Forestry, Faculty of
DSpace
UBCV
Muirragui, Emilia
Master of Science - MSc
University of British Columbia
Sucrose synthase is a glycosyl transferase enzyme responsible for catalyzing the reversible cleavage of sucrose into fructose and UDP-glucose in terrestrial plants. This role in sucrose breakdown has been associated with determining sink strength, modulating phloem loading, and possibly participating in cellulose and starch biosynthesis. In Arabidopsis thaliana, all six SuSy isoforms have been observed to localize to vascular tissues other than the xylem, a major site of cellulose deposition. However, it is highly possible that SuSy plays a more significant role in cellulose biosynthesis in other plant species, such as the aspen family (Populus spp.), where several isoforms have been identified to localize preferentially to the mature xylem. Previous studies have demonstrated that promoters play essential roles in transcriptional regulation, and that the regulatory elements found in their distal and proximal regions could be fundamental to regulating spatiotemporal gene expression. As such, the primary objective of this thesis was to identify differences in the spatiotemporal localization of SuSy in A. thaliana and P. trichocarpa, by assessing the putative contribution of the promoter regions to the functional role(s) of SuSy in plant growth and development. This was investigated by first identifying essential motifs via a bioinformatic analysis of PtSuSy1, PtSuSy2, AtSuSy1, AtSuSy4 promoter sequences, and then comparing enzyme localization via histochemical staining and live-cell imaging across the vascular tissues of A. thaliana and P. trichocarpa when driven by the same promoter (PtSUS1p). Two SNBE-like motifs were identified in the distal region of the poplar SuSy promoters, and given their absence from the Arabidopsis orthologs, were selected for further analysis via promoter truncation experiments. These, along with other GUS transformants, had a sequencing error which caused a frameshift mutation preventing verified transgenic plants from producing visible GUS phenotypes. Instead, expression analysis was conducted to compare transcript abundance across vascular tissues in poplar trees. Results showed significantly higher GUS gene expression in the xylem tissues compared to the phloem, in wild-type poplar where the gene is driven by the native PtSUS1p. Although several questions remain unanswered, these findings partially support PtSUS1p as a spatiotemporal promoter capable of regulating xylem-specific gene expression.
COMPARING SUCROSE SYNTHASE PROMOTERS IN ARABIDOPSIS THALIANA AND HYBRID POPLAR by Emilia MUIRRAGUI B.Sc., University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULLFILMENT 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) April 2023  Emilia Muirragui, 2023 iiThe following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Comparing sucrose synthase promoters in Arabidopsis thaliana and hybrid poplar submitted by Emilia Muirragui in partial fulfilment of the requirements for the degree of Master of Science in Forestry Examining Committee: Dr. Shawn D. Mansfield, Professor, Wood Science, UBC Supervisor Dr. Lacey A. Samuels, Professor, Botany, UBC Supervisory Committee Member Dr. Abel Rosado, Associate Professor, Botany, UBC Supervisory Committee Member Dr. Sean Crowe, Associate Professor, Microbiology and Immunology, UBC Additional Examiner iiiAbstract Sucrose synthase is a glycosyl transferase enzyme responsible for catalyzing the reversible cleavage of sucrose into fructose and UDP-glucose in terrestrial plants. This role in sucrose breakdown has been associated with determining sink strength, modulating phloem loading, and possibly participating in cellulose and starch biosynthesis. In Arabidopsis thaliana, all six SuSy isoforms have been observed to localize to vascular tissues other than the xylem, a major site of cellulose deposition. However, it is highly possible that SuSy plays a more significant role in cellulose biosynthesis in other plant species, such as the aspen family (Populus spp.), where several isoforms have been identified to localize preferentially to the mature xylem. Previous studies have demonstrated that promoters play essential roles in transcriptional regulation, and that the regulatory elements found in their distal and proximal regions could be fundamental to regulating spatiotemporal gene expression. As such, the primary objective of this thesis was to identify differences in the spatiotemporal localization of SuSy in A. thaliana and P. trichocarpa, by assessing the putative contribution of the promoter regions to the functional role(s) of SuSy in plant growth and development. This was investigated by first identifying essential motifs via a bioinformatic analysis of PtSuSy1, PtSuSy2, AtSuSy1, AtSuSy4 promoter sequences, and then comparing enzyme localization via histochemical staining and live-cell imaging across the vascular tissues of A. thaliana and P. trichocarpa