Forestry, Faculty of
DSpace
UBCV
MacKay, Heather A.
Master of Science - MSc
University of British Columbia
Woody biomass utilisation for the production of pulp and paper, specialty cellulose and chemicals, and cellulosic biofuels requires significant investment of chemicals and energy. Lignin, a polyphenolic constituent of the secondary cell walls of vascular plants, is a significant impediment for wood processing. As such, delignification is essential for biomass utilisation, and typically relies on the addition of acid or alkali to promote fibre separation, and the recovery of the chemical fractions of the biomass. Increasing the degree of acylation (the process of adding an acyl group, or RCO– group, to a compound) of lignin and/or integrating unique bonds into the lignin polymer backbone can improve the processability of an otherwise chemically stable polymer. In Salix, Populus, and Palmae species, p-hydroxybenzoic acid (pHBA) acylates the monolignols. When these monolignol p-hydroxybenzoate conjugates are incorporated into lignin, pendant ester groups decorate the lignin polymer, and can be easily ‘clipped off’ by mild chemical treatment. Consequently, pHBA is of particular interest in the pursuit of creating designer lignins. p-HYDROXYBENZOYL-COA MONOLIGNOL TRANSFERASE (pHBMT) is the putative enzyme thought to be responsible for decorating monolignols with pHBA. The gene encoding pHBMT, however, remains elusive. In this study a putative pHBMT gene was identified through in vitro enzyme activity assays, and subsequently overexpressed in both A. thaliana and hybrid poplar under the control of the Cauliflower mosaic virus 35S promoter (CaMV35SP) and the Arabidopsis thaliana CINNAMATE-4-HYDROXYLASE promoter (AtC4HP). Although, overexpression of this putative pHBMT did not result in a significant increase in pHBA in the cell walls of A. thaliana, increases were observed in poplar. This result furthers our understandings of the mechanisms driving acylated monolignol biosynthesis, and identified a promising avenue in the pursuit and development of novel transgenic plant varieties with unique lignin properties and commercial applications.
Identifying a native p-hydroxybenzoyl-CoA monolignol transferase gene in poplar by Heather A. MacKay B.Sc., Queen’s University, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Forestry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2019 © Heather A. MacKay, 2019 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Identifying a native p-hydroxybenzoyl-CoA monolignol transferase gene in poplar submitted by Heather A. MacKay in partial fulfillment of the requirements for the degree of Master of Science in Forestry Examining Committee: Shawn D. Mansfield Supervisor Lacey Samuels Supervisory Committee Member Simone D. Castellarin Supervisory Committee Member Harry Brumer Additional Examiner iii Abstract Woody biomass utilisation for the production of pulp and paper, specialty cellulose and chemicals, and cellulosic biofuels requires significant investment of chemicals and energy. Lignin, a polyphenolic constituent of the secondary cell walls of vascular plants, is a significant impediment for wood processing. As such, delignification is essential for biomass utilisation, and typically relies on the addition of acid or alkali to promote fibre separation, and the recovery of the chemical fractions of the biomass. Increasing the degree of acylation (the process of adding an acyl group, or RCO– group, to a compound) of lignin and/or integrating unique bonds into the lignin polymer backbone can improve the processability of an otherwise chemically stable polymer. In Salix, Populus, and Palmae species, p-hydroxybenzoic acid (pHBA) acylates the monolignols. When these monolignol p-hydroxybenzoate conjugates are incorporated into lignin, pendant ester groups decorate the lignin polymer, and can be easily ‘clipped off’ by mild chemical treatment. Consequently, pHBA is of particular interest in the pursuit of creating designer lignins. p-HYDROXYBENZOYL-COA MONOLIGNOL TRANSFERASE (pHBMT) is the putative enzyme thought to be responsible for decorating monolignols with pHBA. The gene encoding pHBMT, however, remains elusive. In this study a putative pHBMT gene was identified through in vitro enzyme activity assays, and subsequently overexpressed in both A. thaliana and hybrid poplar under the control of the Cauliflower mosaic virus 35S promoter (CaMV35SP) and the Arabidopsis thaliana CINNAMATE-4-HYDROXYLASE promoter (AtC4HP). Although, iv overexpression of this putative pHBMT did not result in a significant increase in pHBA in the cell walls of A. thaliana, increases were observed in poplar. This result furthers our understandings of the mechanisms driving acylated monolignol biosynthesis, and identified a promising avenue in the pursuit and development of novel transgenic plant varieties with unique lignin properties and commercial applications. v Lay Summary Woody biomass utilisation for the production of pulp and paper, specialty cellulose and chemicals, and cellulosic biofuels requires significant investment of chemicals and energy. Lignin, a major constituent of the secondary cell walls of vascular plants, is a significant impediment to chemically processing wood. Manipulation of the secondary cell wall chemical components and the composition of the lignin polymer provides an opportunity to gain insight into secondary cell wall deposition, and to develop genetically modified trees fit for competition within the global lignocellulosic market. vi Preface Evaluation of the vaiability of pHBA in Populus was completed in collaboration with Yaseen Mottiar (Mansfield lab, University of British Columbia). Generation of the master list of all poplar BAHD’s was completed by Drs. Charles Hefer (Mansfield lab, University of British Columbia) and Craig Bingman, (Fox lab, University of Wisconsin–Madison). Generation of 35S::CPL-47 (CPL Arabidopsis) overexpression line that expressed the E. coli ubiC gene which encodes CHORISMATE PYRUVATE-LYASE was established by Yaseen Mottiar (Mansfield lab, University of British Columbia). Drs. Rebecca A. Smith, Steven D. Karlen, John Ralph, Emily Beebe and Brian G. Fox (University of Wisconson-Madison) in collaboration with the US Departement of Energy Joint Genome Institute (JGI) and Great Lakes Bioenergy Research Center (GLBRC) completed in vitro enzyme synthesis and activity assays. The woody tissue used in Aim 1 of this work was collected and prepared for the Optimized Populus feedstocks and novel enzyme systems for a British Columbia bioenergy sector project through the Applied Genomics Innovation Program lead by Drs. Carl Douglas and Shawn D. Mansfield (University of British Columbia). The analysis of the wood properties of these genotypes used in Aim 1 of this work was previously published in Proth et al., 2008a and Proth et al., 2008b. vii RNA-Seq data used in Aim 2 of this work was collected and analysed for the POPCAN- Genetic improvement of poplar trees as a Canadian bioenergy feedstock project (POPCAN) through the 2010 Large-Scale Applied Research Project lead by Drs. Carl Douglas and Shawn D. Mansfield (University of British Columbia). All other work presented in this thesis represents original, unpublished, and independent work conducted by the author, Heather A. MacKay. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ............................................................................................................... xviii Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ................................................................................................................1 1.1 Cell walls ........................................................................................................................ 1 1.1.1 Lignin biosynthesis ................................................................................................. 2 1.1.2 Alteration of lignin content ..................................................................................... 5 1.1.3 Manipulation of lignin composition ........................................................................ 9 1.2 Acylated lignin .............................................................................................................. 12 1.2.1 p-hydroxybenzoylated monolignols ...................................................................... 13 1.2.2 pHBA-monolignol conjugate biosynthesis via BAHD – ATs .............................. 14 1.3 Objectives and rational ................................................................................................. 17 1.3.1 Evaluate the variability of pHBA in Populus ....................................................... 17 1.3.2 Identify putative p-hydroxybenzoyl-CoA monolignol transferase genes from P. trichocarpa (PtpHBMT) ........................................................................................................ 18 ix 1.3.3 Determine the in planta function of putative PtpHBMT in A. thaliana and hybrid poplar (Populus alba x grandidentata) ................................................................................. 18 1.4 Significance ................................................................................................................... 19 Chapter 2: Materials and Methods ............................................................................................20 2.1 Evaluating the variability of pHBA in Populus ............................................................ 20 2.1.1 Tissue growth and collection ................................................................................ 20 2.1.2 Isolation of cell wall bound phenolics .................................................................. 20 2.1.3 HPLC analysis ...................................................................................................... 21 2.1.4 Correlation analyses .............................................................................................. 21 2.1.5 Identify putative PtpHBMT from P. trichocarpa ................................................. 22 2.1.6 Selection of gene sequences .................................................................................. 22 2.1.7 Gene synthesis ...................................................................................................... 23 2.1.8 Transcription and translation ................................................................................ 23 2.1.9 Activity screening ................................................................................................. 24 2.1.9.1 LC-MS analysis ................................................................................................ 25 2.1.10 Putative PtpHBMT identification .......................................................................... 25 2.2 Expressing putative PtpHBMT in A. thaliana ............................................................... 26 2.2.1 Construct development ......................................................................................... 27 2.2.1.1 pK7WG2 constructs .......................................................................................... 27 2.2.1.2 AtC4HP constructs ............................................................................................. 29 2.2.2 A. tumefaciens transformation .............................................................................. 30 2.2.3 Generation of transgenic Arabidopsis ................................................................... 31 2.2.3.1 Genotypic analysis ............................................................................................ 32 x 2.2.3.1.1 Preliminary DNA screening ........................................................................ 32 2.2.3.1.2 Relative RT-PCR ........................................................................................ 33 2.2.3.1.3 qRT-PCR .................................................................................................... 34 2.2.3.2 Phenotypic analysis ........................................................................................... 35 2.2.3.2.1 Tissue harvest .............................................................................................. 35 2.2.3.2.2 Histochemical staining ................................................................................ 36 2.2.3.2.3 Soluble phenolic isolation ........................................................................... 36 2.2.3.2.4 Isolation of cell wall bound phenolics ........................................................ 37 2.2.3.2.5 HPLC Analysis ........................................................................................... 38 2.2.3.3 Statistical analysis ............................................................................................. 38 2.3 Expression of putative PtpHBMT in Hybrid Poplar ..................................................... 39 2.3.1 Agrobacterium transformation .............................................................................. 39 2.3.2 Generation of transgenic poplar ............................................................................ 39 2.3.2.1 Genomic Analysis ............................................................................................. 41 2.3.2.1.1 Preliminary DNA screening ........................................................................ 41 2.3.2.1.2 Relative RT-PCR ........................................................................................ 41 2.3.2.1.3 Tissue growth and collection ...................................................................... 42 2.3.2.1.4 qRT-PCR .................................................................................................... 43 2.3.2.2 Phenotypic analysis ........................................................................................... 44 2.3.2.2.1 Soluble phenolic isolation ........................................................................... 44 2.3.2.2.2 Isolation of cell-wall bound phenolics ........................................................ 45 2.3.2.2.3 Fraction Analysis via HPLC ....................................................................... 45 2.3.2.3 Statistical analysis ............................................................................................. 45 xi Chapter 3: Results ........................................................................................................................46 3.1 Evaluating the variability of pHBA in Populus ............................................................ 46 3.1.1 Quantification of cell wall bound pHBA .............................................................. 46 3.1.2 Correlation of pHBA with cell wall traits ............................................................. 46 3.2 Identify putative pHBMTs from P. trichocarpa ........................................................... 49 3.2.1 BAHD – AT identification and activity assay ...................................................... 49 3.2.2 Putative pHBMT identification ............................................................................. 49 3.3 Expression of putative PtpHBMT1 in A. thaliana ........................................................ 56 3.3.1 Line selection ........................................................................................................ 56 3.3.2 Quantification of soluble and cell wall bound pHBA ........................................... 58 3.3.2.1 Overexpression of PtpHBMT1 in Col-0 Arabidopsis - 35S::pHBMT1 ........... 58 3.3.2.2 Overexpression of PtpHBMT1 in CPL Arabidopsis - 35S::pHBMT1 + CPL .. 59 3.3.2.3 Overexpression of PtpHBMT1 in CPL Arabidopsis - C4H::pHBMT1+ CPL . 60 3.3.3 Supplementary phenotyping ................................................................................. 62 3.4 Expression of putative pHBMT in hybrid poplar ......................................................... 63 3.4.1 Line selection ........................................................................................................ 64 3.4.2 Quantification of soluble and cell wall bound pHBA ........................................... 65 3.4.2.1 Overexpression of PtpHBMT1 in Hybrid Poplar - 35S::pHBMT1 .................. 65 3.4.2.2 Overexpression of PtpHBMT1 in Hybrid Poplar - C4H::pHBMT1 ................ 67 3.4.3 Supplementary phenotyping ................................................................................. 69 Chapter 4: Discussion ..................................................................................................................71 4.1 Evaluating the variability of pHBA in Populus ............................................................ 71 4.2 Identify putative pHBMTs from P. trichocarpa ........................................................... 75 xii 4.3 Expression of PtpHBMT1 in A. thaliana ...................................................................... 78 4.3.1 Overexpression of PtpHBMT1 in Col-0 Arabidopsis ........................................... 78 4.3.2 Overexpression of PtpHBMT1 in CPL Arabidopsis ............................................. 81 4.4 Expression of the putative PtpHBMT in hybrid poplar ................................................ 83 4.4.1 Overexpression of PtpHBMT1 in hybrid poplar ................................................... 83 4.4.2 Overexpression of PtpHBMT1 in hybrid poplar using AtC4HP ........................... 84 Chapter 5: Conclusion .................................................................................................................88 5.1.1 Variability of pHBA in Populus ........................................................................... 88 5.1.2 Putative pHBMTs from P. trichocarpa ................................................................. 88 5.1.3 PtpHBMT1 in A. thaliana ..................................................................................... 89 5.1.4 PtpHBMT1 in hybrid poplar ................................................................................. 89 5.2 Significance ................................................................................................................... 90 5.3 Future experiments ........................................................................................................ 91 5.3.1 Examination of other putative PtpHBMTs in planta ............................................ 91 5.3.2 Exploring pHBA biosynthesis .............................................................................. 91 5.3.3 Optimising pHBA-monolignol synthesis and incorporation into the cell wall ..... 92 5.3.4 CPL Poplar ............................................................................................................ 92 5.3.5 Lignin analysis ...................................................................................................... 93 References .....................................................................................................................................94 Appendices ..................................................................................................................................117 Appendix A : Additional Materials and Methods ................................................................... 117 A.1 pHBA calculations .................................................................................................. 117 A.2 Correlation analysis ................................................................................................ 117 xiii A.3 Putative gene screening ........................................................................................... 119 A.4 T-test calculations ................................................................................................... 122 Appendix B : Additional transgenic lines ............................................................................... 123 B.1 Promoter isolation and swapping ............................................................................ 123 B.2 Overexpression of other putative PtpHBMTs ......................................................... 124 xiv List of Tables Table 1.1. Recent genetic modifications of genes within the lignin biosynthetic pathway in tree species (adapted from Chang et al., 2018). ................................................................................... 6 Table 2.1. Primers employed in this study. ................................................................................. 28 Table 3.1. Activity of putative pHBMTs with non-canonical monolignols. ............................... 52 Table 3.2. Identified lignin biosynthesis genes used in co-expression network analysis (Sundell et al., 2017). ................................................................................................................................. 56 Table 3.3. Summary of pHBA levels observed in the soluble and non-soluble fractions of Arabidopsis overexpression lines and corresponding statistics (n=3). ........................................ 61 Table 3.4. Summary of pHBA levels observed in the soluble and non-soluble fractions of poplar overexpression lines and corresponding statistics (n=9). ............................................................ 69 Appendix Table A.1. BLAST results of the 4 putative PtpHBMT enzymes against identified or suspected FMT and PMT enzymes. ........................................................................................... 120 Appendix Table B.1. Primers employed in this study. .............................................................. 124 Appendix Table B.2. Details of the constructs generated and transformations completed. ..... 