6 plant geno fwd AGCTGACACGTCTTGCTGTT This thesis proSCR plant geno fwd TCACGACCAACAACCCTTCA This thesis proSUC2 plant geno fwd GTCACGAAGATACCCTACGCT This thesis MYB63 plant geno rev TTCCCACACCTCAATAGCCC This thesis Generating gRNA cassettes for Golden Gate Assembly of Cas9 constructs PRX64.3-DT1-BsF ATATATGGTCTCGATTGCAAGTATGGTCGTAGTAGTGGTT This thesis PRX64.3-DT1-F0 TGCAAGTATGGTCGTAGTAGTGGTTTTAGAGCTAGAAATAGC This thesis DT0-BsR2 ATATTATTGGTCTCAATCTCTTAGTCGACTCTACCAAT Wang et al. (2015) PRX64.4-DT2-BsF2 ATATTATTGGTCTCAAGATTGCATCCTCAAAAGCGCAGCAGTT This thesis PRX64.4-DT2-F0 TGCATCCTCAAAAGCGCAGCAGTTTTAGAGCTAGAAATAGC This thesis DT0-BsR3 ATATTATTGGTCTCATCACTACTTCGTCTCTAACCAT Wang et al. (2015) PRX71.3-DT3-BsF3 ATATTATTGGTCTCAGTGATTGGAAACAGGACCAGGCCTTGGTT This thesis PRX71.3-DT3-F0 TGGAAACAGGACCAGGCCTTGGTTTTAGAGCTAGAAATAGC This thesis 88 Primer name Sequence (5' to 3') Source PRX71.4-DT4-R0 AACGGTGCGATTCTGGGGTCAGACAATCACTACTTCGACTCTAGCTGTAT This thesis PRX71.4-DT4-BsR ATTATTGGTCTCTAAACGGTGCGATTCTGGGGTCAGA This thesis PRX72.3-DT1-BsF ATATATGGTCTCGATTGCACGATTGGTCATAAAACTGGTT This thesis PRX72.3-DT1-F0 TGCACGATTGGTCATAAAACTGGTTTTAGAGCTAGAAATAGC This thesis PRX72.4-DT2-R0 AACTGCTCAGACTCCATTTCCACAATCTCTTAGTCGACTCTAC This thesis PRX72.4-DT2-BsR ATTATTGGTCTCGAAACTGCTCAGACTCCATTTCCAC This thesis Confirming Cas9 constructs (colony PCR and sequencing) U6-26p-F TGTCCCAGGATTAGAATGATTAGGC Wang et al. (2015) U6-29p-F TTAATCCAAACTACTGCAGCCTGAC Wang et al. (2015) U6-29p-R AGCCCTCTTCTTTCGATCCATCAAC Wang et al. (2015) U6-1t-F GCTAAGACAAAGTGATTGGTCCGTT Wang et al. (2015) U6-1t-R AACGGACCAATCACTTTGTCTTAGC Wang et al. (2015) Confirming CRISPR/Cas9-induced mutations in plants prx64 CRISPR fwd TCTCACTAACAACTCCATCTCCC This thesis prx64 CRISPR rev CGGCGCAAGAGACGATACC This thesis prx71 CRISPR fwd TCTATCCCCCACAAACACCAAA This thesis prx71 CRISPR rev 2 TCACGAGCGGCTAATGCTAA This thesis prx72 CRISPR fwd GCAACCCTTGGCATCACACA This thesis prx72 CRISPR rev ACGGGCTGAGTTTCTGTTAGG This thesis Confirming T-DNA insertions in plants GabiKat 8474 LB ATAATAACGCTGCGGACATCTACATTTT GabiKat lac4-2 GabiKat LP GTTCGCCACTACAAGTTTAACGT This thesis lac4-2 GabiKat RP TTTCTAACTGGGCCTGGGTGTC This thesis SALK LBb1.3 ATTTTGCCGATTTCGGAAC SALK Institute lac17 SALK LP ATTTCGGAAATTCCCTTCATG This thesis lac17 SALK RP TTTGATCAGAACCTGGTCACC This thesis Quantitative real-time PCR MYB63 qPCR fwd GGGTTCCAACCAACAAGATG Perkins et al. (2020) MYB63 qPCR rev GCTCTTGAGCCTGAGCTGTT Perkins et al. (2020) LAC4 qPCR fwd TCCTTCAAGATACCGGCGTCA Berthet et al. (2011) LAC4 qPCR rev ACCGGATGGAACTCCGATTGT Berthet et al. (2011) LAC17 qPCR fwd CGATAAACGGGCTTCCTGGTC Berthet et al. (2011) LAC17 qPCR rev AACCGTGTGATTTGCGATGCT Berthet et al. (2011) PRX72 qPCR fwd GAAGAAGAGACGCGAGAGGAG This thesis PRX72 qPCR rev TTCCGTTGCCGGATTGATTG This thesis EF1-α qPCR fwd CTGGAGGTTTTGAGGCTGGTAT Berthet et al. (2011) EF1-α qPCR rev CCAAGGGTGAAAGCAAGAAGA Berthet et al. (2011) 89 Supplementary Table 2. Thermal cycles for PCR, cloning, and qRT-PCR. Amplifying promoter elements / Regular colony PCR and genotyping Touchdown PCR 1. 94-98°C, 3 minutes* 2. 94-98°C, 20 seconds* 3. 67°C, 20 seconds 4. 72°C, 20 seconds-3 minutes** 5. Go to step 2, 10×, -1°C per cycle 6. 94-98°C, 20 seconds* 7. 57°C, 20 seconds 8. 72°C, 20 seconds-3 minutes** 9. Go to step 6, 23× 10. 72°C, 5 minutes 11. 12°C, HOLD *The denaturing temperature depends on the polymerase used. **The extension time depends on the size of the amplicon (1 kb/minute). Amplifying gRNA cassettes to be incorporated into the Cas9 construct 1. 98°C, 2 minutes 2. 98°C, 15 seconds 3. 58°C, 30 seconds 4. 72°C, 1 minute 5. Go to step 2, 39× 6. 72°C, 5 minutes 7. 12°C, HOLD Golden Gate Assembly with BsaI and T4 ligase in one reaction 1. 37°C, 5 minutes 2. 16°C, 5 minutes 3. Go to step 1, 29× 4. 60°C, 5 minutes 5. 4°C, HOLD Quantitative real-time PCR 1. 95°C, 2 minutes 2. 95°C, 15 seconds 3. 60°C, 30 seconds, acquire signal 4. Go to step 2, 39× To generate melt curves: 5. 55°C, 5 seconds, acquire signal 6. Go to step 5, 80×, +0.5°C per cycle
Thesis/Dissertation
eng
Botany
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Samuels, Anne Lacey, 1962-
Dissecting the roles of monolignol supply and oxidative enzymes in Arabidopsis thaliana lignification
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