when driven by the same promoter (PtSUS1p). Two SNBE-like motifs were identified in the distal region of the poplar SuSy promoters, and given their absence from the Arabidopsis orthologs, were selected for further analysis via promoter truncation experiments. These, along with other GUS transformants, had a sequencing error which caused a frameshift mutation preventing verified transgenic plants from producing visible GUS phenotypes. Instead, expression analysis was conducted to compare transcript abundance across vascular tissues in poplar trees. Results showed significantly higher GUS gene expression in the xylem tissues ivcompared to the phloem, in wild-type poplar where the gene is driven by the native PtSUS1p. Although several questions remain unanswered, these findings partially support PtSUS1p as a spatiotemporal promoter capable of regulating xylem-specific gene expression. vLay Summary The need for development of strategies and tools that allow for precise regulation of gene expression has led scientific efforts to focus on promoter research, with the goal of eventually developing synthetic promoters capable of addressing specific challenges in biotechnological applications. The sucrose synthase families of poplar trees and Arabidopsis have been found to function in different vascular tissues despite their parallel evolution, making them strong candidates for further investigation. The purpose of this work was to identify components within the SuSy promoter sequences of poplar that could direct gene expression to the xylem tissues. It also aimed to visually compare protein location in Arabidopsis and poplar when controlled by poplar’s SuSy1 promoter. The findings support the hypothesis that poplar’s SuSy1 promoter could act to directing gene expression to the xylem tissue of plants. viPreface The research questions and initial research strategy for this project was conceived by Dr. Shawn Mansfield. Emilia Muirragui was responsible for the design of the research plan, the completion of research objectives, and the collection and interpretation of data. The experiments were conducted by Emilia Muirragui, with technical support and guidance of Dr. Lisanne de Vries. Arabidopsis thaliana positive control specimens for GUS assays were kindly provided by Dr. Lacey A. Samuels and Dr. Abel Rosado. The GUS vector employed in this study was tested by Dr. Mathias Schuetz and supplied by Dr. Lacey A. Samuels. Dr. Lisanne de Vries and Madison Kaplen assisted in harvesting transgenic poplar tree tissues. Dr. EunKyoung Lee provided assistance and training to prepare specimens and capture the live-cell images at UBC Bioimaging Facility. Drs. Shawn Mansfield, Lacey A. Samuels, and Abel Rosado contributed to editing this thesis. All other work presented in this thesis represents original, unpublished, and independent work conducted by the author, Emilia Muirragui. viiTable of Contents Abstract ............................................................................................................... iii Lay Summary ....................................................................................................... v Preface ................................................................................................................. vi Table of Contents ............................................................................................... vii List of Tables ........................................................................................................ x List of Figures .....................................................................................................xi List of Abbreviations ......................................................................................... xii List of Symbols ................................................................................................... xv Acknowledgements ............................................................................................ xvi Dedication ......................................................................................................... xvii Chapter 1: Introduction ....................................................................................... 1 1.1 Plant cell wall.................................................................................................................. 1 1.2 Cellulose .......................................................................................................................... 1 1.2.1 Cellulose synthase ....................................................................................................................... 2 1.3 Phloem structure ............................................................................................................ 3 1.3.1 Sugar translocation ...................................................................................................................... 3 1.4 Sucrose metabolism ....................................................................................................... 