125 xv List of Figures Figure 1.1. Simplified monolignol biosynthesis pathway and a representation of poplar lignin composition, as suggested by NMR-based analysis (adapted from Stewart et al., 2009; Boerjan et al., 2003; Vanholme et al., 2010; Mottiar et al., 2015; Yaseen Mottiar, Mansfield Lab, unpublished). .................................................................................................................................. 4 Figure 1.2. A proposed biosynthetic mechanism for BAHD mediated synthesis and incorporation of pHBA as a monolignol conjugate. .................................................................... 15 Figure 3.1. mg pHBA/g stem xylem tissue extracted from 9-year-old P. trichocarpa trees at 1.3m. ............................................................................................................................................ 47 Figure 3.2. Spearman correlation matrix of statistically significant wood traits with pHBA (mg pHBA/g xylem tissue). ................................................................................................................. 48 Figure 3.3. Clade organisation and LC-MS spectra of putative PtpHBMT1. ............................. 51 Figure 3.4. Spearman correlation matrix of xylem RNA expression levels of the BAHD clade containing the 5 putative pHBMTs and pHBA content (mg pHBA/g xylem tissue). .................. 53 Figure 3.5. The expression profile through cambial growth and wood formation and correlation heatmap of putative pHBMT’s in P. tremula. .............................................................................. 54 Figure 3.6. Co-expression network of lignin biosynthesis genes with putative pHBMT’s (Sundell et al., 2017). ................................................................................................................................. 55 Figure 3.7. Relative expression of PtpHBMT1 in Arabidopsis overoxpression lines. ................. 57 Figure 3.8 Levels of pHBA (mg/g stem tissue) within MeOH-HCL extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from Arabidopsis overexpression lines. ....................................................................................... 58 xvi Figure 3.9. Levels of pHBA (mg/g stem tissue) within MeOH-HCL extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from overexpression CPL Arabidopsis lines. .............................................................................. 59 Figure 3.10. Levels of pHBA (mg/g stem tissue) within MeOH-HCL extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from overexpression CPL Arabidopsis lines using AtC4HP. ....................................................... 60 Figure 3.11. 6-week-old A. thaliana overexpression line 35S::pHBMT1 + CPL-12 displaying no notable growth phenotype or alteration in lignin deposition compared to Col-0 and CPL Arabidopsis. ................................................................................................................................. 63 Figure 3.12. Relative expression of PtpHBMT1 in P39 over-expression lines. Expression normalised to PtTIF5. .................................................................................................................. 64 Figure 3.13. Levels of pHBA (mg/g stem tissue) within MeOH-HCL extracted soluble (post-saponification) fraction and saponified cell wall bound fraction of 4-month-old xylem tissue from overexpression P39 lines. .................................................................................................... 66 Figure 3.14. Levels of pHBA (mg/g stem tissue) within MeOH-HCL extracted soluble (post-saponification) fraction and saponified cell wall bound fraction of 4-month-old xylem tissue from overexpression hybrid poplar lines using AtC4HP. ............................................................. 68 Figure 3.15. 4-month-old P39 overexpression line 35S::pHBMT1-9 displaying no notable growth or diameter phenotype compared to P39. ........................................................................ 70 Figure 4.1. Geographical origins of 9 P. trichocarpa genotypes used for genotypic and phenotypic analysis by Xie et al. and Proth et al. (2009; 2013b) and the corresponding mg pHBA/g of xylem tissue (n ≥ 3). .................................................................................................. 72 Figure 4.2. Heatmap of mg pHBA/g tissue from P. nigra cv. ‘Italica’. ..................................... 73 xvii Appendix Figure A.1. Example of R code used in correlation analysis. .................................. 118 Appendix Figure A.2. Nucleotide sequence alignment showing differences between Potri.001G448000 from P. trichocarpa and the altered Potri.001G448000 sequence synthesized, and identical translated amino acid sequence of both. ................................................................ 119 Appendix Figure A.3. LC-MS and NMR results from in vitro screening. ............................... 121 xviii List of Abbreviations AsFMT - Angelica sinensis FERULOYL-CoA:MONOLIGNOL TRANSFERASE AT - Acyltransferase AtCesA7P - Arabidopsis thaliana CELLULOSE SYNTHASE CATALYTIC SUBUNIT 7 promoter AtC4HP - Arabidopsis thaliana CINNAMATE-4-HYDROXYLASE promoter AtUBQ5 - Arabidopsis thaliana UBIQUITIN 5 BA - 6-benzylaminopurine BdPMT - Brachybodium distachyon P-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE BLAST - Basic Local Alignment Search Tool bp - Base Pairs CAD - CINNAMYL ALCOHOL DEHYDROGENASE CaMV35SP - Cauliflower mosaic virus 35S promoter CCoAOMT - CAFFEOYL-CoA O-METHYLTRANSFERASE CCR - CINNAMOYL-CoA REDUCTASE CER2-LIKE - ECERIFERUM2-LIKE C3¢H - p-COUMARATE 3-HYDROXYLASE C4H - CINNAMATE 4-HYDROXYLASE xix C4H::pHBMT - pK7WG2 with AtC4HP and PtpHBMT1 4CL - 4-COUMARATE:CoA LIGASE COMT - CAFFEIC ACID O-METHYLTRANSFERASE CPL - CHORISMATE PYRUVATE-LYASE CPL Arabidopsis - 35S::CPL-47 DTT - Dithiothreitol F5H - FERULATE 5-HYDROXYLASE G - Guaiacyl GC - Gas Chromatography GLBRC - Great Lakes BioEnergy Research Center H - p-hydroxyphenyl HCT - p-HYDROXYCINNAMOYL-CoA:QUINATE/SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE HMM - Hidden Markov Model HPLC - High Performance Liquid Chromatography IS - Internal Standard JGI - Joint Genome Institute LB - Luria-Bertani LC-MS - Liquid Chromatography Mass Spectrometry miRCg1 - MIR156 family microRNA MS - Mass Spectroscopy MS Media - Murashige and Skoog basal medium xx NAA - 1-naphthaleneacetic acid NMR - Nuclear Magnetic Resonance OsFMT - Oryza sativa FERULOYL-CoA:MONOLIGNOL TRANSFERASE OsPMT - Oryza sativa P-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE P39 - Hybrid poplar genotype Populus alba x grandidentata PCR - Polymerase Chain Reaction pHBA - p-hydroxybenzoic acid pHBMT - p-HYDROXYBENZOYL-CoA MONOLIGNOL TRANSFERASE POPCAN - POPCAN-Genetic improvement of poplar trees as a Canadian bioenergy feedstock project PtCesA8P - Populus trichocarpa CELLULOSE SYNTHASE CATALYTIC SUBUNIT 8 promoter PtpHBMT - Populus trichocarpa pHBMT PtpHBMT1 - Putative PtpHBMT Potri.001G448000 PtpHBMT2 - Putative PtpHBMT Potri.001G447300 PtpHBMT3 - Putative PtpHBMT Potri.001G447400 PtpHBMT4 - Putative PtpHBMT Potri.001G447500 PtTIF5 - Populus trichocarpa TRANSLATION INITIATION FACTOR qPT-PCR - Quantitative real-time PCR xxi RT-PCR - Reverse-Transcriptase PCR S - Syringyl 35S::pHBMT1 - pK7WG2 with PtpHBMT1 TDZ - Thidiazuron TFA - Trifluoroacetic Acid YEP - Yeast Extract Peptone WPM - McCown’s Woody Plant Media ZmPMT - Zea mays P-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE xxii Acknowledgements I would like to thank my supervisor, Dr. Shawn Mansfield, for all of his support and scientific expertise. Additionally, I would like to thank my supervisory committee Drs. Lacey A. Samuels and Simone D. Castellarin for their guidance and support throughout this project. I would like to thank my lab mates, both past and present. Their support and knowledge were fundamental to the development and completion of this work. I would like to specifically acknowledge Drs. Eliana Gonzales-Vigil, Faride Unda, and Letitia Da Ros, and fellow graduate students Yaseen Mottiar and Reilly Ische who were particularly generous with their time, scientific advice, and support. I would also like to thank my collaborators at the University of Wisconsin-Madison who conducted the in vitro activity assays, Nuclear magnetic resonance (NMR) peak analysis, and Liquid chromatography–mass spectrometry (LC-MS) analysis, and the Great Lakes BioEnergy Research Centre for facilitating this partnership. Lastly, I would like to extend my thanks to all of the administrative staff within the Department of Wood Science and to the UBC greenhouse team, all of whom aided in the completion of this work with the provision of resources, time, and knowledge. xxiii Dedication I would like to dedicate this work to my family. Your guidance, support, and generosity have enabled me to pursue my passions and dreams. Your unconditional love has been fundamental to my development both as a scientist but also as an adult. Thank you for always being there for me. 1 Chapter 1: Introduction Woody biomass utilisation for the production of pulp and paper, specialty cellulose and chemicals, and cellulosic biofuels has gained significant attention in recent decades with the rapidly growing need for more sustainable and economical energy and commodity production practices. As a result, there is motivated interest in modifying plant cell wall traits, via selective breeding or genetic engineering, in order to enhance secondary xylem formation and economise the conversion of biopolymers common to woody biomass. Advancements in forest genomics have been critical to elucidating the pathways and mechanisms involved in cell wall biosynthesis. More specifically, the release of the Populus trichocarpa and Eucalyptus grandis genomes has been integral in the selection of candidate genes and pathways for manipulation (Tuskan et al., 2006; Myburg et al., 2014). Lignin, a polyphenolic constituent of the secondary cell walls of vascular plants and the second most abundant biopolymer on earth, has received considerable research attention within the last decade in the pursuit of woody biomass with novel cell wall characteristics. The objective of this research was to identify the BAHD acyltransferase (AT) that is responsible for the biosynthesis of the non-canonical monolignol p-hydroxybenzoic acid (pHBA). Elucidating the function of this gene should help gain a better understanding of lignin biosynthesis in plants, as well as generate transgenic trees with lignin containing altered levels of pHBA, valourising a major fraction of woody biomass. 1.1 Cell walls Plant cell walls are composed primarily of four polymeric macromolecules: cellulose, lignin, hemicellulose, and pectin. Cellulose represents approximately half of the woody biomass produced by trees, and is composed of semi-crystalline microfibril bundles comprised of β-(1-4)-2 linked D-glucan chains. These highly ordered microfibril bundles serve as the scaffold around which other cell wall polymers assemble (Taylor et al., 1992; Mellerowicz et al., 2001). Lignin, the second most abundant biopolymer on earth, is a polyphenolic constituent of the secondary cell walls of vascular plants and is comprised of varying ratios of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) moieties that polymerise within the cell wall by oxidative coupling post-laccase and peroxidase reduction of p-coumaryl, coniferyl, and sinapyl alcohols, respectively (Ralph et al., 2004). Although both cellulose and lignin both play a fundamental role in the vertical growth of trees, lignin is conventionally considered to be the glue providing structural integrity. Additionally, lignin has been shown to aid in water conduction and plant defense. In addition to the major chemical fractions in secondary cell walls, cellulose and lignin, in angiosperms such as poplar and eucalyptus, hemicelluloses are composed predominantly of glucuronoxylan chains. Pectin, the fourth major component of cell walls, is found mainly as a pectic-matrix surrounding the cellulosic-glycan network in the primary cell walls of woody tissues (Willats et al., 2001). Although pectin is not traditionally associated with secondary cell walls specifically, there is mounting evidence that modification of pectin may alter woody biomass processability and saccharification yields (Jarvis 1984; Biswal et al., 2018). 1.1.1 Lignin biosynthesis At a superficial level, lignin is considered to be comprised of varying ratios of H, G and S moieties that polymerise within the cell wall by oxidative coupling (Ralph et al., 2004). However, upon closer inspection we find that there is vast diversity within the polymer composition (Ralph et al., 2004; Morreel et al., 2004; Mottiar et al., 2016). For example, 5-hydroxyconiferyl alcohol, hydroxycinnaldehydes, and hydroxycinnamic acids, which are 3 bioproducts of monolignol biosynthesis, have all been clearly found within the lignin polymer (Vanholme et al., 2008). Additionally, atypical monolignol conjugates like sinapyl p-hydroxybenzoate and coniferyl p-coumarate, amongst others, have been found incorporated in the polymer, revealing great physicochemical diversity and plasticity (Morreel et al., 2004; Ralph et al., 2004; Del Rio et al., 2007). From an economic perspective, the substantive quantity and stability of lignin within woody biomass poses a major obstacle to industrial processing, and is often highlighted as the most significant impediment to the effective recovery of the high-value carbohydrate fractions. Delignification is critical for biomass utilisation, and typically relies on the addition of acid or alkali at high temperatures to remove lignin for fibre separation in pulp and paper production and to facilitate the liberation and subsequent saccharification of lignocellulosic material for biofuel production (Mansfield 2009). To economise biomass utilisation, previous research has focused on understanding lignin biosynthesis and plasticity, and as a result the lignin biosynthetic pathway has largely been mapped and provides biosynthetic mechanisms explaining some of the diversity found within the polymer (Humphreys and Chapple 2002; see Figure 1.1 for an overview of the phenylpropanoid biosynthetic pathway). More recent research endeavours have focused on improving the digestibility by altering the composition of the polymer and its constitutive monomers to engineer lignin with the aim to valorise biomass utilisation. 4 5 Figure 1.1. Simplified monolignol biosynthetic pathway and a representation of poplar lignin as suggested by NMR-based analysis (adapted from Stewart et al., 2009; Boerjan et al., 2003; Vanholme et al., 2010; Mottiar et al., 2015; Yaseen Mottiar, Mansfield Lab, unpublished). Abbreviations are as follows: PAL, PHENYLALANINE AMMONIA-LYASE; C4H, CINNAMATE 4-HYDROXYLASE; 4CL, 4-COUMARATE:CoA LIGASE; C3H, p-COUMARATE 3-HYDROXYLASE; HCT, p-HYDROXYCINNAMOYL-CoA:QUINATE/SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE; CCoAOMT, CAFFEOYL-CoA O-METHYLTRANSFERASE; CCR, CINNAMOYL-CoA REDUCTASE; F5H, FERULATE 5-HYDROXYLASE; COMT, CAFFEIC ACID O-METHYLTRANSFERASE; CAD, CINNAMYL ALCOHOL DEHYDROGENASE. Phenylcoumaran (b-5 or a-O-4) bonds are highlighted in green, resinol (b-b or g-O-a) linkages are highlighted in blue, and b-aryl ether (b-O-4) linkages are highlighted in red within the example polymer. 1.1.2 Alteration of lignin content Poplar, eucalyptus, and pine species are major sources of industrially important woody biomass and, as such, have been the main foci of biotechnological pursuits. Myriad of potential solutions to the lignin challenge have been proposed, but most notable efforts have focused on reducing the lignin content in plants by downregulating critical lignin biosynthetic genes (see Table 1.1 for a summary of genetic modifications of genes within the lignin biosynthetic pathway in tree species). For example, downregulation of CINNAMATE 4-HYDROXYLASE (C4H) resulted in a 30% reduction in lignin content in hybrid poplar (P. tremula x tremuloides; Bjurhager et al., 2010). Further downstream in the pathway, p-COUMARATE 3-HYDROXYLASE (C3¢H) has been downregulated in hybrid poplar (P. grandidentata x alba; P39) and eucalyptus (E. urophylla x grandis), and also resulted in a significant reduction of lignin content (from a conventional 22% and 29.6% to as low as 10% and 21.7% total cell wall content respectively; Coleman et al., 2008a; Sykes et al., 2015). Further analysis of these transgenic varieties revealed alteration in the H:S:G ratios within the polymer, with the most significant alterations being an increase in the H:G ratio (Coleman et al., 2008a; Ralph et al., 2012). Significant reduction in 6 Table 1.1. Recent genetic modifications of genes within the lignin biosynthetic pathway in tree species (adapted from Chang et al., 2018). Genetic modification Gene function Gene Transformant Species Reference Homologous expression cinnamate-4-hydroxylase C4H ↓ Populus tremula x tremuloides Bjurhager et al., 2010 p-coumaroyl-CoA 3-hydroxylase C3'H ↓ Populus grandidentata x alba Coleman et al., 2008a, Ralph et al., 2012 p-coumaroyl-CoA 3-hydroxylase C3'H ↓ Eucalyptus urophylla x grandis Sykes et al., 2015 4-coumarate:coenzyme A ligase 4CL ↓ Populus trichocarpa Hu et al., 1999; Schmidt et al., 2009; 4-coumarate:coenzyme A ligase 4CL ↓ Populus tremula x alba Voelker et al., 2011 4-coumarate:coenzyme A ligase 4CL ↓ Populus tomentosa Carr. Tian et al., 2013 4-coumarate:coenzyme A ligase 4CL ↓ Populus tomentosa Carr. Tian et al., 2013 4-coumarate:coenzyme A ligase 4CL ↓ Populus tomentosa Jia et al., 2004 4-coumarate:coenzyme A ligase 4CL ↓ Populus tremuloides Li et al., 2003 4-coumarate:coenzyme A ligase 4CL ↓ Pinus radiata Wagner et al., 2009 4-coumarate:coenzyme A ligase 4CL ↓ Betula pendula Sutela et al., 2014 hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferase HCT ↓ Populus radiata Wagner et al., 2007 cinnamoly-CoA reductase CCR ↓ Populus trichocarpa Laple et al., 2007 cinnamoly-CoA reductase CCR ↓ Populus radiata Wagner et al., 2007; Wagner et al., 2013 cinnamoly-CoA reductase CCR ↓ Picea abies Wadenbck et al., 2008 peroxidase PtrPO21 ↓ Populus trichocarpa Lin et al., 2016 microRNA Ptr-miR397a ↑ Populus trichocarpa Lu et al., 2013 ferulate/coniferaldehyde-5-hydroxylase F5H ↑ Populus tremula x alba Franke et al., 2000; Huntley et al., 2003; Stewart et al., 2009 caffeic acid O-methyltransferase COMT ↓ Populus trichocarpa x deltoides Van Doorsselaere et al., 1995; Lu et al., 2010 caffeic acid O-methyltransferase COMT ↓ Populus tremula x alba Marita et al., 2001 caffeoyl-coenzyme A O-methyltransferase CCoAOMT ↓ Populus tremula x alba Meyermans et al., 2000 7 Table 1.1. Recent genetic modifications of genes within the lignin biosynthetic pathway in tree species (adapted from Chang et al., 2018). Genetic modification Gene function Gene Transformant Species Reference caffeoyl-coenzyme A O-methyltransferase CCoAOMT ↓ Pinus radiata Wagner et al., 2011 microRNA miRCG1↑ Populus tremula x alba Rubinelli et al., 2013 Heterologous expression feruloyl-coenzymeA(CoA)monolignol transferase Angelica sinensis derived FMT Populus tremuloides Wilkerson et al., 2014 p-coumaroyl-CoA monolignol transferase Oryza sitiva derived PMT Populus trichocarpa Simth et al., 2015 tyrosine cell wall peptide Petroselinum crispum derived TYR Populus nigra x maximowiczii Liang et al., 2008 coniferaldehyde 5 hydroxylase Liquidambar styraciflua derived CAld5H Populus radiata Li et al., 2003 glutamine synthase Pinus pinaster derived GS1a Populus tremula x alba Gallardo et al., 1999; Coleman et al., 2012 Multigene manipulation coniferaldehyde 5 hydroxylase : 4-coumarate:coenzyme A ligase Liquidambar styraciflua derived CAld5H ↑ : 4CL ↓ Populus tremuloides Li et al., 2003; Suzuki et al., 2010 coniferaldehyde 5 hydroxylase : 4-coumarate:coenzyme A ligase Liquidambar styraciflua derived CAld5H ↑ : 4CL ↓ Populus trichocarpa Xiang et al., 2015 ferulate/coniferaldehyde-5-hydroxylase : 4-coumarate:coenzyme A ligase F5H ↓ : 4CL ↑ Populus nigra x maximowiczii Min et al., 2013; Min et al., 2014a; Min et al., 2014b ferulate/coniferaldehyde-5-hydroxylase : caffeic acid O-methyltransferase Liquidambar styraciflua derived F5H ↑: L. styraciflua derived COMT ↑ Populus radiata Wagner et al., 2015 8 lignin content has also been achieved by downregulating 4-COUMARATE:CoA LIGASE (4CL), which resulted in some transgenic lines showing as much as a 45% reduction in lignin and a significant increase in the presence of p-hydroxycinnamic acids, such as p-coumaroyl, caffeic, sinapic and ferulic acids (Hu et al., 1999; Li et al., 2003; Jia et al., 2004; Schmidt et al., 2009; Wagner et al., 2009; Voelker et al., 2011; Tian et al., 2013; Sutela et al., 2014). Downregulation of a putative p-HYDROXYCINNAMOYL-CoA:QUINATE/SHIKIMATE p-HYDROXYCINNAMOYLTRANSFERASE (HCT), a major artery enzyme involved in S and G monolignol biosynthesis was shown to be an effective target for reducing lignin content in P. radiata, where a 42% reduction in lignin content in tracheary elements was observed (Wagner et al., 2007). Downregulation of CINNAMOYL-CoA REDUCTASE (CCR), the penultimate step in monolignol biosynthesis, was shown to result in a 50% reduction in lignin content in P. trichocarpa, 46% reduction in P. radiata, and an 8% reduction in P. abies (Laple et al., 2007; Wagner et al., 2013; Wadenback et al., 2008). Interestingly, downregulation of CCR also resulted in the lignin being significantly altered in S:G ratio, and showed a dramatic shift in levels of p-coumaryl, caffeic acid, and ferulic acid hexoses, and the metabolic profile in general (Wagner et al., 2013). Although there are many ways in which the phenylpropanoid pathway can be modified to reduce lignin content, some researchers have looked outside of the common lignin biosynthetic genes for a solution. For example, Lin et al. (2016) identified and downregulated a class III peroxidase in P. trichocarpa (PtrPO21) that resulted in an average 20% reduction in lignin. This reduction in lignin was attributed to a decrease in the oxidative enzymes, therefore decreasing the degree of radicalisation of monolignols, and thus polymerisation. A similar result was also observed with 9 the upregulation of Ptr-miR397a, a microRNA that targets laccases (Lu et al., 2013). Though these aforementioned transgenic lines do, on average, have reduced lignin and presumably an increased ease of processing, dramatic changes in lignin content have often manifested in significant alterations in the morphology and agronomic potential of the ensuing plants (Coleman et al., 2008b; Min et al., 2012; Sykes et al., 2015). Trees with reduced lignin content tend to lack structural independence, have stunted growth, have reduced water conductivity and vascular stability, and have an increased vulnerability to pathogens and pests (Coleman et al.,2008b; Voelker et al., 2011a; Voelker et al., 2011b; Bonawitz and Chapple 2013). As a result, recent efforts have focused towards altering the physiochemical properties of lignin without significantly altering its content or functionality. 