4 1.4.1 Invertase ....................................................................................................................................... 5 1.4.2 Sucrose synthase in higher plants ................................................................................................ 6 1.5 Promoters........................................................................................................................ 7 1.5.1 Promoter anatomy ........................................................................................................................ 8 1.5.2 Role in regulation ........................................................................................................................ 9 1.6 Research objectives ...................................................................................................... 11 Chapter 2: Materials and Methods .................................................................... 14 2.1 Plant material and growth conditions ........................................................................ 14 2.1.1 Hybrid poplar ............................................................................................................................. 14 2.1.2 Arabidopsis thaliana .................................................................................................................. 14 2.2 Genomic DNA extraction (P. trichocarpa) ................................................................. 14 2.3 Promoter amplification and isolation......................................................................... 15 2.3.1 Primer design ............................................................................................................................. 15 2.3.2 PCR reaction and gel isolation of PtSUS1 promoter ................................................................. 15 2.4 Generation of constructs ............................................................................................. 15 2.4.1 GUS construct ............................................................................................................................ 15 2.5 Preparation and transformation of competent cells ................................................. 16 viii2.5.1 Escherichia coli ......................................................................................................................... 16 2.5.2 Agrobacterium tumefaciens ....................................................................................................... 16 2.6 Agrobacterium-mediated plant transformations ....................................................... 17 2.6.1 Hybrid poplar ............................................................................................................................. 17 2.6.2 Arabidopsis thaliana .................................................................................................................. 18 2.7 Preliminary DNA screening ........................................................................................ 19 2.8 Microscopy.................................................................................................................... 19 2.8.1 Histochemical GUS staining ...................................................................................................... 19 2.8.2 Sectioning and visualization ...................................................................................................... 20 2.9 Expression analysis ...................................................................................................... 20 2.9.1 Poplar greenhouse trial and harvest ........................................................................................... 20 2.9.2 RNA extraction .......................................................................................................................... 21 2.9.3 cDNA synthesis ......................................................................................................................... 22 2.9.4 qRT-PCR analysis ..................................................................................................................... 22 2.10 Bioinformatic analysis ............................................................................................... 23 2.10.1 Sequence analysis and motif identification .............................................................................. 23 2.10.3 Promoter truncations ................................................................................................................ 23 Chapter 3: Bioinformatic analysis of xylem and secondary cell wall motifs identified in select promoters ............................................................................. 25 3.1 Introduction .................................................................................................................. 