1.1.3 Manipulation of lignin composition Due to the pleiotropic effects associated with lignin reductions, efforts have focused on altering the ratio of lignin monomers. The H:G:S ratio varies considerably from species to species, and greatly affects the processability of xylem fibres and vessel elements. For example, S-rich lignins, which are more predominant in angiosperm species, contain more β–O–4 bonds compared to G-rich lignin, which dominants gymnosperm species and contain more β–5, 5-5, and β–β carbon–carbon bonds. The higher instance of β-ethers common to S-rich lignin make it more susceptible to alkali-mediated degradation, and thus increasing the S:G ratio has been a major scientific pursuit within the last few decades (Mansfield et al., 2012). The expression level of F5H (FERULATE 5-HYDROXYLASE/CONIFERALDEHYDE 5-10 HYDROXYLASE), CAFFEIC ACID O-METHYLTRANSFERASE (COMT), CAFFEOYL-CoA O-METHYLTRANSFERASE (CCoAOMT), and MIR156 family microRNA (miRCg1) have all been shown to impact the S:G ratio. For example, overexpression of F5H in hybrid poplar (P. tremula x alba) and P. tremuloides, resulted in an increase in S units within the lignin polymer, to as high as 97.5%, compared to the wild-type level of 68% (Franke et al., 2000; Huntley et al., 2003; Li et al., 2003; Stewart et al., 2009). In addition to upregulating F5H, Li et al. (2003) also downregulated 4CL and found that multigene misregulation significantly increased the S:G ratio, reduced lignin content, altered the phenolic composition, and improved enzyme-mediated saccharification yields (Suzuki et al., 2010; Xiang et al., 2015). Downregulation of COMT in poplar (P. trichocarpa x deltoides and P. tremula x alba) resulted in a significant decrease in S units, an increase in 5-OH-CA linkages within the polymer, an increased number of β–O–4 bonds in G and S end units, and the integration of 5-hydroxyconiferyl moieties within the polymer (Van Doorsselaere et al., 1995; Marita et al., 2001; Lu et al., 2010). Downregulation of CCoAOMT, while resulting in a decrease in the overall lignin content, also led to a significant increase in the S:G ratio of poplar (P. tremula x alba) and an increase in the H:G ratio in P. radiata (Meyermans et al., 2000; Wagner et al., 2011). In addition, downregulation of CCoAOMT resulted in the novel integration of caffeyl alcohol into the lignin polymer (Wagner et al., 2011). Moreover, overexpression of miRCg1 in hybrid poplar (P. tremula x alba) resulted in a 30% reduction in lignin and a significantly lower S:G ratio (Rubinelli et al., 2013). Another interesting avenue of strategic manipulation has been the introduction of S units into conifer species that are largely S deficient. Although increasing the abundance of S units in 11 hybrid poplar has met with great success (overexpression of F5H for example), the same cannot be said for alteration of conifer species. However, a successful proof of principle was achieved by simultaneously expressing Liquidambar styraciflua F5H and COMT in P. radiata tracheary element cell cultures (Wagner et al., 2015). In addition to the aforementioned methods for engineering lignin, there are also examples of altered lignin with reduced carbohydrate coupling. Reducing the linkages between lignin and secondary cell wall carbohydrate components limits the need for intensive pre-saccharification treatment by increasing carbohydrate accessibility for enzyme-mediated saccharification. For example, Min et al. (2013) observed that by concomitantly upregulating F5H and downregulating 4CL in hybrid poplar (P. nigra x maximowiczii), there was an increase in the lignin carbohydrate cross linkages within the secondary cell wall that resulted in less accessible cellulose for saccharification (Min et al., 2014a; Min et al., 2014b). Moreover, Coleman et al. (2012) successfully expressed a pine cytosolic glutamine synthetase in hybrid poplar (P. tremula x alba) and observed an increase in the S:G ratio. Notably, these transgenics also had significantly elevated levels of secondary cell wall carbohydrates, including glucose, galactose, xylose, and mannose. These significant studies have revealed the extensive inherent plasticity of lignification both in terms of the biosynthetic pathway, and the structure of the polymer itself (Vanholme et al., 2008; Stewart et al., 2009; Eudes et al., 2012; Mottiar et al., 2016). As a result of this malleability, there are significant active efforts to identify the genes responsible for the synthesis of favourable monolignol conjugates. For example, monolignol conjugates which, when integrated 12 into the lignin backbone, increase the number of alkali-cleavable bonds and improve delignification rates (Wilkerson et al., 2014). One promising method to increase the number of alkali-cleavable bonds is to increase the degree of acylation of lignin monomers. 1.2 Acylated lignin Increasing the degree of acylation (the process of adding an acyl group, or RCO– group, to a compound) of lignin monomers and/or integrating unique ester bonds (as opposed to ether bonds) into the lignin polymer backbone can increase the alkaline processability of the otherwise chemically stable polymer. Acylated monolignols are found both integrated and decorating the lignin polymer of many plants, and are thought to be synthesised via ATs prior to export to the cell wall and enzyme-mediated oxidation and radical coupling of lignin monomers (Del Rio et al., 2007). To-date, five monolignol ATs have been identified; three P-COUMAROYL-COA:MONOLIGNOL TRANSFERASEs in Oryza sativa (OsPMT), Zea mays (ZmPMT), and Brachybodium distachyon (BdPMT) which are responsible for acylating p-coumaroyl-CoA to monolignols, and two FERULOYL-COA:MONOLIGNOL TRANSFERASEs in Angelica sinensis (AsFMT) and O. sativa (OsFMT) that are responsible for acylating feruloyl-CoA to coniferyl and sinapyl alcohol (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014; Karlen et al., 2016). Successful development of more easily digestible lignin (A.K.A ‘zip-lignin’) was achieved with the integration of ferulate–monolignol conjugates into the lignin of hybrid poplar (P. alba x grandidentata) by heterologous expression of AsFMT (Wilkerson et al., 2014). Integration of ferulate–monolignol conjugates resulted in novel ester bonds within feruloylated lignin units, and increased the efficiency of delignification when subject to a number of pretreatment processes. This approach was also shown to be successful 13 with the expression of OsPMT in hybrid poplar (P. alba x grandidentata), resulting in p-coumarate conjugates incorporated into lignin (Smith et al., 2015). Introducing or increasing the abundance of these hydroxycinnamates (p-coumarate, ferulate, and sinapate) by employing or manipulating ATs is one area of particular interest in the pursuit of more chemically labile lignins (Ralph 2010). 1.2.1 p-hydroxybenzoylated monolignols p-hydroxybenzoic acid (pHBA) is one such hydroxycinnamate of interest, and is found decorating the lignin polymer as a pendant acylated monolignol conjugate at modest levels in Salix, Populus, and Palmae species. Currently, there is little known regarding the biosynthesis and incorporation of pHBA as a monolignol conjugate, and there is significant variability in historical reports of cell wall bound pHBA levels within and between taxa. For example, in Populus species pHBA reports range from 0.56 - 10% by lignin weight (Smith 1955; Nakano et al., 1961; Smith et al., 2015). Additionally, the function of pHBA constituents in lignin remains unclear. Observations of elevated pHBA levels within certain woody tissue types suggest that pHBA may play a role in wood mechanical properties. Closer investigation of hybrid aspen (P. alba × tremula) tension and opposite wood reveal elevated levels of pHBA when compared to normal wood (Gerttula et al., 2015; Mansfield Lab unpublished data). Another proposed function of pHBA is that it plays a significant role in plant defense. Elevated levels of cell wall bound pHBA in root tissue has been observed in a number of angiosperms, including Daucus carrota (Carrot; Sircar et al., 2011; Sircar et al., 2008; Sircar et al., 2007), Phoenix dactylifera (Date palm; Modafar et al., 2000), Solanum lycopersicum L. (Tomato; Mandel and Mitra, 2008), and 14 hybrid poplar (Mansfield Lab unpublished data), and has been correlated with host resilience to microbial infection (Mandel et al., 2009; Hussain et al., 2015). Alterations in lignin processability as a result of pHBA acylation remain unclear. However, current hypotheses suggest that acylation of lignin with pHBA conjugates as pendant moieties reduces the degree of polymerisation of the polymer, resulting in a more linear and chemically labile lignin structure. As such, increasing the incorporation of pHBA conjugates is a promising endeavour in the pursuit of more cost-effective biomass processing, and as a potential high-value cleavable phenolic molecule valourising this fraction of the biomass. Benzoate conjugates have significant commercial applications as they are used for their health and therapeutic effects, and widely applied in the cosmetic and food industries as additives (Eudes et al., 2016; Chedgy 2015; Adams et al., 2005). 1.2.2 pHBA-monolignol conjugate biosynthesis via BAHD – ATs pHBA biosynthesis is thought to occur as a perturbation from p-coumaroyl biosynthesis, however, the mechanism of pHBA-monolignol conjugate biosynthesis remains elusive (See Figure 1.2 for a proposed pHBA conjugate synthesis pathway). The prevailing hypothesis involves an enzyme-mediated cytosolic synthesis employing a BAHD – AT. BAHD – ATs are a notoriously promiscuous class of enzymes, and show vast diversity in both form and function (St-Pierre and De Luca, 2000). The common characteristic of these enzymes is the presence of the HXXXD and DFGWG motifs (Unno et al., 2007). The HXXXD motif serves a highly functional role as the histidine residue is the catalytic acylation site of these enzymes and 15 Figure 1.2. A proposed biosynthetic mechanism for BAHD mediated synthesis and incorporation of pHBA as a monolignol conjugate. Image modified from Yaseen Mottiar, Mansfield Lab, unpublished.Phenylpropanoid PathwayUnknown pathway in plants16 deprotonates the oxygen or nitrogen atom of an acceptor substrate, generating a nucleophile. This nucleophile subsequently attacks the carbonyl carbon of an acyl-CoA donor, forming an unstable tetrahedral intermediate. Reprotonation then occurs to yield an acylalted compound (St-Pierre and De Luca, 2000). To better understand the diversity and function of BAHD – ATs, Tuominen et al. (2011) conducted clade analysis employing the Arabidopsis, Medicago, Oryza, Populus, and Vitis genomes and identified eight functionally distinct clades. Although informative, this analysis reveals the significant diversity in AT function, and in vitro and in vivo characterisation of members of these clades shows substantive diversity in substrate specificity. For example, members of Clade V (closely related to hydroxycinnamoyl transferases responsible for chlorogenic acid and monolignol biosynthesis, and enzymes involved in volatile ester biosynthesis) have been shown to react with a variety of acyl-acceptor substrates, from alcohols to monolignols (D’Auria, 2006). A p-HYDROXYBENZOYL-COA MONOLIGNOL TRANSFERASE (pHBMT) is the putative BAHD – AT thought to be responsible for decorating monolignols with pHBA by facilitating the acylation between pHBA-CoA and canonical monolignols. However, the gene encoding this BAHD – AT remains elusive. 17 1.3 Objectives and rational To better understand the function of pHBA and the role of pHBMT in acylated monolignol biosynthesis, this study had three objectives: 1) Evaluate the variability of pHBA in Populus 2) Identify putative p-hydroxybenzoyl-CoA monolignol transferase genes from P. trichocarpa (PtpHBMT) 3) Determine the in planta function of putative PtpHBMT in A. thaliana and hybrid poplar (Populus alba x grandidentata) 1.3.1 Evaluate the variability of pHBA in Populus As stated previously, there is little known about the variability of pHBA in Populus species and there is significant variability in historical reports (estimates of pHBA by lignin weight have been documented to range from 0.56% to 10%; Smith 1955: Nakano et al., 1961; Smith et al., 2015). Establishing the variability of pHBA in poplar should enable correlations to be established between pHBA levels and other phenotypic and genotypic variables, and this may therefore offer a means to potentially uncover pHBA’s function, and to implicate candidate gene(s) in pHBA synthesis. 18 1.3.2 Identify putative p-hydroxybenzoyl-CoA monolignol transferase genes from P. trichocarpa (PtpHBMT) Monolignol transferases identified thus far all belong to the BAHD – AT enzyme class which has been shown to catalyse the transfer of CoA thioesters to acceptor molecules (D’Auria 2006). Previous research has shown that expression of monolignol transferases from B. distachyon, O. sativa, and A. sinensis in model organisms (e.g., A. thaliana) results in alterations in the conjugate profile of the lignin polymer, and an alteration of the polymer digestibility (Sibout et al., 2016; Petrick et al., 2014; Withers et al., 2012; Wilkerson et al., 2014; Karlen et al., 2016). However, as stated previously, the gene encoding pHBMT in Salix, Populus, and Palmae species has yet to be identified and thus the mechanism for pHBA conjugate synthesis remains largely a mystery. Identification of the pHBMT in P. trichocarpa should aid in identifying and understanding the molecular mechanism behind p-hydroxybenzoylated lignin synthesis. 1.3.3 Determine the in planta function of putative PtpHBMT in A. thaliana and hybrid poplar (Populus alba x grandidentata) A. thaliana is a model organism with vast genotypic and phenotypic information available and standardised transformation methods (Zhang et al., 2006). Additionally, gas chromatography (GC) and mass spectroscopy (MS) analyses of cell wall bound phenolics of A. thaliana have not shown appreciable levels of pHBA moieties (Ralph 2008). Therefore, expression of putative PtpHBMT in A. thaliana and an evaluation of associated phenotypes should be relatively straightforward. Expression of PtpHBMT in A. thaliana should aid in identifying the enzymes driving p-hydroxybenzoylated lignin synthesis and any phenotypic impact these moieties may confer. However, the conclusive experiment to determine PtpHBMT’s functionality in planta 19 will be to alter its expression in hybrid poplar (genotype P39). Many of the physiological properties of P39 are understood and there are reliable transformation methods available, therefore it is possible to express and identify physiological changes in response to the altered expression of putative PtpHBMTs. Overexpression of PtpHBMT in P39 should aid in confirming the enzyme(s) responsible for the production of p-hydroxybenzoylated lignin and the phenotypic impact these moieties have in poplar. 1.4 Significance pHBA has been found to decorate lignin as pendant acyl groups within woody tree species and is thought, by some, to reduce the degree of polymerisation of the lignin polymer and increase the processability of lignified tissue. Additionally, pHBA incorporated via ester linkages results in a valuable phenolic that can be easily “clipped off” by mild alkaline treatment, and can be extracted and applied in the pharmaceutical, cosmetic, and food industries, as well as a component in the production of certain synthetic polymers. Consequently, pHBA is of particular interest in the pursuit of designer lignin with enhanced value from both an industrial and commercial perspective. Identification of the pHBMT and manipulation of its expression in planta should allow us to better understand the molecular mechanisms controlling the acylation of monolignol biosynthesis in woody tree species and develop novel transgenic plants with unique lignin properties and commercial applications. 20 Chapter 2: Materials and Methods 2.1 Evaluating the variability of pHBA in Populus 2.1.1 Tissue growth and collection A 10 mm increment core was harvested at breast height from 118 unrelated P. trichocarpa genotypes grown under natural conditions for 9 years at the Ministry of Forests and Range Nursery in Surrey, British Columbia, Canada. These core samples were dried and analysed to determine the diversity in microfibril angle, fibre length, crystallinity, wood density. Samples were then ground using a Wiley® Mini-mill (Thomas®, USA) to pass a 40-mesh (0.4 mm) screen and analysed to determine the diversity in total lignin, acid-insoluble lignin, acid-soluble lignin, xylan, hemicellulose content, and S:G ratio among these unrelated genotypes (see Porth et al. 2013a; 2013b for full methodology of these analyses). Tissue was then left at room temperature for long term storage and eventual analyses. 84 of these 118 unrelated P. trichocarpa genotypes were selected for further pHBA analysis. 2.1.2 Isolation of cell wall bound phenolics To determine pHBA levels inherent to these P. trichocarpa genotypes, dried and ground xylem tissue was first subject to an acetone mediated extraction for 24 hours using a Soxhlet apparatus. 30 mg of extractive-free core tissue was then weighed into triplicate samples for each genotype. To liberate cell wall bound pHBA, tissue was then subject to base-mediated saponification using 1 mL of 2 M NaOH, in a Thermomixer comfort (Eppendorf, Germany) at 500 rpm and 30˚C for 24 hours. After this incubation period, saponification reactions were halted with the addition of 21 100 µL of 72% (w/w) H2SO4-, and placed on ice for 10 minutes. Tissue was then pelleted in a centrifuge at 13000 rpm for 5 minutes, and only the supernatant was retained and filtered through 0.45 µm Nylon Syringe Filters into 1.5 mL glass vials (Chromatographic Specialties, Canada) for high performance liquid chromatography (HPLC) analysis. 2.1.3 HPLC analysis Quantification of cell wall bound pHBA content was achieved using a Summit HPLC (Dionex, USA) fit with a Symmetry C18 column (Waters, USA) and PDA-100 Photodiode Array Detector (Dionex, USA). Samples were eluted through the column at a flow rate of 0.7 mL/minute using a gradient from 95% of a 0.1% trifluoroacetic acid (TFA) solution, to 75% acetonitrile:methanol:TFA (75:25:0.1) solution over 30 minutes. This elution gradient was followed by a 10-minute wash consisting of 75% of the 0.1% TFA and a column re-acclimation with 95% acetonitrile:methanol:TFA (75:25:0.1) solution for 15 minutes. Peak identification was achieved using standards of pHBA to act as references for the retention time and UV-spectra (at 255l). Peaks were manually integrated and pHBA concentrations were calculated using standard curves created by injecting serially diluted pHBA standards (Appendix A.1). All samples were run in triplicate with an internal standard of o-anisic acid (quantified at 296l) to account for sample injection error. 2.1.4 Correlation analyses Pair-wise comparisons of pHBA levels to previously determined wood traits (total lignin, acid-insoluble lignin, acid-soluble lignin, xylan, hemicellulose, microfibril angle, fibre length, 22 crystallinity, wood density, and S:G ratio; Porth et al., 2008a) was conducted to identify any potential statistically significant correlations. Statistical analysis, was performed using R version 3.5.1. (RStudio Inc., USA). Spearman correlation analysis was conducted using the statistical packages car v.2.1-4 (Fox and Weisberg, 2011) and an adjusted significance level was determined for each padj comparison using the adjustment formula !adj= #.#%# () *+,-./0/1(23. An example of the R code employed can be found in Appendix A.2. 2.1.5 Identify putative PtpHBMT from P. trichocarpa 2.1.6 Selection of gene sequences The entire complement of protein sequences from P. trichocarpa was obtained from NCBI GenBank (National Center for Biotechnology Information, USA) and matched against the hidden Markov model (HMM) profile for the Pfam 02458 family of CoA-dependent transferases to identify 148 proteins of interest. The protein sequences were organised into clades, where one gene was identified as an outlier. The remaining proteins were examined for a number of characteristics. Firstly, clades of highly similar sequences were examined for completeness where sequences that failed to cover at least 80% of the HMM profile were excluded from further analysis. The sequences were then tested for a number of characteristics that are known to provide a poor prognosis in protein expression systems. Specifically, they were evaluated for the presence of signal peptides or transmembrane domains, and for low complexity regions. No sequences contained signal peptides or possessed extensive low-complexity regions. Putative transferases that passed all of these tests were matched with their nucleotide sequences, and sent for synthesis. 