25 3.2 Selection of motifs of interest ...................................................................................... 26 3.3 Scope and limitations ................................................................................................... 27 3.4 Results ........................................................................................................................... 28 3.4.1 Analysis of the poplar SuSy1 and SuSy2 promoters ................................................................. 28 3.4.2 Analysis of the Arabidopsis SuSy1 and SuSy4 promoters ........................................................ 30 3.4.3 Analysis of the poplar CesA4, CesA7 and CesA2 promoters .................................................... 32 3.5 Discussion...................................................................................................................... 34 3.5.1 Core promoter ............................................................................................................................ 34 3.5.2 Proximal promoter ..................................................................................................................... 34 3.5.3 Distal promoter .......................................................................................................................... 36 3.6 Promoter truncations design ....................................................................................... 37 3.7 Protein localization in truncated promoter transformants ...................................... 40 Chapter 4: Investigating the role of PtSUS1 promoter in spatiotemporal expression ........................................................................................................... 41 4.1 Introduction .................................................................................................................. 41 4.2 Expression analysis results .......................................................................................... 42 4.3 Discussion...................................................................................................................... 46 4.4 Background troubleshooting....................................................................................... 48 4.4.1 GUS staining and imaging ......................................................................................................... 48 4.4.2 Promoter and intron amplification ............................................................................................. 54 4.4.3 Frameshift mutation ................................................................................................................... 55 4.4.4 Arabidopsis experiments ............................................................................................................ 55 Chapter 5: Conclusion ....................................................................................... 57 ix5.1 Thesis summary ........................................................................................................... 57 5.2 Relevance ...................................................................................................................... 58 5.3 Future research ............................................................................................................ 59 5.3.1 Intron removal and evaluation of influence on gene expression ................................................ 59 5.3.2 Promoter swapping Arabidopsis and poplar SuSy homologs .................................................... 60 5.3.3 Comparing SuSy and secondary CesA expression levels in vascular tissues ............................ 61 References........................................................................................................... 62 Appendices .......................................................................................................... 79 Appendix A: Additional Materials and Methods ............................................................ 79 Appendix B: Additional motif information ..................................................................... 80 Appendix C: GUS staining controls and additional troubleshooting information ...... 81 xList of Tables Appendix Table A.1. PCR primers employed in this study ................................................... 79 Appendix Table A.2. qRT-PCR primers employed in this study ........................................... 80 Appendix Table B.1. Motifs identified in this study .............................................................. 80 Appendix Table C.1. GUS positive controls employed in this study .................................... 81 xiList of Figures Figure 3.2: Motif comparison between AtSUS1, AtSUS4 and PtSUS1 promoters ............... 31 Figure 3.3: Motif comparison between P. trichocarpa’s CesA 4, 7, and 2 promoters and the SuSy1 promoter ....................................................................................................................... 