23 2.1.7 Gene synthesis BAHD genes were synthesised at the US Department of Energy Joint Genome Institute (JGI) through a collaboration with the Great Lakes Bioenergy Research Center (GLBRC). As many of the known acyltransferase genes are GC-rich (>60%) and potentially problematic for DNA amplification and recombinant protein expression, the genes were codon-altered for ease of synthesis. The gene synthesis platform produced open reading frames in the wheatgerm cell-free expression vector pEU7 (Takai et al., 2010), which contains an SP6 promoter and omega enhancer sequence from the Tobacco Mosaic Virus. Plasmid DNA was purified from Escherichia coli using a commercial purification kit, then treated with proteinase K, and re-purified to remove residual RNAse activity and concentrate the DNA. 2.1.8 Transcription and translation Messenger RNA was prepared by adding 1.6 U of SP6 RNA polymerase and 1 U of RNase inhibitor (Promega Corporation, USA) to plasmid DNA solutions containing sub-cloned pEU7 plasmid DNA, 2.5 mM of UTP, CTP, ATP, and GTP, 20 mM magnesium acetate, 2 mM spermidine HCl, 10 mM dithiothreitol (DTT), and 80 mM HEPES-KOH. Reactions were then incubated at 37°C for 4 hours. This mRNA was then employed as the template for cell free translation using the WEPRO1240 Series Expression Kit (CellFree Sciences, Japan). In essence, this protocol involves taking the translation reaction and diluting it 4x using a solution of wheat germ extract, 0.04 mg/mL creatine kinase, 0.3 mM of each amino acid, 12.6 mM HEPES-KOH (pH 7.8), 52.6 mM potassium acetate, 1.3 mM magnesium acetate, 0.2 mM spermidine HCl, 2.1 mM DTT, 0.6 mM ATP, 0.13 mM GTP, 8.4 mM creatine phosphate, and 0.003% sodium azide. 125 µL of a solution containing 0.3 mM of each amino acid, 24 mM HEPES-KOH, 100 mM 24 potassium acetate, 2.5 mM magnesium acetate, 0.4 mM spermidine HCl, 4 mM DTT, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, and 0.005% sodium azide, was added to 96-well plates. 25 µL of the translation reaction mixture was carefully underlaid below 125 µL aliquots forming a bilayer. Plates were then incubated at 22°C for 18 hours. After translation, the reactions were sampled and evaluated for expression and protein characteristics using sodium dodecyl sulphate-phlyacrylamide gel electrophoresis. Trp-fluorescence or densitometric quantification of protein bands, migration position, and solubility of each reaction was analysed to approximate yield and indicate if the protein is properly folded and is a good candidate for scale-up purification. Purified products were analysed for in vitro activity. 2.1.9 Activity screening Translated enzymes were screened for activity with acetyl-CoA, benzoyl-CoA, p-hydroxybenzoyl-CoA, p-coumaroyl-CoA, and feruloyl-CoA, and all three monolignols (p-coumaryl, coniferyl, and sinapyl alcohol). 20 μL of cell-free translation product was added to a 30 µL solution comprised of 50 mM sodium phosphate buffer, 1 mM DTT, 1 mM CoA thioester, 1 mM monolignol mixture containing each monolignol. After a 1-hour incubation at room temperature, reactions were stopped with the addition of 50 µL of 0.1 M HCL. Finally, 50 µL of MeOH was added to each reaction and filtered through 0.2 µm Nylon Syringe Filters and analysed for product by liquid chromatography mass spectrometry (LC-MS). 25 2.1.9.1 LC-MS analysis Identification of enzymes with positive in vitro activity were identified on a Shimadzu LC-MS 8040 (Prominence LC with a triple-quadrupole mass spectrometer) equipped with a Kinetex 5u XB-C18 column (Phenomenex; 250 mm×4.6 mm×5μm, 100 Å, P/N:00G-4605-E0) held at 50 °C. The mobile phase was a binary gradient of water (A) and methanol (B) at a flow rate of 1 mL/min. The gradient protocol ran as follows: 0 min, 5% B; 2 min 5% B; 30 min, 100% B; 34 min, 100% B; 35 min, 5% B; 45 min 5% B. The eluent was first analysed by a diode array detector (scanning from 250-400 nm), and then by MS. The eluent was ionised using a DUIS probe head operating in electrospray ionization and atmospheric pressure chemical ionization modes (nebulizing gas 2.5 L/min, drying gas 15 L/min) and subject to a voltage of 4.5 kV to negatively ionise the samples scanning in both positive and negative mode from 120–600 m/z. Enzymes showing positive in vitro activity with the production of expected nuclear magnetic resonance (NMR) spectra (relative to known dimer standards) were identified as putative pHBMTs and subject to a final screening to identify the gene of interest for further analysis. 2.1.10 Putative PtpHBMT identification Using previously generated and publicly available datasets, identified genes of interest were further analysed. Leaf and xylem RNA-seq data (POPCAN-Genetic improvement of poplar trees as a Canadian bioenergy feedstock project, 2010) and publicly available microarray data (Sundell et al., 2017; Yang et al., 2008) was employed to determine the tissue-specific expression levels and patterns in poplar during various stages of growth. The National Center for Biotechnology Information basic local alignment search tool (BLAST) was used to assess 26 sequence similarity between candidates and conduct gene cluster analysis. Finally, using the pHBA levels determined in the previously described P. trichocarpa survey, correlation analyses were completed to compare observed cell wall bound pHBA with xylem RNA-seq data (POPCAN, 2010) following correlation statistical methods described previously. The identified candidate (PtpHBMT1) was then expressed in A. thaliana and P39 to determine its putative role in planta. 2.2 Expressing putative PtpHBMT in A. thaliana To identify the phenotypic impact of PtpHBMT1 expression on A. thaliana, three transgenic lines were created. First, PtpHBMT1 was expressed in wild-type A. thaliana Col-0 using the plant overexpression vector pK7WG2 and Gateway™ cloning methods (Karimi et al., 2002). Transformation, growth, and selection of transgenic lines were established following Agrobacterium-mediated transformation method outlined by Zhang et al. (2006). Additionally, PtpHBMT1 was expressed in a previously generated 35S::CPL-47 (CPL Arabidopsis) overexpression line that expressed the E. coli ubiC gene which encodes CHORISMATE PYRUVATE-LYASE (CPL; Yaseen Mottiar, Mansfield Lab, unpublished). CPL converts chorismate to pHBA and pyruvate (Viitanen et al., 2004). CPL Arabidopsis lines contain high levels of pHBA in the soluble fraction as an esterified glucoside, and contain detectable levels of pHBA in the cell wall bound fraction (Mansfield lab, unpublished data). The current hypothesis is that although pHBA is being synthesised in these lines, it may have limited incorporation into the cell wall due to one of two possible reasons: the lack of the yet unidentified CoA ligase (which produces the pHBMT substrate hydroxybenzoyl-CoA) or this CoA ligase may be rate 27 limiting the reaction resulting in conjugate synthesis but subsequent glycosylation and vacuolar compartmentalisation. It is certainly possible that neither the pHBMT or CoA ligase are highly expressed or even present in A. thaliana as the cell walls are not known for their pHBA-conjugate content. Expression of PtpHBMT1 in CPL Arabidopsis will firstly reduce the occurrence of false negatives (i.e., rejection of PtpHBMT1 due to lack of substrate availability and not due to alternative function), and aid in understanding the biosynthetic mechanism of pHBA-conjugate synthesis. In addition, PtpHBMT1 was expressed in CPL Arabidopsis, using the pK7WG2 backbone containing the A. thaliana CINNAMATE-4-HYDROXYLASE promoter (2900 base pairs (bp) upstream of At2G30490; AtC4HP) vs. the standard pK7WG2 Cauliflower mosaic virus 35S promoter (CaMV35SP). 35S::pHBMT1, 35S::pHBMT1 + CPL, and C4H::pHBMT1 + CPL Arabidopsis lines were phenotypically characterised by monitoring the growth and development (e.g., height and growth rate), examining lignin content and distribution in transverse stem sections (via histochemical staining), and identifying alterations in the phenolic profile of transformants compared to wild-type Col-0 and CPL backgrounds by HPLC analysis. 2.2.1 Construct development 2.2.1.1 pK7WG2 constructs Amplification of the full-length coding sequence of PtpHBMT1 was achieved using primers 1 and 2 (Table 2.1), and the purified pEU7 plasmid containing the synthesised putative PtpHBMT1 as the template in Bestaq™ DNA Polymerase (Applied Biological Materials, Vancouver) mediated polymerase chain reactions (PCR). The PCR product was evaluated by electrophoresis 28 on a 1% agarose gel in TAE buffer to ensure appropriate amplicon size, and purified via the QIAquick PCR Purification Kit (Qiagen, Netherlands) protocol according to the manufacturer's instructions. Purified pHBMT1 containing the appropriate 5¢ and 3¢ overhangs was then ligated into the Gateway™ pDONR™/Zeo Vector (Thermo Fisher Scientific, USA) following the standardised Gateway™ cloning methods, and transformed into E. coli One Shot™ competent cells following the suggested protocol (Invitrogen, USA). Successfully transformed competent cells were selected by screening on low-sodium Luria-Bertani (LB)-agar petri plates containing 50 μg/mL Zeocin™, colony PCR, and sequencing using primers 3 and 4 (Table 2.1) of sub-cloned purified plasmid (Sequencing + Bioinformatics Consortium, Vancouver). Once confirmed, PtpHBMT1 was then ligated into the plant overexpression vector pK7WG2 using Gateway™ LR Clonase according to the manufacturer's instructions and transformed into E. coli One Shot™ competent cells (Invitrogen, USA). Successfully transformed competent cells were selected by screening on LB-agar petri plates containing Spectinomycin (100 μg/mL), colony PCR, and sequencing using primers 3 and 4 (Table 2.1) of sub-cloned purified plasmid (Sequencing + Bioinformatics Consortium, Vancouver). The final construct, henceforth referred to as 35S::pHBMT1, was transformed into Agrobacterium tumefaciencs strain GV3101 and EHA105 for subsequent plant transformations. Table 2.1. Primers employed for plasmid construction and gene identification. No. Name Use and Properties Sequence (5¢ to 3¢) 1 pHBMT_RV3 Amplification of the full reading frame of synthesised potri.001G448000 for GATEWAYTM cloning. Contains attB recognition sites. GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAGACGATGACGAATTGGC 2 pHBMT_FW3 GGGGACAAGTTTGTACAAAAAAGCAGGCT-ATGCCAACCCCAACCAG 3 pHBMT_Seq_RV Sequencing primers CAG AAT ACT TGG GGC TTG GG 4 pHBMT_Seq_FW GTG AAT GCC CGT AGT AAA TTT AAT CCA C 29 5 AtC4HP + XbaI RV Clone out AtC4HP from QSUB plasmid and insertion of SpeI and XbaI restriction sites, and sections of pK7WG2 plasmid GCTCTAGAGCGGCCGCCTGCAGGTCGACCTAGGGGGCGAGAGTAATTG 6 AtC4HP + SacI FW GTGAGCTCTCCCATATGGTCGACGGAATGAGAGACGAGAGC 7 AtC4HP Seq RV Sequencing primers CTCCTGAAATGATGAAGTTTTCCCG 8 AtC4HP Seq FW GATATTGAAAGAGTTCATGGACGGATGTG 9 AtC4HP Seq RV2 CACATCCGTCCATGAACTCTTTCAATATC 10 AtC4HP Seq FW2 CGGGAAAACTTCATCATTTCAGGAG 11 UBQ5_RT_R Amplification of AtUBQ5 for relative and semi-quantitative PCR CAAGCCGAAGAAGATCAAGCACAAG 12 UBQ5_RT_F CAGCTCCACAGGTTGCGTTA 13 pHBMT4-qPCR RV Amplification of PtpHBMT1 for relative and semi-quantitative PCR GGTACAGTTGACGATCAATATCACTC 14 pHBMT4-qPCR FW GGCCTTTAATGTGCGTCG 15 Tif_qPCR_R Amplification of PtTIF5 for relative and semi-quantitative PCR CTGATAACACAAGTTCCCTGC 16 Tif_qPCR_F GACGGTATTTTAGCTATGGAATTG 2.2.1.2 AtC4HP constructs Amplification of the 2900 bp promoter sequence of AtC4HP was achieved using primers 5 and 6 (Table 2.1) from purified pA6pC4HsQsuB plasmid. This plasmid was previously generated and promoter expression analysis was conducted by collaborators (Eudes et al., 2015). Primer 6 (Table 2.1) contained a SacI restriction site at the 5¢ end, while primer 5 contained a XbaI restriction site at the 3¢ end to enable enzyme mediated promoter ligation. Bestaq™ DNA Polymerase was used to minimise errors during amplification (Applied Biological Materials, Vancouver). The PCR product was then electrophoresed on a 1% agarose gel in TAE buffer and the anticipated 2900 bp band was excised and purified. Gel extraction was completed following the PureLink Quick Gel Extraction Kit (Invitrogen), where DNA isolation was achieved by filtering the dissolved gel sample through a silica membrane. Both the purified AtC4HP and the plant overexpression vector pK7WG2 were subject to SacI digestion following the optimised enzyme FastDigest™ protocol. To generate the compatible overhangs and prevent the ligation of 30 alternative constructs due to the presence of multiple restriction sites, the purified AtC4HP product was then subject to XbaI digestion, while the vector pK7WG2 was subject to SpeI digestion following optimised enzyme FastDigest™ protocol. The pK7WG2 digested product was then electrophoresed on a 1.5% agarose gel in TAE buffer and the band of expected size (~11,000 bp) was extracted and purified. The complementary overhangs of purified pK7WG2 backbone (lacking the CamV35SP) and digested AtC4HP were subsequently ligated together using T4 DNA Ligase according to the manufacturer's instructions (Thermo Fisher Scientific, USA) and transformed into E. coli One Shot™ ccdB Survival™ competent cells. Successfully transformed competent cells were selected by screening on LB-agar petri plates, colony PCR, and sequencing using primers 7, 8, 9, and 10 of purified plasmid (Table 2.1; Sequencing + Bioinformatics Consortium, Vancouver). Identical methods were employed for PtpHBMT1 ligation and construct development as described for the pK7WG2 construct. The final construct, henceforth referred to as C4H::pHBMT1, was transformed into A. tumefaciencs strain GV3101 and EHA105 for subsequent plant transformations. 2.2.2 A. tumefaciens transformation A. tumefaciens strain GV3101 containing 35S::pHBMT1 and C4H::pHBMT1 constructs was employed to transform both Col-0 and CPL Arabidopsis. A. tumefaciens transformation was completed using the freeze-thaw method, whereby 1 μg of plasmid DNA was added to thawed 50 µL Agrobacterium aliquots and incubated on ice for 30 minutes. Cells were then subject to a 37 °C water bath heat shock for 5 minutes and allowed to recover in 1mL of Yeast Extract Peptone (YEP) medium at 28 °C and 200 rpm for 4 hours. Cells were then concentrated by 31 centrifugation at 13,000 rpm for 1 minute and resuspended in 100µL of fresh YEP media. Reactions were then plated and incubated at 28 °C for 48 hours. Successfully transformed cells were selected by screening on YEP-agar plates containing 25 µg/mL rifampicin and 50 µg/mL spectinomycin, and PCR of purified plasmid using primers 1 and 2 for cells transformed with 35S::pHBMT1, and primers 1, 2, 5, and 6 for cells transformed with C4H::pHBMT1. 2.2.3 Generation of transgenic Arabidopsis Transformation, growth, and selection of transgenic A. thaliana was completed by following the Agrobacterium-mediated transformation method outlined by Zhang et al. (2006). Sterilised wild-type Arabidopsis Col-0 seeds were stratified in the dark at 4 °C for 72 hours and plated on ½ concentration Murashige and Skoog basal medium (MS media) – agar plates, and allowed to germinate in tissue culture for 2 weeks. Sterilised CPL Arabidopsis seeds were stratified in the dark at 4 °C for 72 hours and plated on ½ concentration MS media– agar plates containing 25g/mL hygromycin, and allowed to germinate in tissue culture for 2 weeks. Germinated seedlings were then planted, five to a pot, in autoclaved potting soil (Westcreek Farms Ltd., Fort Langely, BC), and grown covered in a growth chamber under long-day conditions (16/8-h light/dark cycle) at 21 °C for 2 weeks. Covers were then removed and plants allowed to grow for an additional 4 weeks under controlled long-day conditions. Transformed Agrobacterium containing 35S::pHBMT1 and C4H::pHBMT1 constructs were grown in YEP media with 25 µg/mL rifampicin and 50 µg/mL spectinomycin at 28 °C for 16 hours at 200 rpm. Once cell cultures reached an optical density (OD600) = 1-1.5, cells were concentrated by centrifugation at 13,000 rpm for 1 minute and resuspended in 50mL of a 5% sucrose and 0.02% SYLGARD® 309 (Dow Corning, USA) solution. The floral portions of Arabidopsis plants were submerged in the 32 aforementioned Agrobacterium solutions, and agitated for approximately 20 seconds. Once excess solution was removed, plants were wrapped in opaque cellophane, placed horizontally, and left for 24 hours. After this recovery period, plants were placed vertically, uncovered, and left for 2 weeks, and thereafter the identical transformation procedure was repeated. Plants were then left to senescence and seeds were collected. These T1 seeds were subsequently sterilised, stratified, and plated following the methods described above. However, to screen for successful transformants, ½ concentration MS media–agar plates containing 50 mg/mL kanamycin were employed. Germinated seedlings which developed true leaves after two weeks in tissue culture were planted in autoclaved soil and grown in growth chambers for 6 weeks for preliminary DNA screening and quantitative genotypic and phenotypic analyses. 2.2.3.1 Genotypic analysis 2.2.3.1.1 Preliminary DNA screening 6-week-old leaf tissue was used for the isolation of genomic DNA to confirm successfully transformed lines. T1 Arabidopsis leaves were harvested and ground with a sterile micro-pestle in 400 μL of a DNA extraction buffer containing 25 mM NaEDTA, 250 mM NaCl, 200 mM Tris HCl, and 0.5% Sodium Dodecyl Sulfate. Homogenised samples were then pelleted by centrifugation at 13,000 rpm for 3 minutes and the supernatant retained for isopropanol mediated DNA precipitation which involved adding 300 µL isopropanol, a brief agitation by vortexing, and a 5-minute incubation at room temperature. Samples were then pelleted by centrifugation at 13,000 rpm for 5 minutes and the supernatant discarded. To wash the residual pellets, 500 µL of 70% EtOH was added, samples were briefly vortexed, and re-pelleted at 13,000 rpm for 2 33 minutes. The supernatant was removed, and the pellets were left to air dry to remove residual EtOH and finally resuspended in autoclaved distilled water. This genomic DNA was employed as the template in Taq DNA Polymerase (Applied Biological Materials, Vancouver) mediated PCR reactions with primers 1 and 2 (Table 2.1) for Arabidopsis transformed with 35S::pHBMT1, and primers 1, 2, 5, and 6 for Arabidopsis transformed with C4H::pHBMT1. 2.2.3.1.2 Relative RT-PCR To identify lines of interest for further analysis, PtpHBMT1 expression levels were estimated in 6-week-old T2 leaf tissue using the BioRad Image Lab Software (Bio-Rad Laboratories, USA). Leaf tissue was ground in a mortar and pestle under liquid nitrogen and approximately 100 mg of tissue was allowed to thaw at room temperature in 1 mL of TRIzol™ Reagent (Thermo Fisher Scientific, USA). Excess tissue remained frozen and was stored at -80°C. RNA isolation was achieved by following the TRIzol™ Reagent protocol with the addition of a second EtOH wash using 95% EtOH. To remove contaminating DNA, isolated RNA was subjected to TURBO™ DNase treatment following the manufacturer’s instructions (Thermo Fisher Scientific, USA). To ensure successful RNA isolation, the sample concentration and purity was determined spectrophotometrically using a Nanodrop 1000 (Thermo-Fischer Scientific, USA). Samples with reasonable concentrations and A260/280 ratios were diluted in autoclaved distilled water to a working concentration of 100 ng/µL and used as templates for cDNA synthesis using the EasyScript PlusTM cDNA Synthesis Kit according to manufacturer’s instructions (Applied Biological Materials, Vancouver) and stored at -80°C. It is worth noting that both Oligo(dT) and random primers were employed in cDNA synthesis reactions. Synthesised cDNA was used as the 34 template in Taq DNA Polymerase (Applied Biological Materials, Vancouver) mediated reverse-transcriptase PCR (RT-PCR) reactions using primers 11, 12, 13 and 14 (Table 2.1). The PCR product was then electrophoresed on a 1% agarose gel in TAE buffer and imaged using the BioRad Image LabTM software. Using the Relative Quantity tool, expression level was estimated by normalising the band brightness of PtpHBMT1 compared to A. thaliana UBIQUITIN 5 gene (AT3G62250; AtUBQ5) in each reaction. Lines showing the highest relative expression were then left to senescence and seeds were collected. The ensuing T2 seeds were subsequently sterilised, stratified, and plated following the methods described previously. Germinated seedlings were planted in autoclaved soil and grown in growth chambers for 6 weeks for eventual quantitative genotypic and phenotypic analyses. 2.2.3.1.3 qRT-PCR Quantitative real-time PCR (qPT-PCR) techniques were employed to determine the relative transcript abundance in the stem tissue of T2 Arabidopsis overexpression lines. T2 seeds were sterilised, stratified, and plated following the methods described previously. Germinated seedlings were planted in autoclaved soil and grown in growth chambers for 6 weeks. The bottom 10 cm of the main stems were harvested from 6-week-old Arabidopsis, and cDNA was synthesised following methods as described above. The cDNA was then used as the template in qPCR reactions using BrightGreen Express 2X qPCR MasterMix (Applied Biological Materials, Vancouver) and a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, USA) according to the manufacturer's instructions. To determine primer efficiency and an appropriate template concentration, cDNA from all transgenic lines and background lines 35 was pooled and serial diluted 5 times. This dilution series was then applied in qRT-PCR reactions using gene specific primers 13 and 14, and reference gene primers 11 and 12 (Table 2.1). Appropriate dilutions of cDNA stock were then established for each line using autoclaved distilled water and used in qRT-PCR analysis. Relative gene expression levels were determined using the formula: 2−(45678!9: − 45;:<:;:=>:). All qRT-PCR reactions were run with 3 technical replicates and 3 biological replicates. 