33 Figure 3.4: Truncation design for PtSUS1 promoter based on the bioinformatic analysis of select motifs ............................................................................................................................. 38 Figure 4.1: Fold change of GUS expression levels in the phloem tissue of greenhouse-grown transgenic PtSUS1p::GUS poplar lines ................................................................................... 43 Figure 4.2: Fold change of GUS expression levels in the root tissue of greenhouse-grown transgenic PtSUS1p::GUS poplar lines ................................................................................... 44 Figure 4.3: Fold change of GUS expression levels in the xylem and phloem tissues of greenhouse-grown transgenic PtSUS1p::GUS poplar lines ..................................................... 45 Figure 4.4: Fold change of GUS expression levels in the xylem tissue of select greenhouse-grown high expressing transgenic PtSUS1p::GUS poplar lines and external P39 controls .... 46 Figure 4.5: Two-week old positive control RPL4A::GUS seedling ....................................... 49 Figure 4.6: Two-week old positive control PrABCB11::GUS seedling ................................. 50 Figure 4.7: Four- and six-week old positive controls PrABCB11::GUS stems and leaf ........ 51 Figure 4.8: Stem cross-sections from six-week old positive controls for GUS assays .......... 51 Figure 4.9: Stem cross-sections from the top 7cm of the main stem of the PtSUS1p::GUS greenhouse-grown lines after GUS assay ................................................................................ 52 Figure 4.10: Stem cross-sections from the bottom 5cm of the main stem of the PtSUS1p::GUS greenhouse-grown lines after GUS assay ................................................................................ 52 Figure 4.11: Root cross-sections from the main root of the PtSUS1p::GUS greenhouse-grown lines after GUS assay ............................................................................................................... 53 Figure 4.12: GUS artifacts present in stem and root tissue from PtSUS1p::GUS greenhouse-grown lines ............................................................................................................................... 53 Appendix Figure C.1. Vector map of the PtSUS1p::GUS construct displaying the two main ORFs ........................................................................................................................................ 81 Appendix Figure C.2. Amino acid sequence comparison of the resulting protein translated from ORF-1 and ORF-2 ........................................................................................................... 82 xiiList of Abbreviations aa amino acid ADP Adenosine diphosphate At Arabidopsis thaliana BA 6-benzylaminopurine bp base pairs CaCl2 calcium chloride CC companion cells cDNA synthetic DNA CesA cellulose synthase ChIP-seq Chromatin immunoprecipitation sequencing CIN neutral invertase cm centimeters Col-0 Columbia-0 ecotype CPT core promoter truncation CRE cis-regulatory element CRM cis-regulatory module CSC cellulose synthase complex Ct cycle threshold CTAB cetrimonium bromide CWIN cell wall -bound invertase DEPC diethyl pyrocarbonate DNA deoxyribonucleic acid DPT distal promoter truncation E. coli Escherichia coli EDTA ethylenediaminetetraacetic acid FAA formalin acetic acid alcohol g g-force / unit of gravitational force GFP green fluorescent protein GUS glucuronidase H+ protons / hydrogen atoms HCl hydrochloric acid HDR homology dependent repair Hz Hertz IAA isoamyl alcohol IME intron mediated enhancement INV invertase kb kilobase kDa kilo Daltons L liter LB lysogeny broth LiCl lithium chloride M molar mg milligrams min minute(s) mL milliliters mM millimolar MPa mega pascals xiiiMS Murashige & Skoog medium MYB myeloblastosis (transcription factor family) NAA naphthaleneacetic acid NAC acronym derived from NAM, ATAF and CUC genes (TF family) NaCl sodium chloride NaPO4 sodium phosphate ng nanogram nm nanometers NST NAC secondary wall thickening factor OD optical density ORF open reading frame P39 hybrid poplar genotype Populus alba x gradidentata PBS phosphate buffered saline PCR polymerase chain reaction pH potential hydrogen PPT proximal promoter truncation Pt Populus trichocarpa PtrEF1B Populus trichocarpa ELONGATION FACTOR 1-BETA PtrUBQ11 Populus trichocarpa UBIQUITIN 11 PVPP polyvinylpolypyrrolidone QD quantitative dialysis qRT-PCR quantitative real-time PCR RNA ribonucleic acid rpm revolutions per minute RT reverse transcriptase s second(s) SCW secondary cell wall SDS sodium dodecyl-sulfate SE sieve tube elements SNBE secondary-wall NAC binding element SOC super optimal broth with catabolite repression SS splice site SUS sucrose synthase SuSy sucrose synthase SUT/SUC sucrose transporter T1 transgenic generation 1 T3 transgenic generation 3 TDZ thidiazuron TE buffer tris-EDTA TERE tracheary element-regulating cis-element TF transcription factor TFBS transcription factor binding site TSS transcription start site UDP uridine diphosphate UDPG uridine diphosphate-glucose UTP uridine triphosphate UTR untranslated region VIN vacoular invertase VND vascular-related NAC domain VNS gene family consisting of VND, NST, and SMB groups xivw/v weight per volume WPM McCown’s Woody Plant Media WT wildtype YEP yeast extract peptone xvList of Symbols  beta  p-value C Celsius % percentage < less than △ Delta ™ trademark µ micro xviAcknowledgements I would like to start by expressing my gratitude to my supervisor Dr. Shawn Mansfield for providing me with the opportunity and resources to successfully complete this Master’s degree. Additionally, I would also like to thank my supervisory committee Drs. Lacey A. Samuels and Abel Rosado for their scientific guidance and unwavering support throughout this project. To my mentors, Dr. Faride Unda, Heather Mackay and Dr. Lisanne de Vries, thank you for taking me under your wing and always being so generous with your time, advice, and support. Within a short period of time, you’ve taught me things that would have taken me years to learn. Thank you for the difference you have made in my career. To the past and current Mansfield lab members, thank you for making the day-to-day grind much lighter and brighter with your friendship. I wish nothing but sunny days and beautiful PCR gels for all of you. I would like to specifically acknowledge Dr. Elizabeth Mahon, Madison Kaplen, Michael Bilek, Sydne Guevara, Zimou Sun and Melike Karaca Bulut for their encouragement and kindness through the hills and valleys of the road. My sincere gratitude to the UBC Bioimaging Facility staff (Drs. Miki Fujita and EunKyoung Lee) for giving me access to their skills, knowledge, and passion for microscopy. My further gratitude to the administrative staff in the Department of Wood Science, as well as the UBC Greenhouse team for providing me with their resources, time, and knowledge. To my parents, Dr. Aldo Muirragui and Tania Torres, you have poured your love and trust over me from the day I was born. Thank you for believing in me, for believing in my dreams, for instilling in me the love for science, and for allowing me the opportunity to pursue higher education. Most importantly, thank you for teaching me that perseverance, commitment, and hard work never go unrewarded. Without you I would not be the person I am today. To my husband, Juan Esteban Bustamante, thank you for your endless patience and loving encouragement. You have reminded me of my strength and courage when I needed them most. Thank you for seeing the best in me, for being my most enthusiastic cheerleader, and for making me laugh amidst the chaos that academia brings. You have been a ray of sunshine in the stormiest days. Lastly, to my little Manitoba. Thank you for providing me with your warmth, comfort, and loud purrs during my restless nights. My mental health would’ve not been the same, especially during the pandemic, without your silliness and loyal companionship. Your clueless innocence makes me smile. xviiDedication To my parents, for giving me wings and allowing me to fly. To my husband, who encouraged me to soar. “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.” -Charles Darwin 1 Chapter 1: Introduction Sucrose is the primary transportable form of carbon in terrestrial plants, which can supply energy for metabolic processes and plant growth. Moreover, plant cell walls are the largest sink for fixed carbon, thus relying heavily on sucrose for their development. Sucrose synthase (SuSy) could potentially play a key role in the biosynthesis of complex carbohydrates, such as cellulose, however, SuSy’s spatiotemporal gene expression differs in Populus compared to that of the model species Arabidopsis thaliana. For this reason, the role of these SuSy promoters in gene expression needs to be investigated further. 1.1 Plant cell wall Cell walls are essential components of plants that provide structural support to the cell and protection against biotic and abiotic stresses. Many plant cell walls have three main layers: the primary cell wall, the secondary cell wall, and the middle lamellae, which differ in their chemical and physical properties. The middle lamella is rich in pectin and is found between two adjoining primary cell walls regulating intercellular adhesion (Willats et al. 2001). Primary cell walls are synthesized during cell division and expansion, while secondary cell walls form in specific cells between the primary cell wall and the plasma membrane during cell differentiation (Nakano et al. 2015). Primary cell walls are inherently thin and flexible, while secondary cell walls are relatively thick, rigid, and provide additional hydrophobicity to the cell (Nakano et al. 2015). Secondary cell walls provide significant mechanical strength to cells and are typically composed of cellulose, hemicellulose, and most often lignin (Zhong and Ye 2007). Overall, the plant cell wall is an intricate matrix largely composed of polysaccharides and phenolic-derived constituents, and their abundance and relative quantities will vary between plant tissues and plant species (Bauer et al. 2006). Plant cell walls are one of the main sink tissues in terrestrial plants, thus requiring a large amount of the fixed carbon produced during photosynthesis to be mobilized and deposited into the polysaccharide-rich cell walls (Verbančič et al. 2018). 