2.2.3.2 Phenotypic analysis 2.2.3.2.1 Tissue harvest T2 seeds were sterilised, stratified, and plated following the methods described previously, and germinated seedlings were planted in autoclaved soil and grown in growth chambers for 6 weeks. For histochemical analysis, the bottom 5 cm of the stem base was harvested and placed in distilled water for sectioning. For phenolic analysis the bottom 10 cm of the main inflorescence of each line was harvested and pooled. Tissue was then dried in a 50˚C oven for 48 hours and homogenised using a 2000 Geno/Grinder® (SPEX® SamplePrep, USA) at 500 strokes/minute for 2 minutes. To ensure tissue was completely dehydrated, samples were lyophilised overnight in a FreeZone 4.5 Liter Benchtop Freeze Dry System (Labconco, USA). This tissue was then weighed, and allocated into screw cap tubes for phenolic analysis, while the remaining tissue was stored at room temperature in a desiccator containing DrieriteTM desiccant (W.A. Hammond, USA). 36 2.2.3.2.2 Histochemical staining On the same day of tissue harvest, the 5 cm of base stem tissue was hand sectioned and subject to toluidine blue O and phloroglucinol-HCL staining to examine lignin content and distribution in the overexpression lines following the protocol outlined by Mitra and Loqué (2014). Fresh phloroglucinol stain was prepared by dissolving 0.2 g of phloroglucinol (Sigma-Aldrich, USA) in 10 mL of absolute ethanol and then 5 mL of concentrated 37 M HCl was added. 1 mL of phloroglucinol-HCL was added to single stem segments and immediately pipetted onto microscope slides for visualisation using a DMR light microscope (Leica Microsystems, Germany). Toluidine blue O stain was achieved by adding 500 mL of a 0.02% solution of toluidine blue O (Thermo Fisher Scientific, USA) to another aliquot of stem sections. Sections were gently mixed by inversion and left to stain for 5 minutes at room temperature. The toluidine blue O stain was removed, and sections gently subject to 5 rounds of washing with distilled water. Washed sections were then pipetted onto microscope slides for visualisation. Images were captured remotely using OpenLab software (Agilent Technologies, USA) with a Canon EOS Rebel T5 and QImaging Qcapture camera (Roper Technologies, USA), and EOSUtility software. 2.2.3.2.3 Soluble phenolic isolation 30 mg of dried stem tissue was weighed out in triplicate for each transgenic line and subject to a MeOH extraction for determination of phenolics. Extraction was achieved by adding 1.5 mL of a methanol/water/HCl (48.5:48.5:1) solution and incubating samples at 50°C for 4 hours. Extracted samples were then pelleted by centrifugation at 15,000 g for 10 minutes. The 37 supernatant was retained and divided into two approximate 500 µL aliquots. To liberate ester-bound phenolics, one aliquot was subject to base-mediated saponification using 2 M NaOH, in a Thermomixer comfort (Eppendorf, Germany) at 500 rpm and 30˚C for 24 hours. The reactions were then stopped with the addition of 100 µL of 72% (w/w) H2SO4- and placed on ice for 10 minutes. The ether phase of these samples was then isolated by ethyl ether phase separation. 1 mL of nano-pure water and 1 mL of ethyl ether were added to both the saponified and non-saponified samples, briefly vortexed, and left to separate for approximately 10 minutes. The upper phase was retained and subject to a second ethyl ether phase separation. The upper phase was again retained and pooled with the first phase separation, concentrated using a 5301 Vacufuge (Eppendorf, Germany) at 1,400 rpm for 15 minutes. Pellets were resuspended in 1 mL MeOH and filtered through 0.45 µm Nylon Syringe Filters into 1.5 mL glass vials (Chromatographic Specialties, Canada) for HPLC analysis. 2.2.3.2.4 Isolation of cell wall bound phenolics To determine ester-bound cell wall bound phenolics, 30 mg of dried stem tissue was weighed out in triplicate for each transgenic line and subject to a methanol/water/HCl (48.5:48.5:1) wash, as previously described. The supernatant was discarded while the pellet was retained. Pellets were then re-suspended in acetone, briefly vortexed, and re-pelleted by centrifugation at 4,000 g for 15 min. The supernatant was discarded and pellets were washed with acetone a second time. Pellets were then air dried at 50˚C for 24 hours. Samples were then re-weighed and subject to saponification following the methodology described above with two alterations; after the termination of the saponification reaction, samples were centrifuged at 13,000 rpm for 2 minutes 38 and only the supernatant was retained HPLC analysis, and samples were not subject to phase separation. 2.2.3.2.5 HPLC Analysis Analysis pHBA content and phenolic profiles of transgenic Arabidopsis lines was achieved on a Summit HPLC (Dionex, USA) fit with a Symmetry C18 column (Waters, USA) and PDA-100 Photodiode Array Detector (Dionex, USA). Samples were eluted through the column at a flow rate of 0.7 mL/minute using a gradient from 95% of a 0.1% TFA, to 75% of a acetonitrile:methanol:TFA (75:25:0.1) solution over 30 minutes. This gradient was followed by a 10-minute wash with 75% of the 0.1% TFA and a re-acclimation phase consisting of 95% acetonitrile:methanol:TFA (75:25:0.1) solution for 15 minutes. Peak identification was achieved using standards of pHBA. Peaks were manually integrated and pHBA concentrations were calculated using standard curves created using the serially diluted pHBA standards. All samples were run in triplicate with an internal standard of either 3,4,5 trimethoxycinnamic acid, or o-anisic acid to account for sample injection error. A MeOH wash was periodically run through the system to ensure no sample-to-sample contamination. 2.2.3.3 Statistical analysis Significant differences between observed pHBA levels between both backgrounds (Col-0 and CPL Arabidopsis) and overexpression lines was completed using a standard Student’s t-test for samples of equal size and variance (Appendix A.3). Significant p-values ≥ 0.05. 39 2.3 Expression of putative PtpHBMT in Hybrid Poplar PtpHBMT1 was expressed in P39 using the overexpression constructs 35S::pHBMT1 and C4H::pHBMT1. Successfully transformed lines were then evaluated using the genotypic and phenotypic analyses employed for the A. thaliana overexpression lines to determine the effect of PtpHBMT1 expression on lignin composition. P39 transformation and growth was completed following the method outlined in Wilkerson et al. (2014). 2.3.1 Agrobacterium transformation A. tumefaciens strain EHA105 was employed to transform P39 with both 35S::pHBMT1 and C4H::pHBMT1 constructs. Agrobacterium transformation was completed using the freeze-thaw methods as previously outlined. Successfully transformed colonies were selected by screening on YEP-agar plates containing 25 µg/mL rifampicin and 50 µg/mL spectinomycin, and PCR of purified plasmid using primers 1 and 2 for cells transformed with 35S::pHBMT1, and primers 1, 2, 5 and 6 (Table 2.1) for cells transformed with C4H::pHBMT1. 2.3.2 Generation of transgenic poplar Transformation, growth, and selection of transgenic P39 was completed following the methods outlined in Wilkerson et al. (2014). Transformed Agrobacterium containing 35S::pHBMT1 and C4H::pHBMT1 were grown in YEP media with 25 µg/mL rifampicin and 50 µg/mL spectinomycin at 28 °C for 16 hours at 200 rpm. These cultures were then diluted with a 1:100 solution of McCown’s Woody Plant Media (WPM):acetosyringone. Cultures were then left at 28 °C for 16 hours at 200 rpm. After which the cultures were diluted to an OD600 = 0.2 using liquid 40 WPM and aliquoted into 25 mL solutions in 50 mL conical centrifuge tubes. Using sterile techniques, approximately 400 leaf disks were cut from 6-week-old tissue culture grown P39 using a generic single-hole punch. Approximately 50 leaf disks were added to each 25 mL aliquot of Agrobacterium. Tubes were then placed horizontally in a rotary shaker at 28 °C at 100 rpm for 30 minutes. Leaf discs were then removed from the Agrobacterium and excess media was removed by blotting disks with sterile filter paper. Dried disks were subsequently transferred onto WPM-agar petri plates containing 0.1 μM 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and thidiazuron (TDZ) abaxial side up, and allowed to recover in dark conditions for 48 hours. Leaf discs were then transferred (again, abaxial side up) onto WPM agar containing 0.1 μM NAA, BA, TDZ supplemented with 250 mg/L cefotoxine and 500 mg/L carbenicillin to kill remaining Agrobacterium, and 50 mg/L kanamycin to allow the identification of positively transformed poplar lines. These plates were left in reduced light conditions for approximately 4 weeks to enable the development of callus. Disks displaying substantial callus were transferred to WPM-agar petri plates containing 0.01 μM BA, 250 mg/L cefotoxine, 500 mg/L carbenicillin, and 50 mg/L kanamycin. These plates were again left in reduced light for approximately 4 weeks to enable the development of shoots. One shoot from each successful calli were excised and transferred to WPM-agar petri plates containing 0.01 μM NAA, 250 mg/L cefotoxine, 500 mg/L carbenicillin, and 50 mg/L kanamycin. These plates were left in reduced light conditions for approximately 4 weeks to enable the development of roots. Shoots with well-established roots were excised and transferred to WPM-agar Magenta™ GA-7 vessels containing 0.01 μM NAA and 50 mg/L kanamycin and moved to tissue culture. After 4 weeks of growth, plants were removed from selection media and transferred to fresh GA-7 with WPM-agar containing 0.01 μM NAA. After the plants reached approximately 6 weeks of 41 growth, leaf tissue was harvested for preliminary DNA screening and sub-cultured onto fresh media. 2.3.2.1 Genomic Analysis 2.3.2.1.1 Preliminary DNA screening 6-week-old leaf tissue from tissue culture was employed for the isolation of genomic DNA to confirm successfully transformed lines, following the genomic DNA isolation protocol describes previously with no modifications. Genomic DNA was used as the template in Taq DNA Polymerase (Applied Biological Materials, Vancouver) PCR reactions with primers 1 and 2 (Table 2.1) for P39 transformed with 35S::pHBMT1, and primers 1, 2, 5 and 6 (Table 2.1) for P39 transformed with C4H::pHBMT1. The PCR product was then electrophoresed on a 1% agarose gel in TAE buffer and imaged using the BioRad Image LabTM Software. Confirmed transformants were then sub-cultured onto fresh GA-7’s containing WPM-agar with 0.01 μM NAA and left for 6 weeks in tissue culture conditions. 2.3.2.1.2 Relative RT-PCR To identify lines of interest for further analysis, PtpHBMT1 expression levels were estimated in 6-week-old leaf tissue of confirmed transformants using the BioRad Image Lab Software (Bio-Rad Labratories, USA). RNA was isolated from ground leaf tissue following the TRIzol™ Reagent protocol described previously with no modifications. Isolated RNA was subject to TURBO™ DNase treatment following the manufacturer’s instructions (Thermo Fisher Scientific, USA). Once the concentration and purity were determined spectrophotometrically, the 42 samples were diluted in nanopure water to a working concentration of 100 ng/µL. RNA from each successfully transformed line was used as the template in cDNA synthesis reactions using the EasyScript PlusTM cDNA Synthesis Kit according to manufacturer’s instructions (Applied Biological Materials, Vancouver) and stored at -80°C. Both Oligo(dT) and random primers were used in cDNA synthesis reactions, and used as the template in Taq DNA Polymerase (Applied Biological Materials, Vancouver) RT-PCR reactions using primers 13, 14, 15 and 16 (Table 2.1). The PCR product was then electrophoresed on a 1% agarose gel in TAE buffer and imaged using the BioRad Image LabTM Software. Using the Relative Quantity tool, expression level was estimated by normalising the band brightness of PtpHBMT1 compared to the P. trichocarpa Translation Initiation Factor 5A housekeeping gene (PtTIF5; Potri.006G185000) in each reaction. Tops from the 3 lines showing the highest relative expression were then sub-cultured onto fresh GA-7’s containing WPM-agar with 0.01 μM NAA and left for 6 weeks in tissue culture for eventual planting in the greenhouse. 2.3.2.1.3 Tissue growth and collection Once at least 8 plants of each of the three highest expressing lines were available, 6-week-old plantlets were transferred to 2-gallon pots containing perennial mix (Westcreek Farms Ltd., Fort Langley, BC) in a greenhouse. To minimise mortality, high humidity conditions were maintained by covering plants with clear plastic cups and giving each plant a bi-weekly misting with distilled water for 2 weeks. Plants were grown with 18 hours of light provided by a mix of deep red/white to low blue lights, and deep red/white to medium blue lights at 20-26 °C, and watered with fertilised water 4 times per week. After 4 months of growth, the stem diameter was 43 measured 10 cm above the root collar, and the height of the tree recorded (from the apex of the tree to the soil level). Stem tissue was harvested by cutting 10 cm above root collar and removing the top to yield 100 cm of stem. Leaves and bark tissue was simultaneously harvested and flash frozen in liquid nitrogen at -80 °C for storage. Xylem scrapings were also collected using sterile technique and flash frozen in liquid nitrogen at -80 °C for eventual qRT-PCR analysis. The bottom 15 cm of the debarked stems were harvested and placed at 50˚C to dry for 72 hours for eventual phenotypic analysis, while the top 85 cm was left at room temperature to dry for long term storage. All lines were grown with a minimum of 8 biological replicates, with 3 replicates being harvested and 5 remaining in the greenhouse for future analysis. All lines were grown parallel to wild-type P39. 2.3.2.1.4 qRT-PCR qRT-PCR techniques were employed to determine the relative transcript levels in the xylem tissue of 4-month-old poplar overexpression lines. cDNA was synthesised following identical methods as described previously. This cDNA was then used as the template in qPCR reactions using BrightGreen Express 2X qPCR MasterMix (Applied Biological Materials, Vancouver) and a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, USA) according the manufacturer's instructions. As previously completed, cDNA from all transgenic lines and background lines was pooled and serial diluted 5 times to determine primer efficiency and an appropriate template concentration. This dilution series was then used in qRT-PCR reactions using gene specific primers 13 and 14, and reference gene primers 15 and 16 for PtTIF5 amplification (Table 2.1). Appropriate dilutions of cDNA stocks were then achieved for 44 each line using autoclaved distilled water and used in qRT-PCR analysis. Relative gene expression levels were determined using the formula: 2−(45678!9: − 45;:<:;:=>:). All qRT-PCR reactions were run with 3 technical replicates and 3 biological replicates. 2.3.2.2 Phenotypic analysis Once the bottom 15 cm of debarked stems had dried for 72 hours at 50˚C the pith was removed and stems were cut into smaller matchstick-sized pieces. This xylem was then ground in a Wiley® Mini-mill (Thomas®, USA) to pass a 40-mesh (0.4 mm) screen. Ground xylem tissue was then re-dried at 50˚C for 72 hours. This tissue was then weighed into screw cap tubes for phenolic analysis. The remaining tissue was stored at room temperature in a desiccator containing DrieriteTM desiccant (W.A. Hammond, USA). 2.3.2.2.1 Soluble phenolic isolation 30 mg of dried xylem tissue was weighed out in triplicate for each transgenic line and subject to a MeOH extraction and NaOH-mediated saponification for determination of ester-bound soluble phenolics following the methodology described previously. Both the saponified soluble fraction and the non-saponified fractions were subjected to phase separation, and re-suspended samples were filtered into glass vials for HCLC analysis. 45 2.3.2.2.2 Isolation of cell-wall bound phenolics To determine ester-bound cell wall bound phenolics, 30 mg of dried stem tissue was weighed out in triplicate samples for each transgenic line and extracted and saponified following the methods described for cell-wall bound phenolic analysis of Arabidopsis lines. 2.3.2.2.3 Fraction Analysis via HPLC Analysis of pHBA content and phenolic profile of transgenic poplar lines was achieved using a Summit HPLC (Dionex, USA) fit with a Symmetry C18 column (Waters, USA) and PDA-100 Photodiode Array Detector (Dionex, USA), using the same protocol employed for Arabidopsis overexpression lines in section 2.3.3.2.5. 2.3.2.3 Statistical analysis Significant differences between observed pHBA levels between P39 and overexpression lines was assessed using a standard Student’s t-test for samples of equal size and variance (Appendix A.3). Significant p-values £ 0.05. 46 Chapter 3: Results 3.1 Evaluating the variability of pHBA in Populus 3.1.1 Quantification of cell wall bound pHBA HPLC analysis of cell wall bound pHBA levels clearly showed substantial variability among the 84 P. trichocarpa genotypes (Figure 3.1), with the pHBA content ranging from 0.399 ± 0.004 mg (genotype: SKWC) to 2.753 ± 0.016 mg pHBA/g xylem tissue (genotype: QCTN). 3.1.2 Correlation of pHBA with cell wall traits Spearman correlation analysis of pHBA levels with previously determined wood properties revealed that cell wall bound pHBA has a significant negative correlation with xylan (;@ = −0.28, ! = 3.17:HI), hemicellulose (;@ = −0.26, ! = 1.84eH%), density (;@ = −0.27, ! =8.65eHI), microfibril angle (;@ = −0.33, ! = 3.02eHO), acid soluble lignin (;@ = −0.26, ! =1.19eH%), and S:G (;@ = −0.26, ! = 1.23:H%) within this P. trichocarpa population (Figure 3.2). Significance was determined using a !adj £ 0.000238 and = = 210. 47 Figure 3.1. mg pHBA/g stem xylem tissue of 9-year-old P. trichocarpa trees at breastheight (1.3m). The five genotypes with the highest observed pHBA levels are shown in yellow, while the five genotypes with the lowest observed pHBA levels are shown in blue. The pHBA level of P. trichocarpa Nisqually-1 is shown in pink. Error bars represent standard error of the mean (n ≥ 3).HRSOKTMALAFYLILBLILCMCHANisqually-1PHLAQCTNSKWCSLMC00.511.522.53mg pHBA/g xylem tissueP. trichocarpa Provanance48 Figure 3.2. Spearman correlation matrix of statistically significant wood traits with pHBA (mg pHBA/g xylem tissue). x marks correlations with ! > 0.000238 (i.e., not statistically significant). pHBADensitySoluble LigninS:GMicrofibril AngleXylanDensitySoluble Lignin S:GMicrofibril AngleXylanHemicelluloseVar1Var2−1.0−0.50.00.51.0Corr49 3.2 Identify putative pHBMTs from P. trichocarpa 3.2.1 BAHD – AT identification and activity assay 116 BAHD – ATs were identified via BLAST and motif analysis, and organised into clades based on the phylogenetic relatedness of their nucleotide sequences (Figure 3.3a). Of the 116 genes, only 5 showed in vitro activity with p-hydroxybenzoyl-CoA. It is worth noting that these 5 genes clustered to a single clade made up of 8 BAHD – ATs (Figure 3.3b). Characteristically, 4 of the 5 BAHD – ATs with in vitro activity displayed promiscuity and also displayed activity with acetyl-CoA, benzoyl-CoA, p-coumaroyl-CoA and feruloyl-CoA as substrates (Figure 3.3c, Table 3.1). 3.2.2 Putative pHBMT identification Further screening of these 5 candidate PtpHBMTs reveals one candidate of particular interest, Potri.001G448000. Expression patterns of the putative PtpHBMTs was examined using previously generated leaf and xylem RNA-seq data (POPCAN, 2010) and correlated to the pHBA survey results. This analysis showed that only the expression of Potri.001G448000 had a significant positive correlation with observed pHBA levels (Figure 3.4; $% = 0.41, ! =7.84-./). A BAHD-AT shown to act as an ECERIFERUM2-LIKE (CER2-LIKE) protein involved in fatty acid chain elongation and cuticular wax biosynthesis in planta was previously identified as a candidate pHBMT using similar phenotypic and genotypic screening methods (von Loessl, 2016). Its inclusion in the correlation analysis was to act as a control, and was found to have a negative correlation with pHBA levels. An examination of these candidates using 50 publicly available and previously published data further identified Potri.001G448000 (PtpHBMT1) as being of particular interest. Specifically, using publicly available microarray data, PtpHBMT1 was the only candidate of the five to have a higher expression in P. trichocarpa internode tissue (absolute expression = 9.99) compared to mature leaf tissue (absolute expression = 7.27) and had the highest expression level during xylem deposition (Figure 3.5a; Sundell et al., 2017; Yang et al., 2008). It is worth noting that Potri.011G153500 was excluded from further analysis due to lack of available expression data. When examining the sequencing similarity of the candidates, PtpHBMT1 was the most unique compared to the other candidate genes; ~99% protein similarity between Potri.001G447300 (PtpHBMT2), Potri.001G447400 (PtpHBMT3),, Potri.001G447500 (PtpHBMT4),, compared to PtpHBMT1 which only had ~88% protein similarity. This result is also highlighted in gene cluster analysis with identified lignin biosynthesis genes (Figure 3.6; Table 3.2). PtpHBMT1 shows strong expression correlation to other early lignin biosynthesis-related genes vs. PtpHBMT2, 3, and 4 that cluster tightly together and only directly correlate with the expression of HCT and CAD (Sundell et al., 2017). Taken together, PtpHBMT1 was identified as the most promising putative pHBMT, and therefore was selected for overexpression in Arabidopsis and poplar for in planta analysis. 