1.2 Cellulose Cell wall polysaccharides can be divided into 3 main groups: cellulose, hemicellulose, and pectin. Cellulose is the main structural component of cell walls. It is an unbranched 2homopolysaccharide, composed of a chain of -1,4-linked glucose that is robust and inflexible due to the presence of inter- and intra-molecular hydrogen bonds (Wohlert et al. 2022). Cellulose chains can have a relatively high degree of polymerization, and can form crystalline regions(O’Sullivan 1997). Cellulose is the primary component of the secondary cell wall. Using UDP-glucose as a precursor, this polymer is composed of parallel, unbranched chains of -1,4-linked glucose, which line up to form cellulose microfibrils (O’Sullivan 1997). Microfibrils are 2-5 nm in diameter, and allow the cell wall to withstand up to 1 MPa of turgor pressure due to their high tensile strength (Anderson et al. 2010). It is estimated that each microfibril is composed of 18 cellulose polymer chains (Kubicki et al. 2018; Purushotham et al. 2020). 1.2.1 Cellulose synthase In higher plants, cellulose is synthesized by a large “rosette”-structured, membrane-bound protein complex, made up of six distinct lobes (Nixon et al. 2016). The cellulose synthase complexes (CSCs) are composed of individual cellulose synthase proteins (CesAs). In Arabidopsis thaliana, ten CesA isoforms have been identified. Analyses of CesA protein sequences suggest these can be classified into six separate groups. CesA1, CesA3, and CesA6 have been linked to cellulose synthesis of the primary cell wall (Desprez et al. 2007), and CesA4, CesA7, and CesA8 are essential for the synthesis cellulose in lignified secondary cell walls (Taylor et al. 2003). From the secondary CesAs, CesA8 seems to be play a particular role in SCW formation, as it is more highly expressed in aspen tension wood than isoforms CesA4 and CesA7 (Purushotham et al. 2020). CesA2 antisense mutants display milder morphological changes, but have phenotypic characteristics consistent with a role in primary cell wall deposition (Burn et al. 2002). CesA5 is essential for the cellulose biosynthesis in seed mucilage (Sullivan et al. 2011). Also highly expressed in the seeds, CesA9 is vital for secondary cell wall biosynthesis and proper function of the epidermal seed coat (Stork et al. 2010). Both CesA9 and CesA10 are expressed at the base of rosette leaves, however, the latter has not been fully characterized (Doblin et al. 2002). The CesA proteins are transported from the Golgi apparatus to the plasma membrane via vesicles, where upon delivery, will assemble and mediate the deposition of cellulose (Watanabe et al. 2015). 31.3 Phloem structure While xylem tissue is responsible for the transport of water and other minerals from the roots to other tissues, phloem tissue translocates sugars made from photosynthetic organs (source tissue) to sink organs for growth and storage (Kühn and Grof 2010), as well as other vital nutrients and compounds. The phloem consists of sieve tube elements (SE), companion cells (CC), phloem parenchyma cells, and phloem fibers (Schuetz et al. 2013). SEs and CCs are derived from the same mother cell, yet the development and differentiation of SEs is independent from the presence of CCs (Oparka and Turgeon 1999). SE cells undergo incomplete autolysis that is highly selective and results in the loss of several organelles. However, mature SEs will retain their endoplasmic reticulum, plasma membrane, modified mitochondria, and plastids (Oparka and Turgeon 1999). These cells will differentiate in vertical files and connect to each other via sieve plates to form the sieve tube for long distance transport. Sieve plates tend to have a lining of callose around the sieve pores, which is deposited in response to damage from wounding of the SEs (Barratt et al. 2009). CCs have dense cytoplasms and relatively low starch contents. These cells can form plasmodesmatal connections with the SEs, better known as the sieve element-companion cell (SE-CC) complex (Cronshaw 1981). The SE-CC complexes serve as long distance conduits in the transportation of sucrose from source tissues to sites of utilisation or storage in sink organs (Kühn and Grof 2010). 