51 Figure 3.3. (a) A Maximum likelihood phylogenetic tree of identified BAHD – ATs from P. trichocarpa. (b) A Maximum likelihood phylogenetic tree of the clade containing the 5 BAHD – ATs with pHBMT in vitro activity. (c) LC-MS spectra of in vitro product of putative PtpHBMT1. 52 Table 3.1. Activity of putative PtpHBMTs with non-canonical monolignols. Potri.011G153500 Potri.001G447500 Potri.001G447400 Potri.001G447300 Potri.001G448000 Benzoic acid + + + + - p-hydroxybenzoic acid + + + + + p-coumaric acid - - - + - Ferulic acid - - - + - Acetic acid - + + + - OHOOHOHOOHSCoAOHOOHHOOMeOMeOOHHOOMeOOHHOOMeO53 Figure 3.4. Spearman correlation matrix of xylem RNA expression levels of the BAHD clade containing the 5 putative PtpHBMTs and pHBA content (mg pHBA/g xylem tissue). x marks correlations with ! > 0.000556 (i.e., not statistically significant). * BAHDs of high amino acid similarity to candidate PtpHBMTs with non-detectable in vitro activity. ** BAHD identified as an ECERIFERUM2-LIKE (CER2-LIKE) involved in fatty acid chain elongation and wax biosynthesis. 54 Figure 3.5. (a) The expression profile of putative PtpHBMTs in P. tremula woody tissue throughout defined phases of cambial growth and wood formation according to Sundell et al. (2017). (b) A correlation heatmap of putative PtpHBMTs expression through phases of cambial growth and wood formation in P. tremula woody tissue with red indicating positive correlations and blue indicating negative correlations. 55 Figure 3.6. Co-expression network of lignin biosynthetic genes with putative PtpHBMTs (Sundell et al., 2017). Linkage thickness is proportional to the strength of expression correlation, while linkage colour is representative of correlation sign, where red linkages represent a negative correlation and grey linkages represent a positive correlation. 56 Table 3.2. Identified lignin biosynthesis genes used in the co-expression network analysis (Sundell et al., 2017). Genes with a direct positive correlation with PtpHBMT1 are shown in bold. 3.3 Expression of putative PtpHBMT1 in A. thaliana In order to test if putative PtHBMT1 expression can drive pHBA production in Arabidopsis, it was overexpressed using strong constitutive promoters. 3.3.1 Line selection 6-week-old leaf tissue from T1 Arabidopsis plants was used for the isolation of genomic DNA to confirm successfully transformed lines for each construct (i.e., 35S::pHBMT1, 35S::pHBMT1 + CPL, and C4H::pHBMT1 + CPL) . 12 DNA-confirmed lines were then re-grown for 6 weeks, Stage of lignin biosynthesis Enzyme Function Gene Identification Early Late PAL Potri.006G126800, Potri.008G038200, Potri.010G224100, Potri.010G224200, Potri.016G091100 C4H Potri.006G078100, Potri.013G157900, Potri.018G146100, Potri.019G130700 C3H Potri.006G033300, Potri.016G031000 4CL Potri.001G036900, Potri.003G099700, Potri.003G188500, Potri.004G102000, Potri.005G248500, Potri.006G169600, Potri.010G057000, Potri.019G049500 HCT Potri.001G042900, Potri.003G183900, Potri.018G104700, Potri.018G104800 CSE Potri.001G175000, Potri.003G059200 CCR Potri.001G045500, Potri.001G046100, Potri.001G046400, Potri.003G181400, Potri.004G230900, Potri.009G076300 CCoAOMT Potri.001G304800, Potri.008G136600, Potri.008G136700, Potri.009G099800 F5H Potri.005G117500, Potri.007G016400 COMT Potri.001G451100, Potri.002G076800, Potri.011G150500, Potri.012G006400, Potri.014G106500, Potri.014G106600, Potri.015G003100, Potri.016G101600 CAD Potri.001G268600, Potri.001G300000, Potri.001G307200, Potri.002G018300, Potri.005G243700, Potri.009G062800, Potri.009G063100, Potri.009G063300, Potri.009G063400, Potri.009G095800, Potri.011G148100, Potri.011G148200, Potri.016G078300 57 and the leaf tissue was used for the isolation of RNA to estimate the relative transcript abundance of pHBMT1 (compared to AtUBQ5) for each construct. Using these relative expression levels (data not shown), 3 lines of each construct were selected for further qRT-PCR and phenotypic analysis. qRT-PCR of the 6-week-old stem tissue of the three highest expressing lines confirmed the overexpression of PtpHBMT1 within lignifying tissue in all lines selected for phenotypic analysis (Figure 3.7). Figure 3.7. Relative expression of PtpHBMT1 in Arabidopsis overexpression lines. Expression normalised to AtUBQ5. n=3. 00.050.10.150.20.250.3Col-035S::pHBMT-335S::pHBMT-535S::pHBMT1-235S::CPL-4735S::pHBMT1 + CPL-1035S::pHBMT1 + CPL-1235S::pHBMT1 + CPL-7C4H::pHBMT1 + CPL-11C4H::pHBMT1 + CPL-9C4H::pHBMT1 + CPL-4Realtive expression (2^(-∆Cq))11.051.158 3.3.2 Quantification of soluble and cell wall bound pHBA 3.3.2.1 Overexpression of PtpHBMT1 in Col-0 Arabidopsis - 35S::pHBMT1 HPLC analysis of the acid-methanol extracted soluble phenolics from the stem tissue of the three highest expressing lines showed that expression of PtpHBMT1 in Col-0 resulted in a significant increase in soluble pHBA compared to Col-0 (Figure 3.8). The soluble fraction that was later subject to alkaline hydrolysis showed that approximately 1⁄3 of the pHBA is ester-linked with yet unidentified conjugates. This analysis also revealed that esterified pHBA is present within the soluble fraction of Col-0, a finding not previously reported. Analysis of the cell wall bound phenolics reveals that the expression of PtpHBMT1 in Col-0 does not result in a significant increase in non-soluble pHBA. In addition, contrary to the current dogma, this analysis also revealed that pHBA is incorporated in the cell wall of Col-0, albeit at very low levels. See Table 3.3 for summary statistics. Figure 3.8. Levels of pHBA (mg/g stem tissue) in the methanol-extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from overexpression A. thaliana lines. * = ! < 0.05 while ** = ! < 0.01 compared to Col-0 pHBA levels. Error bars represent standard error of the mean. (n=3). ********** **0.0000.0050.0100.0150.0200.0250.0300.0350.0400.0450.050Col-035S::pHBMT1-235S::pHBMT1-335S::pHBMT1-5mg pHBA/g stem tissueSoluble pHBA Soluble pHBA (post-alkaline hydrolysis) Cell Wall Bound pHBA59 3.3.2.2 Overexpression of PtpHBMT1 in CPL Arabidopsis - 35S::pHBMT1 + CPL HPLC analysis of the methanol extracted soluble phenolics from stem tissue revealed that expression of PtpHBMT1 in CPL Arabidopsis results in a two-fold decrease in soluble pHBA when compared to levels in the CPL Arabidopsis background without the expression of putative PtpHBMT1 (Figure 3.9). Alkaline hydrolysis of the soluble fraction clearly showed that approximately 2⁄3 of this pHBA is also ester-linked with yet unidentified conjugates. Although there is a notable increase when compared to Col-0, a similar level of esterification is also observed in the CPL Arabidopsis lines. Analysis of the cell wall bound phenolics demonstrated that expression of PtpHBMT1 in CPL does not result in an increase in non-soluble pHBA, and unexpectedly results in a decrease in the cell wall bound pHBA compared to CPL Arabidopsis. See Table 3.3 for summary statistics. Figure 3.9. Levels of pHBA (mg/g stem tissue) in methanol extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from overexpression A. thaliana CPL lines. * = ! < 0.05 while ** = ! < 0.01 compared to background pHBA levels. Significant decreases in the pHBA content of fractions is highlighted in red. Error bars represent standard error of the mean. (n=3). ****************** *0.0000.0100.0200.0300.0400.0500.0600.070Col-035S::CPL-4735S::pHBMT1 + CPL-735S::pHBMT1 + CPL-1235S::pHBMT1 + CPL-10mg pHBA/g stem tissueSoluble pHBA Soluble pHBA (post-alkaline hydrolysis) Cell Wall Bound pHBA60 3.3.2.3 Overexpression of PtpHBMT1 in CPL Arabidopsis - C4H::pHBMT1+ CPL HPLC analysis of the methanol extracted soluble phenolics from stem tissue showed that expression of PtpHBMT1 driven by AtC4HP in the CPL Arabidopsis background results in a two-fold increase in soluble pHBA when compared to CPL Arabidopsis. Alkaline hydrolysis of the soluble fraction indicated that approximately 1⁄2 of this pHBA is ester-linked with yet unidentified conjugates (vs. 2⁄3 in CPL Arabidopsis). Analysis of the cell wall bound phenolics revealed that overexpression of PtpHBMT1 using the AtC4HP in the CPL Arabidopsis background does indeed result in an increase in non-soluble pHBA and similarly to observed pHBA within the cell wall fraction of 35S::pHBMT1 + CPL lines, there is a significant decrease in the cell wall bound pHBA compared to CPL Arabidopsis. See Table 3.3 for summary statistics. Figure 3.10. Levels of pHBA (mg/g stem tissue) within methanol extracted soluble (pre- and post-saponification) fraction and saponified cell wall bound fraction of 6-week-old stem tissue from overexpression CPL Arabidopsis lines using AtC4HP. * = ! < 0.05 while ** = ! < 0.01 compared to background pHBA levels. Significant decreases in the pHBA content of fractions is highlighted in red. Error bars represent standard error of the mean. (n=3). ** *************** ** **0.0000.0500.1000.1500.2000.250Col-035S::CPL-47C4H::pHBMT1 + CPL-4C4H::pHBMT1 + CPL-9C4H::pHBMT1 + CPL-11mg pHBA/g stem tissueSoluble pHBA Soluble pHBA (post-alkaline hydrolysis) Cell Wall Bound pHBA61 Table 3.3. Summary of pHBA levels observed in the soluble and non-soluble fractions of Arabidopsis overexpression lines and corresponding statistics (n=3). Construct-line Soluble pHBA Soluble pHBA (post-alkaline hydrolysis) Cell Wall Bound pHBA mg pHBA/g stem tissue ! " mg pHBA/g stem tissue ! " mg pHBA/g stem tissue ! " Col-0 0.00000 0.02203 0.01597 35S::pHBMT1-2 0.03198 <0.00001 -27.6 0.03587 0.00308 -5.3 0.01674 0.29957 -0.6 35S::pHBMT1-3 0.02824 <0.00001 -45.4 0.04166 0.00002 -20.7 0.01604 0.48388 0.0 35S::pHBMT1-5 0.01924 <0.00001 -30.7 0.04324 0.00006 -15.0 0.00948 0.10129 1.5 35S::CPL-47 0.06409 <0.00001 -34.0 0.10660 0.00007 -19.3 0.05990 0.00831 -11.4 35S::pHBMT1 + CPL-7 0.00677 0.00013 12.3 0.01806 <0.00001 24.0 0.01258 0.01493 3.3 35S::pHBMT1 + CPL-12 0.01588 0.02478 2.8 0.04649 0.00642 4.3 0.02920 0.18604 1.0 35S::pHBMT1 + CPL-10 0.01023 0.00036 9.4 0.02976 0.00009 13.4 0.01553 0.02221 2.9 C4H::pHBMT1 + CPL-4 0.05101 0.00050 8.6 0.06337 0.00212 5.9 0.03523 0.00088 7.4 C4H::pHBMT1 + CPL-9 0.08527 0.01599 -3.2 0.12452 0.03651 -2.4 0.04146 0.00052 8.5 C4H::pHBMT1 + CPL-11 0.10051 0.01380 -3.4 0.19598 0.00010 -13.0 0.03910 0.00041 9.0 62 3.3.3 Supplementary phenotyping An examination of the height growth and growth rate of the three highest expressing lines for each construct (35S::pHBMT1, 35S::pHBMT1 + CPL, and C4H::pHBMT1 + CPL) of 6-week-old plants (Figure 3.11g) did not shown any alterations in the growth and/or development compared to wild-type (Figure 3.11b) and CPL Arabidopsis (Figure 3.11a), respectively (data not shown). Further histochemical examination with phloroglucinol-HCl and toluidine blue O staining of 6-week-old stem base cross sections did not reveal any alterations in the cell morphology or lignin deposition in overexpression lines (Figure 3.11h and f) compared to wild-type (Figure 3.11e and f) and CPL Arabidopsis (Figure 3.11c and d), respectively. 63 Figure 3.11. (g) 6-week-old Arabidopsis overexpression line 35S::pHBMT1-12 displaying no notable growth phenotype compared to Col-0 (b) and CPL Arabidopsis (a). Histochemical lignin staining of 6-week-old stem base cross sections using phloroglucinol-HCl (h) and toluidine blue O stain (i) revealing similar lignin deposition in overexpression lines compared to Col-0 (e and f) and CPL Arabidopsis (c and d). 3.4 Expression of putative pHBMT in hybrid poplar In order to test if putative PtHBMT1 expression can drive pHBA production in P39, it was expressed using strong constitutive promoters. 50µm200µm 200µm50µmc)b)50µm 50µm50µm50µma) b)e)g)d)f)h) i)35S::pHBMT1-12 35S::pHBMT1-12 35S::pHBMT1-12 35S::pHBMT1-12 35S::pHBMT1-12 Col-0 Col-0Col-0CPLCPL CPL64 3.4.1 Line selection 6-week-old leaf tissue from T1 plants was employed for the isolation of genomic DNA to confirm successful transformants for each construct (35S::pHBMT1 and C4H::pHBMT1). 12 independent DNA-confirmed lines were then re-grown for 6 weeks and the leaf tissue was used for isolation of RNA to estimate the relative expression level of PtpHBMT1 (compared to AtUBQ5) for each construct. Using these relative expression levels (data not shown), 3 lines of each construct were selected for qRT-PCR and phenotypic analyses. qRT-PCR of 6-week-old stem tissue of the three highest expressing lines confirmed the overexpression of PtpHBMT1 within lignifying tissue in all lines selected for phenotypic analysis (Figure 3.12). Figure 3.12. Relative expression of PtpHBMT1 in P39 overexpression lines. Expression normalised to PtTIF5. n=3. 0.000.010.020.030.040.050.060.070.080.09P3935S::pHBMT1-1535S::pHBMT1-1835S::pHBMT1-23C4H::pHBMT1-20C4H::pHBMT1-21C4H::pHBMT1-9Realtive expression (2^(-∆Cq))65 3.4.2 Quantification of soluble and cell wall bound pHBA 3.4.2.1 Overexpression of PtpHBMT1 in Hybrid Poplar - 35S::pHBMT1 HPLC analysis of the methanol extracted soluble phenolics from xylem tissue of the three highest expressing lines showed that expression of PtpHBMT1 in P39 results in a significant increase in soluble pHBA post saponification in all lines when compared to wild-type P39 (Figure 3.13). pHBA was not detected in the non-hydrolysed soluble fraction in wild-type or overexpression lines. Analysis of the cell wall bound phenolics revealed that the expression of PtpHBMT1 in P39 results in a significant increase in cell wall bound pHBA. See Table 3.4 for summary statistics. 66 Figure 3.13. Levels of pHBA (mg/g stem tissue) in methanol extracted soluble (post-saponification) fraction and saponified cell wall bound fraction of 4-month-old xylem tissue from overexpression of 35S::pHBMT1 in P39 lines. * = ! < 0.05 while ** = ! < 0.01 compared to background pHBA levels in P39. Error bars represent standard error of the mean (n=9). *** **0.0000.0050.0100.0150.020P3935S::pHBMT1-1535S::pHBMT1-1835S::pHBMT1-23Soluble (post-alkaline hydrolysis) Cell Wall Bound pHBA****0.9501.0001.0501.1001.1501.2001.250mg pHBA/g xylem tissue67 3.4.2.2 Overexpression of PtpHBMT1 in Hybrid Poplar - C4H::pHBMT1 HPLC analysis of the methanol extracted soluble phenolics from xylem tissue of the three highest expressing lines indicated that expression of PtpHBMT1 driven by AtC4HP in P39 resulted in a significant increase in soluble pHBA post saponification in lines 21 and 9 when compared to wild-type trees (Figure 3.14). A significant decrease in alkaline hydrolysed soluble pHBA was observed in line 20. pHBA was not detected in the non-hydrolysed soluble fraction in wild-type or overexpression lines. Analysis of the cell wall bound phenolics reveals that expression of PtpHBMT1 driven by AtC4HP in P39 results in a significant increase in cell wall bound pHBA in lines 21 and 9 when compared to P39. See Table 3.4 for summary statistics. 68 Figure 3.14. Levels of pHBA (mg/g stem tissue) in methanol extracted soluble (post-saponification) fraction and saponified cell wall bound fraction of 4-month-old xylem tissue from C4H::pHBMT1 P39 lines using AtC4HP. * = ! < 0.05 while ** = ! < 0.01 compared to background pHBA levels in P39. Significant decreases in the pHBA content of fractions is highlighted in red. Error bars represent standard error of the mean. (n=9). ****0.0000.0050.0100.0150.020P39C4H::pHBMT1-9C4H::pHBMT1-20C4H::pHBMT1-21Soluble (post-alkaline hydrolysis) Cell Wall Bound pHBA****0.7000.7500.8000.8500.9000.9501.0001.0501.1001.1501.200mg pHBA/g xylem tissue69 Table 3.4. Summary of the pHBA levels observed in the soluble and non-soluble fractions of P39 35S::pHBMT1 and C4H::pHBMT1 overexpression lines and corresponding statistics (n=9). Construct-line Soluble pHBA post-alkaline hydrolysis Cell Wall Bound pHBA mg pHBA/g xylem tissue ( ) % whole cell wall mg pHBA/g xylem tissue ( ) P39 0.004561 0.718858 35S::pHBMT1-15 0.008210 0.034033 -2.0 0.11 1.119091 0.032004 -2.0 35S::pHBMT1-18 0.016370 < .00001 -7.7 0.12 1.211918 0.002299 -3.3 35S::pHBMT1-23 0.016803 < .00001 -7.8 0.122 1.218750 0.013382 -2.4 C4H::pHBMT1-9 0.006924 0.001679 -3.4 0.116 1.164364 <0.00001 -6.2 C4H::pHBMT1-20 0.003020 0.005256 2.9 0.071 0.711510 0.420197 0.2 C4H::pHBMT1-21 0.004151 0.253829 0.7 0.089 0.891897 0.000526 -4.0 3.4.3 Supplementary phenotyping An examination of height (m) and diameter (mm) of the three highest expressing lines harbouring each construct (35S::pHBMT1, and C4H::pHBMT1) in 4-month-old P39 did not reveal any alterations in the growth compared to wild-type (Figure 3.15), with one exception. A significant decrease in the diameter was observed in line 35S::pHBMT1-18 (Figure 3.15b; * = −2.46, ! = 0.019567).70 Figure 3.15. (a) 4-month-old overexpression line 35S::pHBMT1-9 displaying no notable phenotype compared to wild-type P39 (b). (c) Recorded height (m) and diameter (mm) of 4-month-old P39 overexpression lines. * = ! < 0.05. Error bars represent standard error of the mean (n=5).b)a) c)00.511.522.5 P39C4H::pHBMT1-20C4H::pHBMT1-21C4H::pHBMT1-935S::pHBMT1-1535S::pHBMT1-1835S::pHBMT1-23Height (m)*12.51313.51414.51515.51616.5Diameter (mm)71 Chapter 4: Discussion 4.1 Evaluating the variability of pHBA in Populus Currently, there is little known regarding the variability of cell wall bound pHBA among taxa, species, genotypes, and tissues. Additionally, there is little known regarding the biosynthesis and biological function of this phenolic compound in planta. To better understand the variability in cell wall bound pHBA, the xylem tissue of 84 unrelated P. trichocarpa genotypes was analysed and correlated to a suite of previously collected cell wall and morphological data (Porth et al., 2008a; 2008b) to offer a means to potentially uncover pHBA’s relationship to other cell wall traits, and to implicate the candidate genes in pHBA’s biosynthesis. This analysis revealed substantial variability between P. trichocarpa genotypes (Figure 3.1), ranging from 0.399 ± 0.004 (genotype: SKWC) to 2.753 ± 0.016 mg pHBA/g xylem tissue (genotype: QCTN). Although the source of this variability still remains unclear, correlation analysis allows us to indirectly suggest possible biological roles for pHBA in P. trichocarpa. Xie et al. (2009) conducted a phenotypic analysis on genetically distinct P. trichocarpa genotypes from a broad latitudinal range and found significant differentiation among genotypes across latitude (Figure 4.1). P. trichocarpa genotypes originating from more northern latitudes had higher mortality, grew slower, and were more susceptible to Valsa and Melampsora pathogens. Some of the provenances evaluated by Xie et al. (2009) were also analysed for cell wall bound pHBA, and when compared we find that the more northern genotypes had some of the lowest levels of cell wall bound pHBA observed, while the more southern genotypes (which had lower mortality and were less susceptible to Valsa and Melampsora infection) had higher 72 levels of cell wall bound pHBA. From this we can suggest that pHBA may be linked with pathogen defence, with pHBA content being higher in genotypes occurring at more southern latitudes where there is an increased prevalence of pathogens. Figure 4.1. (a) Geographical origins of 9 P. trichocarpa genotypes used for genotypic and phenotypic analysis by Xie et al. (2009) and Porth et al. (2013b). (b) mg pHBA/g xylem tissue of these same 9 genotypes (n ≥ 3). pHBA has also been shown to be an effective herbicide against many grasses through the inhibition of seedling growth and germination rates (Hussain et al., 2015). Although the exact mechanism of its herbicide properties remains unclear, pHBA had been shown to affect the uptake of potassium and inorganic phosphate in roots and impact membrane depolarisation (Glass 1975). pHBA is found in the soil environment, where it is thought to originate via active secretion as a root exude or as a passive product of plant tissue decomposition. As the NORTH SOUTH Provenance mg pHBA/g xylem tissue DEND 0.048 KLNE 0.55 HOMA 0.64 LILC 0.42 HRSO 2.60 HRSP 1.55 “No Cottonwood Belt” LONG 1.65 NBON 1.86 LAFY 2.10 - - - - - - - DENDKLNEHOMALILCHRSO HRSPHOMBLONGNBONLAFY50 kma) b)73 allelopathic properties of phenolics, including pHBA, have been shown to be concentration dependent, we can also suggest that the concentration of pHBA in a specific tissue may contribute to its susceptibility to pathogen attack (e.g., leaf tissues or seed endocarps), where tissues directly interacting with herbaceous parasites (e.g., roots) should have the highest levels of pHBA if a significant biological function is to aid in plant defense (Mandal et al., 2009; Dixon and Paiva 1995). Although tissue specific pHBA diversity has been studied in a variety of species (e.g., Rencoret et al., 2018; Sircar et al., 2007; Chakraborty et al., 2006), its variability in P. trichocarpa tissues has yet to be determined. Preliminary analysis of the pHBA variability among different tissues of the closely related poplar species P. nigra cv. ‘Italica’ has been conducted (Mansfield Lab, unpublished data: Figure 4.2), and supports the suggested biological role of pHBA as an allopathic agent, however, further analysis is warranted. Figure 4.2. Heatmap of mg pHBA/g tissue in P. nigra cv. ‘Italica’ (Data gathered and image generated by Yaseen Mottiar, Mansfield lab; unpublished data). mg pHBg tissue100.11Lombardy poplar(Populus nigra L. cv ‘Italica’)Young roots9.36 mg pHB/gJuvenile roots1.92 mg pHB/gMature roots1.95 mg pHB/gMature stem4.75 mg pHB/gJuvenile stem4.02 mg pHB/gYoung stem1.18 mg pHB/gYoung leaves0.61 mg pHB/gMature leaves0.37 mg pHB/gmg pHBg tissue100.11Lombardy poplar(Populus nigra L. cv ‘Italica’)Young roots9.36 mg pHB/gJuvenile roots1.92 mg pHB/gMature roots1.95 mg pHB/gMature stem4.75 mg pHB/gJuvenile stem4.02 mg pHB/gYoung stem1.18 mg pHB/gYoung leaves0.61 mg pHB/gMature leaves0.37 mg pHB/gmg pHBg tissue100.11Lombardy poplar(Populus nigra L. cv ‘Italica’)Young roots9.36 mg pHB/gJuvenile roots1.92 mg pHB/gMature roots1.95 mg pHB/gMature stem4.75 mg pHB/gJuvenile stem4.02 mg pHB/gYoung stem1.18 mg pHB/gYoung leaves0.61 mg pHB/gMature leaves0.37 mg pHB/gmg pHBg tissue100.11Lombardy poplar(Populus nigra L. cv ‘Italica’)Young roots9.36 mg pHB/gJuvenile roots1.92 mg pHB/gMature roots1.95 mg pHB/gMature stem4.75 mg pHB/gJuvenile stem4.02 mg pHB/gYoung stem1.18 mg pHB/gYoung leaves0.61 mg pHB/gMature leaves0.37 mg pHB/g74 While the biological role of pHBA has been examined as it relates to the surrounding environment, little work has been done regarding the biological impact of pHBA in planta, and more specifically, its effect on the properties of woody tissue. Using previously determined wood properties and conducting correlation analysis with pHBA levels revealed that cell wall bound pHBA has a significant negative correlation with xylan (34 = −0.28, ! = 3.17789), hemicellulose (34 = −0.26, ! = 1.84e8;), density (34 = −0.27, ! = 8.65e89), microfibril angle (34 = −0.33, ! = 3.02e8=), acid soluble lignin (34 = −0.26, ! = 1.19e8;), and S:G (34 = −0.26, ! = 1.2378;) within the P. trichocarpa population surveyed (Figure 3.2). Some of these significant correlations are expected considering what we know about cell wall biosynthesis and carbon allocation. For example, the negative correlation between pHBA and secondary cell wall polysaccharides (hemicellulose and xylan) could simply be a result of carbon allocation, where the inverse relationship could simply be indicative of the inverse balance between lignin and cell wall polysaccharides (i.e., cell walls with lower polysaccharides generally contain higher lignin contents, and thus higher pHBA). Similarly, the observation of the negative correlation between wood density and microfibril angle is anticipated, as both of these wood characteristics have been found to have an inverse relationship with the lignin content (Porth et al., 2013a; Sheng-zuo et al., 2004). Provenances with lower densities and higher microfibril angles have lower total lignin and therefore lower pHBA incorporated in the cell wall. One unexpected observation is the significant inverse correlation between pHBA and the soluble lignin fraction and S:G ratio. The current hypothesis is that pHBA conjugates are mainly incorporated into lignin as pendant ester groups on the β-position of S lignin (Ralph 2010). Contrary to previous suggestion, this result suggests that pHBA is more favourably coupled to guaiacyl monolignols compared to syringyl. Alternatively, this 75 observation may be a result of limitations in our lignin analysis techniques. Thioacidolysis, the technique employed to determine monomer composition, relies on the cleavage of β–O–4-ether bonds within the polymer. If pHBA does couple with syringyl monolignols as an ester bond, cleavage of these moieties and subsequent quantification of S lignin may be limited (i.e., increases in pHBA-syringyl conjugates results in decreased observed S lignin, and decreased estimation of S:G ratio and soluble lignin (Grabber et al., 1996; Robinson and Mansfield 2009). 4.2 Identify putative pHBMTs from P. trichocarpa 116 BAHD – ATs were identified via BLAST and motif analysis, and organised into clades based on the phylogenetic relatedness of their nucleotide sequences (Figure 3.3a). From in vitro activity assays 5 enzymes displayed positive activity with p-hydroxybenzoyl-CoA. These 5 genes were found to be members of one clade consisting of 8 highly similar BAHD – ATs (Figure 3.3b and c). Characteristically, 4 of the 5 BAHD-AT displayed promiscuity and had in vitro activity with several substrates, including acetyl-CoA, benzoyl-CoA, p-coumaroyl-CoA, and feruloyl-CoA (Table 3.2). This promiscuity within the BAHD family has been observed previously, where certain members of the BAHD superfamily display a wide substrate specificity (D’Auria et al., 2002; D’Auria, 2006; Lou et al., 2007; Landmann et al., 2011; Tuominen et al., 2011; Weng and Noel, 2012; Fan et al., 2017; Moghe et al., 2017; Kreis and Munkert, 2019). For example, Eudes et al. (2016) identified 10 BAHD-AT enzymes which used hydroxycinnamoyl-CoAs and/or benzoyl-CoAs as donor molecules in vitro, and found their expression in Saccharomyces cerevisiae resulted in the synthesis of 30 different products, including rosmarinic acid; quinate hydroxycinnamate esters and amides; malate and fatty alcohol hydroxycinnamate 76 esters; tropane alkaloids; and benzoate alcohol esters. Although this property is valuable in the development of engineered systems for the production of beneficial compounds (e.g., compounds of interest in the pharmaceutical industry), it hinders our ability to succinctly identify putative genes of interest in the pursuit of designer lignin as analysis of transgenic plants with modified BAHD-AT expression suggests that the product synthesised by the BAHD-AT is largely dependent on substrate availability (D’Auria, 2006). As a result, further analysis of these 5 candidate genes was warranted. Further screening of these putative PtpHBMTs using previously generated leaf and xylem RNA-seq data (POPCAN, 2010) and publicly available datasets and software identified one candidate of particular interest, Potri.001G448000. This gene had a significant positive correlation between xylem expression and observed pHBA levels (Figure 3.4). Three other candidates displayed non-significant, albeit positive correlations, while Potri.011G153500 had an expression pattern that was negatively correlated with pHBA levels. Examination of these candidates using publicly available and previously published data further highlighted Potri.001G448000 as being of particular interest. Specifically, using publicly available microarray data, and comparing that data with the preliminary tissue specific pHBA levels, Potri.001G448000 was the only candidate to have a higher expression in P. trichocarpa internode tissue (absolute expression = 9.99) compared to mature leaf tissue (absolute expression = 7.27), and had the highest expression level during xylem deposition (Figure 3.5; Sundell et al., 2017; Yang et al., 2008). An interesting observation from this analysis is that for all 5 candidate genes, the highest level of expression was found in the root tissue (data not shown). This could 77 further be an indication of pHBAs biological role as a potent allelopathic agent in root exudates, as previously described. It is worth noting that Potri.011G153500 was excluded from analysis due to lack of expression data available. This lack of expression may be an indication of gene selection and historical duplication events. It is possible that the putative gene located on chromosome 11 is an evolutionary relic, and underwent duplication during a genome-wide duplication event. This duplicated gene then underwent tandem duplication on chromosome 1 yielding 4 highly similar putative genes of interest (Yu et al., 2009; Tuskan et al., 2006). It is not outlandish to suggest that these duplicate genes underwent genetic selection and diversification yielding highly similar genes with diverse functions. This hypothesis is supported when we examine the sequence similarity between these candidates. For example, Potri.001G448000 shows the lowest protein similarity when compared to Potri.001G447500, Potri.001G447400, and Potri.001G447300 (88%, compared to 99% similarity). Although Potri.011G153500 was excluded from analysis, it is worth noting that Potri.011G153500 had the most unique protein sequence compared to the other candidates (data not shown). Toumin et al. (2011) came to a similar conclusion and identified both Salicoid duplication and local duplication events as being the major source of the large number of members found within this clade. Their analysis of the P. trichocarpa genome revealed that Potri.011G153500 and Potri.001G448000 are Salicoid duplicates, while Potri.001G447500 and Potri.001G447300 are tandem duplicates of Potri.001G448000 (Potri.001G447400 was not included in their analysis). This hypothesis is further supported with the results from gene cluster analysis (Figure 3.6; Table 3.2). Potri.001G448000 shows strong expression correlation to other early lignin biosynthetic genes compared to Potri.001G447500, Potri.001G447400, and Potri.001G447300 which display a strong correlation to each other. 78 4.3 Expression of PtpHBMT1 in A. thaliana Previous research has shown that expression of monolignol transferases from B. distachyon, O. sativa, and A. sinensis in model organisms (e.g., A. thaliana) results in alterations in the lignin polymer and an alteration of the polymer digestibility (Sibout et al., 2016; Petrick et al., 2014; Withers et al., 2012; Wilkerson et al., 2014; Karlen et al., 2016). Over-expression of PtpHBMT1 in A. thaliana (a background lacking previous reports of cell wall bound pHBA) was thought to be a straightforward initial examination of PtpHBMT1 in planta function. 4.3.1 Overexpression of PtpHBMT1 in Col-0 Arabidopsis HPLC analysis of the methanol extracted soluble phenolics from 6-week-old base stem tissue revealed that expression of PtpHBMT1 in Col-0 results in a significant increase in soluble pHBA compared to Col-0 (Figure 3.8). As the proposed function of pHBMT suggests that it uses a pHBA CoA-thioester as a donor molecule and acylates pHBA to a monolignol acceptor substrate, the increased occurrence of pHBA in the soluble fraction of transgenic Arabidopsis is peculiar. This finding could be due to a number of factors. First, it is possible that as a result of overexpressing PtpHBMT1 there is an upregulation of upstream enzymes within the currently unknown pHBA synthesis pathway, however, the yet-unidentified CoA-ligase may be the rate-limiting enzyme, resulting in a low concentration of the required CoA substrate for PtpHBMT1. Alternatively, it is possible that this observed increase in free soluble pHBA may be due to an upregulation of ubiquinone synthesis and the β-oxidative pathway in response to oxidative stress associated with the overexpression of PtpHBMT1. Ubiquinone is a fundamental compound used in the prevention of oxidative stress and serves as an antioxidant within the peroxisome of plant 79 cell cytosols. Importantly, pHBA provides the critical benzoic ring in the final stages of its synthesis (Block et al., 2014; Qualley et al., 2012). When the soluble fraction was further subject to alkaline hydrolysis it was shown that approximately 1⁄3 of this pHBA is esterified with yet unidentified conjugates. This analysis also demonstrated that esterified pHBA is inherently present within the soluble fraction in Col-0 in low concentrations, a finding contrary to the dogma. Although there are a several potential conjugates that could be esterified to pHBA within the soluble fraction, one likely esterified form is as a glycosylated hydroxycinnamate (ex. as pHBA 4-O-glucoside). Many plants employ UDP-glycosyltransferases to regulate the toxicity, stability, and bioavailability of phenolic compounds (Roy et al., 2016). More specifically, analysis of various transgenic A. thaliana lines that have augmented lignin biosynthesis genes usually accumulate intermediates that are glycosylated and compartmentalised in the vacuole (Vanholme et al., 2012). Alternatively, it is possible that the expression of PtpHBMT1 in Col-0 is in fact resulting in the synthesis of pHBA-monolignol conjugates in the cytosol, however, this conjugate may not being exported to the cell wall for lignin polymerisation as the mechanism of monolignol export in Arabidopsis is still not fully understood (Ehlting et al., 2005; Alejandro et al., 2012). However, this is likely not the case, as both p-coumarate-monolignol and ferulate-monlignol conjugates were shown to cross the membrane and participate in lignification in the wall (Smith et al., 2015; Wilkerson et al., 2014). If there are indeed limitations to the export of pHBA monolignol-conjugates, it is likely that the cell would employ native UDP-glycosyltransferases to avoid the detrimental effects associated with its cytosolic accumulation. However, it is important to note that the conjugate would need be glycosylated as an ether linkage to the monolignol for this hypothesis to ring true, as HPLC 80 analysis of the aqueous soluble fraction post-phase separation did not reveal the presence of canonical monolignols within the soluble fraction. There is uncertainty regarding the bioavailability of monolignols and monolignol-conjugates post-glycosylation and vacuolar storage. Previously, it was thought that glycosylation was necessary in the bioavailability of monolignol intermediates and monolignols in lignin biosynthesis (Roy et al., 2016; Freudenberg and Torres-Serres, 1967). However, more recent experiments using tracer-techniques to observe the subcellular localisation of phenolics, and co-expression analysis in transgenic lines suggest that monolignol glycosylation arises in response to prevent phenolic pressures, and not as an intrinsic mechanism in lignin biosynthesis in A. thaliana (Dima et al., 2015; Chapple et al., 2012). Analysis of the cell wall bound phenolic fraction of PtpHBMT1 overexpression lines supports this argument, as the expression of PtpHBMT1 in Col-0 yields a significant increase in ester-modified pHBA (likley as pHBA 4-O-glucoside) but does not result in a significant increase in cell wall bound pHBA. Although lignin specific analysis was not conducted in this work and limits our ability to make conclusions about the amount of pHBA-monolignol conjugates incorporated into the lignin polymer (as the saponification and HPLC method employed in analysing non-soluble samples revealed the phenolic profile of ester linked groups within the whole cell wall which includes cell wall associated polysaccharides), previous works demonstrated that analysis of stem tissue from Arabidopsis and hybrid poplar overexpressing the OsPMT pre- and post-enzyme hydrolysis did not yield differences in the levels of soluble monolignol p-coumarate conjugates detected (Smith et al., 2015). This suggests that the overexpression of monolignol transferases can 81 manifest in altered phenolic composition of the lignin polymer, and not the polysaccharide fraction of the cell wall. As such, using the pHBA content determined via saponification of tissue as a proxy for pHBA found on the lignin polymer, is believed to be appropriate. 4.3.2 Overexpression of PtpHBMT1 in CPL Arabidopsis As previously mentioned, previous analysis of cell wall bound phenolics in A. thaliana did not show pHBA moieties (Ralph et al., 2008). Although our current results suggest otherwise (likely a result of employing more sensitive techniques), there was uncertainty if PtpHBMT1 expression would result in alterations in pHBA as a result of incorrect putative gene identification or lack of substrates availability (p-hydroxybenzoate-CoA). Therefore, PtpHBMT1 was expressed in previously generated CPL Arabidopsis which had been found to contain significantly high levels of cell wall bound and soluble pHBA. While observation of pHBA in the soluble fraction of CPL Arabidopsis was expected, observation of pHBA in the cell wall bound fraction was not expected to be overwhelmingly high. The suggested mechanism for pHBA-monolignol conjugate synthesis involves a BAHD – AT enzyme catalysing the transfer of a pHBA-CoA thioester, synthesised by a Co-A ligase, to acceptor monolignol (D’Auria 2006). Therefore, expression of CPL without the required monolignol transferase was not expected to yield cell wall bound pHBA due to lack of a conjugate synthesis mechanism. However, in retrospect, this observation in CPL Arabidopsis supports the observation of pHBA in the cell wall bound fraction of Col-0 as it suggests that there is a native mechanism for pHBA-monolignol conjugate synthesis in A. thaliana. 82 Interestingly, expression of PtpHBMT1 in CPL plants resulted in a significant decrease in the soluble and cell wall bound pHBA compared to the CPL alone. This result could be due to the overexpression of two genes using the strong CamV35S promoter is resulting in potential gene silencing with a double transformation employing identical vectors. And, this is supported by the relatively low expression levels of PtpHBMT1 in CPL Arabidopsis lines (Figure 3.7). In an attempt to avoid gene silencing alternative overexpression vectors and promoters were considered. Employing AtC4HP for CPL Arabidopsis transformation aided in two ways. First, it minimised the potential for gene silencing, and allowed the exploration of the effect of a lignin specific promoter potentially minimising pHBA glycosylation and vacuolar sequestration. HPLC analysis of the methanol extracted soluble phenolics from stem tissue revealed that the expression of PtpHBMT1 driven by AtC4HP in CPL Arabidopsis results in a significant increase in soluble free – and esterified-pHBA when compared to CPL alone. Similar to the results of PtpHBMT1 Col-0 lines, it is possible to suggest that the observed esterified-pHBA is due to increased glycosylation and sequestration of excess pHBA in the vacuole. Analysis of the cell wall bound phenolics showed that overexpression of PtpHBMT1 using AtC4HP in CPL Arabidopsis does not result in an increase in cell wall bound pHBA. This finding has two potential explanations. First, there is the potential that PtpHBMT1 does not serve as a monolignol transferase and was incorrectly identified as the putative enzyme responsible for pHBA-monolignol conjugate synthesis. Or, there is a yet unidentified mechanism in the synthesis of pHBA in A. thaliana limiting the incorporation of pHBA moieties in the cell wall. Considering the computational and in vitro evidence supporting PtpHBMT1’s proposed function, and the 83 uncertainty surrounding non-canonical monolignol biosynthesis in A. thaliana, expression of PtpHBMT1 in poplar was deemed as an alternative to examine PtpHBMT1’s function in planta. 4.4 Expression of the putative PtpHBMT in hybrid poplar To-date, five monolignol transferases have been identified and biochemically confirmed; three p-coumaroyl-CoA:monolignol transferases in O. sativa, Z. mays, and B. distachyon, and two feruloyl-CoA:monolignol transferases in A. sinensis and O. sativa (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014; Karlen et al., 2016). Misregulation and heterologous expression of these acylating enzymes has proven to be an effective approach in the pursuit of novel monolignol conjugate profiles. Although the identification of acylating enzymes in various model grass species has been successful, the same cannot be said for identification in woody tree species. As such, many of the mechanisms involved in acylated monolignol biosynthesis in woody tree species remains unclear. While model species like A. thaliana are useful in gene discovery and characterisation studies, there are limitations when considering heterologous gene expression as critical biosynthetic mechanisms may be lacking. As such, the determinative test includes the manipulation of homologous gene expression. 4.4.1 Overexpression of PtpHBMT1 in hybrid poplar HPLC analysis of the methanol extracted soluble phenolics from xylem tissue of the three highest expressing transgenic P39 lines revealed that expression of PtpHBMT1 results in a significant increase in soluble pHBA post-saponification, in all lines when compared to wild-type trees (Figure 3.13). Similar to the A. thaliana overexpression lines, this result suggests that 84 the overexpression of PtpHBMT1 leads to an upregulation of the pHBA biosynthetic pathway resulting in an increase in pHBA within the cytosol of xylem cells, however, the unidentified endogenous CoA-ligase may be the rate limiting step in the synthesis of the CoA-benzoate substrate for PtpHBMT1. In an attempt to prevent potential toxic effects associated with the accumulation of cytosolic phenolics, it is reasonable to suggest that a portion of this soluble pHBA is occurring as an ester-bound glycoside and sequestered to the vacuole. Free pHBA was not detected in the non-hydrolysed soluble fraction in wild-type or overexpression poplar lines. Furthermore, the analysis of the cell wall bound phenolics revealed that the expression of PtpHBMT1 in P39 results in a significant increase in cell wall bound pHBA. Interestingly, the highest expressing line (line 23) does not display a substantive increase in soluble or cell wall bound pHBA compared to the second highest expressing line (line 13), although it is expressing PtpHBMT1 in xylem tissue roughly 8x higher. This could be due to limitations in the biosynthetic pathway leading to the precursor molecule and/or carbon availability. As suggested in Figure 1.2, a yet unidentified Co-A ligase must be involved in the synthesis of a pHB-CoA thioester ‘donor’. This enzyme could also be the rate limiting step in pHBA-monolignol conjugate synthesis, and be limiting the synthesis and incorporation of pHBA into the cell wall. Regardless, this result suggests that PtpHBMT1 may be a monolignol transferase gene responsible for synthesising ester linked pHBA-monolignol conjugates in poplar lignin. 4.4.2 Overexpression of PtpHBMT1 in hybrid poplar using AtC4HP Similar to the strategy of overexpressing PtpHBMT1 in A. thaliana, AtC4HP was concurrently employed to explore the effect of overexpressing PtpHBMT1 using a lignin specific promoter. 85 Although tissue specific analysis of AtC4HP expression has been done in A. thaliana and revealed localisation to xylem and sclerified parenchyma between vascular bundles, a tissue expression survey of AtC4HP in poplar has yet to be conducted (Bell Le-Long et al., 1997). However, preliminary analysis of transgenic P39 employing AtC4HP suggests that it is an effective promoter in the overexpression of genes linked to lignin biosynthesis (see Huntley et al., 2003). An interesting observation by Bell Le-Long et al. (1997) examining AtC4HP-GUS constructs showed that the highest expression was found within the root tissue of Arabidopsis. Linking this observation with the hypothesis of pHBA’s biological role in plant defense in root tissue suggests that analysis of pHBA levels in the root tissue of C4H::pHBMT1 lines is worthwhile. HPLC analysis of the methanol extracted soluble phenolics from xylem tissue of the three highest expressing lines showed that expression of PtpHBMT1 driven by AtC4HP in P39 results in a significant increase in soluble pHBA post saponification in lines 21 and 9 when compared to wild-type P39 (Figure 3.14). Interestingly, line 20 saw a significant reduction in the soluble pHBA compared to wild type. It is worth noting that line 20 also had extremely low levels of PtpHBMT1 expression in xylem tissue (Figure 3.12). Such low expression is unlikely to be due to gene silencing, as PtpHBMT1 is a poplar gene, that has been codon optimised for gene synthesis. As a result of this altered nucleotide sequence, its unlikely that silencing would be occurring by interference. Although, the highest expressing lines were identified pre-greenhouse planting by relative RT-PCR analysis, there are a number of assumptions made in this technique and may not be truly indicative of transgene expression levels. In regard to line 20’s pHBA levels, the significant reduction could be linked to a reduced growth rate as this line displayed the largest difference (although not statistically significant) in height and diameter when compared to wild type grown in parallel. Line 20 highlights the benefits of conducting gene 86 manipulation studies in species with more modest resource requirements (e.g., E. coli or A. thaliana), however, serves as a good control. Similar to the observations in A. thaliana overexpression lines and 35S::pHBMT1 P39 lines, the increase in soluble pHBA may be a result of upregulation of the pHBA biosynthetic pathway resulting in an increase in pHBA within the soluble fraction of xylem tissue, and the unidentified endogenous CoA-ligase is limiting the synthesis of the benzoate substrate for PtpHBMT1, and endogenous UDP-glycosyltransferases are employed for the eventual sequestration to the vacuole. pHBA was not detected in the free form in the soluble fraction in wild-type or any overexpression lines. Analysis of the cell wall bound phenolics, the conclusive analysis conducted in this work, reveals that overexpression of PtpHBMT1 driven by AtC4HP in P39 results in a significant increase in cell wall bound pHBA in lines 21 and 9. This result further supports the implication of PtpHBMT1 as the monolignol transferase gene responsible for synthesising ester linked pHBA-monolignol conjugates in poplar lignin. When examining the differences between phenotypes observed in overexpression lines employing CamV35SP and AtC4HP, we see that expression using AtC4HP yields similar pHBA levels in the cell wall bound fraction and reduced pHBA in the soluble fraction (post-saponification). It is worth noting that the expression levels observed in the xylem tissue of AtC4HP constructs was noticeably reduced compared to CamV35SP constructs. Comparing the two highest expressing lines 35S::pHBMT1-23 and C4H::pHBMT1-9 (which also displayed the most significant phenotype) we see that although PtpHBMT1 expression using CamV35SP is roughly double that AtC4HP, the two lines contained similar levels of cell wall bound pHBA and 87 AtC4HP contained less soluble pHBA. These findings suggest that AtC4HP may be a more efficient promoter in the pursuit of modified cell walls. Although overexpression strategies using CamV35SP in plants is a very common technique in gene discovery experiments, its ubiquitous expression may be resulting in pHBA being synthesised in non-lignifying tissue with the absence of necessary machinery for cell wall export and incorporation, yielding higher levels of soluble pHBA, glycosylation, vacuolar sequestration, and thus reduced bioavailability for pHBA in non-canonical monolignol biosynthesis. 88 Chapter 5: Conclusion 5.1.1 Variability of pHBA in Populus HPLC analysis of cell wall-bound pHBA levels reveals substantial variability among P. trichocarpa genotypes (Figure 2.1), with a pHBA content ranging from 0.399 ± 0.004 (genotype: SKWC) to 2.753 ± 0.016 mg pHBA/g xylem tissue (genotype: QCTN). Spearman correlation analysis revealed that cell wall-bound pHBA significantly negatively correlates with xylan, hemicellulose, density, microfibril angle, acid soluble lignin and S:G ratio within this P. trichocarpa population (Figure 2.2). These findings both reveal the inherent level of pHBA variability found in the cell wall of P. trichocarpa genotypes growing in a common garden, and offer a means to propose in planta functions of pHBA moieties. 5.1.2 Putative pHBMTs from P. trichocarpa Identification of all P. trichocrapa BAHD-ATs genes was achieved with the use of a suite of bioinformatic techniques and yielded a list of 116 candidates. By employing qualitative in vitro protein synthesis and activity assay techniques, 5 putative pHBMTs were identified from the 116 P. trichocrapa BAHD-ATs. Further in silico analysis of these candidates enabled the identification of Potri.001G448000 (AKA PtpHBMT1) as the most promising monolignol transferase candidate responsible for the synthesis of pHBA-monolignol conjugates, and was therefore selected for in planta characterisation. 89 5.1.3 PtpHBMT1 in A. thaliana Analysis of successfully transformed Arabidopsis lines demonstrated that only when overexpressing PtpHBMT1 using CamV35SP in Col-0, and using AtC4HP in CPL Arabidopsis lines is a significant increase in the soluble pHBA content in stem tissue apparent. This analysis also revealed that esterified pHBA is present within the soluble fraction and found incorporated in the cell wall of Col-0 stem tissue, a finding not previously reported and contrary to the axiom. None of the Arabidopsis lines displayed a significant increase in the cell wall bound pHBA, with some overexpression lines actually displaying a significant decrease. Although this result suggests that PtpHBMT1 may not serve as the monolignol transferase anticipated, expression of PtpHBMT1in poplar was deemed as a better test to examine PtpHBMT1’s in planta function. 5.1.4 PtpHBMT1 in hybrid poplar Analysis of successfully transformed P39 lines showed that overexpression of PtpHBMT1 using CamV35SP resulted in a significant increase in the soluble pHBA content in xylem tissue. This analysis also revealed that overexpression of PtpHBMT1 using CamV35SP and AtC4HP in P39 resulted in a significant increase in the cell wall bound pHBA content in xylem tissue. Although only qualitatively explored, these results also suggest that the use of cell wall specific promoters (such as AtC4HP) may be a more effective approach in developing novel cell wall traits compared to the ubiquitous promoters typically employed for overexpression experiments. In conclusion, the objective and aims of this research were achieved. We identified a BAHD-AT responsible for the biosynthesis of the non-canonical monolignol p-hydroxybenzoic acid in 90 poplar cell walls, and generated transgenic P39 trees with lignin containing altered levels of pHBA. 5.2 Significance Selectively engineering the composition of the lignin polymer by increasing the degree of acylation of lignin monomers and/or integrating unique ester bonds into the lignin polymeric backbone can increase the alkaline processability of the otherwise chemically stable polymer, while potentially avoiding major phenotypic impairments. pHBA is one such phenolic found acylating monolignols where its incorporation as a pendant ester group decorating the lignin polymer yields chemically labile moieties. While potentially effecting the degree of polymerisation (and thus the processability of woody tissue), these moieties also have major commercial significance within the pharmaceutical, cosmetic, food, and biomaterial industries. As such, pHBA is of particular interest in the pursuit of lignins designed to have enhanced value. Previously the genetic mechanism in pHBA-monlignol conjugate synthesis was unknown. The results from this work suggest that pHBA-monolignol conjugates in P. trichocarpa could be synthesised by the monolignol transferase gene Potri.001G448000 (PtpHBMT1). This finding is highly significant as it furthers our understanding of acylated lignin biosynthesis, while also having the potential to improve the cost-effectiveness and sustainability of biomass utilisation. 91 5.3 Future experiments 5.3.1 Examination of other putative PtpHBMTs in planta Although PtpHBMT1 was selected for in-depth analysis in this work, four other putative PtpHBMTs were identified via in vitro activity assays. Of these four, three putative genes, Potri.001G447300, Potri.001G447400, Potri.001G447500 (PtpHBMT2, 3, and 4, respectively), showed substrate promiscuity and future experiments could be dedicated to determining their in planta function. It is worth noting that these three putative PtpHBMTs have already been overexpressed in both A. thaliana and P39, and have undergone preliminary genomic screening for the identification of successful transformants (see Appendix B). Future experiments should be focused on characterising the biochemical outcomes of their overexpression in planta, while also developing transgenic lines which overexpress multiple putative PtpHBMTs. 5.3.2 Exploring pHBA biosynthesis Analysis of A. thaliana overexpression lines yielded two hypotheses; that PtpHBMT1 does not serve as a monolignol transferase and was incorrectly identified as the putative enzyme responsible for pHBA-monolignol conjugate synthesis, or, there is a yet unidentified mechanism in the synthesis of pHBA in A. thaliana limiting the incorporation of pHBA moieties in the cell wall. This first hypothesis is rejected with analysis of P39 overexpressing PtpHBMT1, and suggests that pHBA synthesis in Arabidopsis is still an enigma. As such, these overexpression A. thaliana lines provide a valuable opportunity to potentially reveal the biosynthetic mechanism for pHBA synthesis. The identification, isolation, and overexpression of other enzymes proposed 92 in the synthesis of pHBA-monolignol conjugates (e.g., a CoA ligase which is thought to produce pHBMT’s substrate hydroxybenzoyl-CoA) may be a valuable endeavour. 5.3.3 Optimising pHBA-monolignol synthesis and incorporation into the cell wall In these experiments, the CamV35SP and AtC4HP were both employed to drive the overexpression of PtpHBMT1, and the results suggest that use of the lignin-specific promoter yields a more substantial lignin phenotype. Further exploration of other suitable lignin specific promoters, or cell wall specific promoters should be completed to determine optimal gene-promoter combinations in A. thaliana and P39 where overexpression results in the highest incorporation of pHBA in the cell wall. It is worth noting that PtpHBMT1, 2, 3, and 4 have already been overexpressed in A. thaliana using the A. thaliana CELLULOSE SYNTHASE7 promoter (AtCesA7P) and in P39 using the CELLULOSE SYNTHASE8 promoter (PtCesA8P). Employing the cellulose synthase promoters limits putative PtpHBMT overexpression to all tracheary elements and fibers in xylem tissue (Smith et al., 2013). These overexpression lines have undergone preliminary genomic screening for the identification of successful transformants (see Appendix B). 5.3.4 CPL Poplar Although expression of PtpHBMT1 in previously generated CPL Arabidopsis did not result in a significant increase in cell wall bound pHBA, expression in P39 overexpressing CPL may yield different results. Future work should be dedicated to the generation of overexpression lines simultaneously containing CPL and PtpHBMT1 to maximise the incorporation of pHBA moieties into poplar lignins by ensuring substrate availability. P39 lines that overexpress CPL 93 have already been developed using both the CamV35SP and the PtCesA8P. Analysis of these overexpression lines is ongoing, however, preliminary evidence suggests that expression of the monolignol transferase may increase the incorporation of pHBA in the cell wall of poplar (data not shown). 5.3.5 Lignin analysis Transgenic A. thaliana and P39 are in the process of being further examined by chemical analyses. Derivatisation followed by Reductive Cleavage (Regner et al., 2018), alkaline hydrolysis (Smith 1955; Karlen et al., 2017), gel permeation chromatography (of isolated lignin untreated, base pretreated, and acid pretreated), and 2D-NMR (Mansfield et al., 2012) will be used to quantify the amount of pHBA found on the lignin polymer. 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Nature Protocols DOI:10.1038/nprot.2006.97. 117 Appendices Appendix A : Additional Materials and Methods A.1 pHBA calculations mg pHBA/g of tissue was determined using an internal standard (IS) in each sample to calculate a correction factor to account for injection error and a standard curve to estimate mass from area. The following equation was applied to determine the correction factor for saponification reactions: >?337>*@?A BC>*?3 =DEFGH C37CDEFGH ICJJDE K7AJ@*L DEFGH ICJJDE K7AJ@*L + N4EOP FGH ICJJN4EOP K7AJ@*L + QCONFGH ICJJQCON K7AJ@*LDERFSTUH C37CDERFSTUH ICJJDE K7AJ@*L DERFSTUH ICJJDE K7AJ@*L + N4EOP RFSTUH ICJJN4EOP K7AJ@*L + QCONRFSTUH ICJJQCON K7AJ@*L Iterations of this equation were used to determine the correction factors in soluble samples and for samples used in the generation of a standard curve. This correction factor was used to adjust the observed area of pHBA, which was subsequently plotted along the standard curve to estimate concentration. A.2 Correlation analysis Correlation analysis done for this thesis was completed by running iterations on the example R-code shown in Appendix Figure A.1 in R version 3.5.1. 118 #Import data and identify variables of interest library(car) scatterplotMatrix(~PROV +ARA +RHA +GAL +GLU +XYL +MAN +INSLIG + SOLLIG +totallignin + holocellulose +alphacellulose + hemicellulose + FibreL + MFA + MFA1 + Density + CRYST + S2G + pHBA, data= PHBAclean) #subset and clean data PHBASIG <- PHBAclean[c("XYL", "SOLLIG", "hemicellulose", "MFA", "Density", "S2G", "pHBA")] #Generate a correlation matrix and associated p-values for each variable cormat2 <- round(cor(PHBASIG, method="spearman"), 2) library(reshape2) melted_cormat2 <-melt(cormat2) library(ggcorrplot) p.mat <- cor_pmat(PHBASIG, method="spearman") ##correlation p-values head(p.mat) melted_pmat <-melt(p.mat) melted_pmat ggcorrplot(cormat2, hc.order = TRUE,outline.col = "white", colors = c("indianred2", "white", "darkseagreen3"), type = "lower", sig.level= 0.000238, p.mat = p.mat, insig = "pch", pch=4, pch.cex=1) + theme_minimal() + theme(axis.text.x = element_text(angle = 90, vjust = 1, size = 10, hjust = 1)) Appendix Figure A.1 Example of R code used in correlation analysis. 119 A.3 Putative gene screening 120 Appendix Figure A.2. Nucleotide sequence alignment showing differences between Potri.001G448000 from P. trichocarpa (a) and the altered Potri.001G448000 nucleotide sequence synthesised by JGI (b). Nucleic acid mis-matches and gaps are highlighted in red. (c) The translated amino acid sequence from Potri.001G448000 in P. trichocarpa and synthesised Potri.001G448000. Appendix Table A.1. BLAST results of the 4 putative PtpHBMT enzymes against identified or suspected FMT and PMT enzymes. Data and figure generated by Drs. Rebecca Smith, Steven Karlen, and John Ralph. AsFMT BdPMT OsFMT OsPMT ZmPMT PtpHBMT2 23.6% 74/314 3.65e-13 28.0% 118/422 6.67e-43 29.3% 119/406 1.05e-40 31.4% 128/408 3.98e-46 30.0% 124/413 1.22e-43 PtpHBMT3 23.6% 74/314 3.45e-12 28.4% 118/415 6.48e-44 29.1% 118/406 6.29e-42 31.6% 129/408 1.94e-47 30.3% 125/416 3.66e-45 PtpHBMT4 23.2% 73/314 3.97e-12 28.0% 116/415 8.78e-44 28.2% 117/406 1.08e-41 31.4% 128/408 1.52e-47 30.0% 124/413 1.01e-44 PtpHBMT1 21.3% 90/422 3.36e-11 26.9% 114/424 5.81e-47 29.1% 120/413 1.17e-43 29.6% 126/426 1.14e-44 27.8% 112/403 1.79e-46 121 Appendix Figure A.3. LC-MS and NMR results from in vitro screening. (a) Negative mode Total Ion Chromatogram mass spec from PtpHBMT1 activity assay. (b) Extracted Ion Chromatogram at 329 m/z confirming the retention time and mass of sinapyl p-hydroxybenzoate when compared to synthetic standard. (c) Sinapyl p-hydroxybenzoate peak fragments (positive). (d) Sinapyl p-hydroxybenzoate peak fragments (negative). Data and figure generated by Drs. Rebecca Smith, Steven Karlen, and John Ralph. 122 A.4 T-test calculations To determine statistical significance between observed pHBA levels (both in mg pHBA/g wood tissue and expression levels between transformants) I completed multiple one-tailed t-tests. Once data had been screened to ensure appropriate distribution, variation, and independence, t-test analysis was completed using the standard equation to determine the test statistic and associated p-value: * = VWXXX − V4XXXY(AW − 1)J[4W + (A4 − 1)J[44AW + A4 − 2 ∗ ] 1AW + 1A4 VX = I7CA ?B JCI!^7 A = JCI!^7 J@_7 J[4W = 7J*@IC*7K `C3@CA>7 123 Appendix B : Additional transgenic lines As described in the main text of this thesis, multiple constructs and lines were generated in addition to those analysed. All four putative PtpHBMTs have been overexpressed in Col-0 and CPL Arabidopsis, and P39 using CamV35SP, AtCesAP, PtCesA8P, and AtC4HP, totaling 24 overexpression lines in A. thaliana, and 12 in P39. B.1 Promoter isolation and swapping Amplification of the 1107 bp promotor sequence found directly upstream of A. thaliana CELLULOSE SYNTHASE CATALYTIC SUBUNIT 7 (AtCesA7P; AT5G17420) was achieved from a previously developed and purified pCesA-mcherry-CesA-BASTA plasmid using primers 20 and 21 (Appendix Table B.1). Sequencing of isolated promoter was confirmed using primers 22 and 23. Amplification of the 1476 bp promotor sequence found directly upstream of the P. trichocarpa CELLULOSE SYNTHASE CATALYTIC SUBUNIT 8 (PtCesA8P; Potri.004G059600) was achieved from genomic DNA of previously harvested xylem scrapings from P39 provided by Dr. Eliana Gonzales-Vigil (Mansfield Lab) using primers 24 and 25. Although promoter isolation was achieved from P39, sequencing of this promoter region using primers 26 and 27 revealed that there is 100% identity with the confirmed P. trichocarpa Nisqually-1 promoter region. Identical band extraction, SacI, XbaI and SpeI digestion, enzyme mediated sticky end promoter ligation, and sub-cloning methods described in section 2.3.1.2 were used in the construction of pK7WG2 constructs employing PtCesA8P and AtCesA7P. Additionally, AtC4HP previously isolated was also ligated into the overexpression vector pH7WG2 following identical experimental methods. 124 B.2 Overexpression of other putative PtpHBMTs Once these overexpression backbones were sequence confirmed, other putative PtpHBMTs were ligated using Gateway™ cloning methods. PCR amplification from the PUC7 plasmid, PCR purification, Gateway™ cloning, sub-cloning, and sequencing for PtpHBMT2, PtpHBMT3, and PtpHBMT4 was completed following identical methods described in section 2.3.1.1 using the primers listed in Appendix Table B.1. Primer 17 and 18 were used to amplify PtpHBMT2 and PtpHBMT3, while primers 17 and 19 were used to amplify PtpHBMT4. Sequencing was completed using primers 3 and 4 (Table 2.1). Sequence confirmed constructs, were transformed into appropriate A. tumefaciencs strains for plant transformations (Appendix Table B.2). Appendix Table B.1. Primers employed in this study. No. Name Use and Properties Sequence (5¢ to 3¢) 17 pHBMT_RV1 Amplification of the full reading rame of synthesised Potri.001G447300, Potri.001G447400 and Potri.001G447500 for GATEWAYTM cloning. Contains attB recognition sites. GGGGACCACTTTGTACAAGAAAGCTGGGTTCACAGACTACTTGGAATCATTTC 18 pHBMT_FW4 Amplification of the full reading frame of synthesised Potri.001G447300 and Potri.001G447400 for GATEWAYTM cloning. Contains attB recognition sites. GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCCACCCCAACCAG 19 pHBMT_FW5 Amplification of the full reading frame of synthesised Potri.001G447500 for GATEWAYTM cloning. Contains attB recognition sites. GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCCACCCCACCAAG 20 AtCesA7 + SacI FW Clone out AtCesA7P from pCesA-mcherry-CesA-BASTA plasmid and insert SpeI and XbaI restriction sites, and sections of pK7WG2 plasmid GTGAGCTCTCCCATATGGTCGAGGCTCCAACGTTT TCAGTTTGG 21 AtCesA7 + XbaI RV GCTCTAGAGCGGCCGCCTGCAGGTCGAATAAGTTTGGTTTTTATGTGTGAAAAGC 125 22 AtCesA7P Seq RV Sequencing primers CCAAGTTACGTTCCCTGTCC 23 AtCesA7P Seq FW CGTTGTGCTTCTGAACCTGC 24 PtCesA8 + SacI FW Clone out PtCesA8P from genomic DNA from P39 and insert SpeI and XbaI restriction sites, and sections of pK7WG2 plasmid GTGAGCTCTCCCATATGGTCGAAAGCGTAGTGAGAAAAACAGAAAATTAGG 25 PtCesA8 + XbaI RV GCTCTAGAGCGGCCGCCTGCAGGTCGACTTGCTTATTACCACTTTACCAGCTC 26 PtCesA8 Seq FW Sequencing primers GACGCAAAAAAAGTAGTAGAGGTC 27 PtCesA8 Seq RV CCATTCAACGAACCGGAAAC Appendix Table B.2. Details of the constructs generated and transformations completed. Name Backbone Promoter Gene Transformed into Col-0 Arabidopsis Transformed into CPL Arabidopsis Transformed into P39 1 pK7WG2 CamV35SP pHMBT1 + + + 2 pHMBT2 3 pHMBT3 4 pHMBT4 pK7WG2 + AtCesA7P pK7WG2 AtCesA7P 5 pK7WG2 AtCesA7P pHMBT1 + + 6 pHMBT2 7 pHMBT3 8 pHMBT4 pK7WG2 + PtCesA8P pK7WG2 PtCesA8P 9 pK7WG2 PtCesA8P pHMBT1 + 10 pHMBT2 11 pHMBT3 12 pHMBT4 pH7WG2 + AtC4HP pH7WG2 AtC4HP pK7WG2 + AtC4HP pK7WG2 AtC4HP 13 pK7WG2 AtC4HP pHMBT1 + + + 14 pHMBT2 15 pHMBT3 16 pHMBT4
Thesis/Dissertation
eng
Forestry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Identifying a native p-hydroxybenzoyl-CoA monolignol transferase gene in poplar
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