1.3.1 Sugar translocation The SE-CC complex contributes many roles in planta, including phloem loading and unloading, as well as long distance photosynthate transport (Oparka and Turgeon 1999). Phloem loading can occur via symplastic transport through the plasmodesmata when significant concentrations of sucrose accumulate and create mass flow towards the sink organs due to a hydrostatic pressure gradient (Van Bel 2003), and by active transport through specific carriers in the apoplast (Riesmeier et al. 1994). Sucrose transporters (identified as SUTs or SUCs interchangeably), located on the plasma membrane of the SE-CC complex, play an essential role in phloem loading, sucrose retrieval, and transferring sucrose to storage cells (Milne et al. 2017). Several SUT genes have been discovered and studied across numerous plant species including potato (Riesmeier et al. 1994), Arabidopsis (Srivastava et al. 2008), maize (Slewinski et al. 2010), rice (Aoki et al. 2003), pomegranate (Poudel et al. 2020) and others. To date, nine SUT genes have been identified in Arabidopsis (Sivitz et al. 2007), and several of these are 4expressed in sink tissues including AtSUC1 in pollen (Stadler et al. 1999), AtSUC3 in trichomes, guard cells, developing seeds and root tips (Meyer et al. 2000), AtSUC5 in seeds (Baud et al. 2005; Pommerrenig et al. 2013), AtSUC6 in reproductive tissues, AtSUC7 in roots and pollen tubes (Rottmann et al. 2018), and AtSUC8 and AtSUC9 in flowers (Sivitz et al. 2007). The AtSUC2 Sucrose/H+ symporter is highly expressed in the companion cells of mature leaves, and studies have shown that it is involved in sucrose retrieval, but its essential role is related to phloem loading (Srivastava et al. 2008). Atsuc2 loss-of-function mutants showed growth reduction and starch accumulation, which can be explained by reduced levels of sucrose transport (Srivastava et al. 2008). Passive phloem loading through the symplast occurs when there is a thermodynamically favourable concentration gradient between the mesophyll cells and the phloem, allowing for passive transport of carbohydrates through abundant plasmodesmata (Slewinski and Braun 2010). Studies have shown that in many plants, especially trees like poplar, the passive loading mechanism seems to be the primary method driving sucrose transport throughout the plant (Zhang et al. 2014). Six SUT genes have been identified in poplar (Payyavula et al. 2011; Mahboubi et al. 2013), however, there seems to be enough evidence to suggest that apoplastic phloem loading does not play a significant role in woody plants (Zhang et al. 2014). It is important to note that even in symplastic loading species, there is a need for SUTs to retrieve excess sucrose along the transport phloem to maintain the chemiosmotic gradient (Braun et al. 2014). 1.4 Sucrose metabolism Sucrose is the main product of photosynthesis and is essential for terrestrial plants, as it serves as the main source of carbon and energy. Photo-assimilated carbon is transported in the form of sucrose (minor species transport raffinose) from sites of photosynthesis (source tissues) to sink tissues. At sink organs, sucrose can be cleaved by either invertases or sucrose synthases to provide monomeric building blocks for immediate energy and/or growth, or to be used to synthesize starch, which can then be stored (Koch 2004). Additionally, sucrose is necessary for signalling pathways related to gene expression and physiological responses (Wind et al. 2010). There are two enzymatic pathways for the degradation of sucrose: Invertases (INV) are capable of irreversibly hydrolysing sucrose into glucose and fructose, while sucrose synthases (SuSy) are responsible for catalysing the formation of UDP-glucose and fructose, a process 5which is reversible (Wang et al. 1993; Koch 2004). Glucose and fructose can then be converted into glucose-6-phosphate and fructose-6-phosphate respectively by hexokinases, and then into glucose-1-phosphate via phosphoglucomutase (Egli et al. 2010; Verbančič et al. 2018). Uridine diphosphate-glucose (UDPG) is a nucleotide sugar essential for the biosynthesis of callose, cellulose, and sucrose, as well as for the formation of key structural components of the cell walls, such as hemicelluloses and pectins (Amor et al. 1995; Johansson et al. 2002; Kleczkowski et al. 2010). UDPG can also be produced directly by sucrose synthase or by UDP-glucose pyrophosphorylase (UGPase), the latter reaction consisting of UTP and glucose-1-phosphate (Kleczkowski 1994; Kleczkowski et al. 2010). 1.4.1 Invertase Plant invertases are an enzyme family of -fructosidases which can be classified based on their subcellular localization, pH optima , solubility, and isoelectric points (Sturm and Chrispeels 1990). Based on these criteria, three different groups have been identified: vacuolar (VIN), cell wall-bound (CWIN), and neutral (CIN) invertases (Roitsch and González 2004). These enzymes irreversibly degrade sucrose into glucose and fructose (Koch 2004; Wang et al., 1993). Prior to entering other metabolic pathways, glucose must first be phosphorylated by hexokinase or glucokinase (Huber and Akazawa 1986). Vacuolar and cell wall invertases are phylogenetically related, and are considered acidic invertases as their optimum pH is between 4.5 and 5 (Sturm 1999; Wan et al. 2018). These invertases are constantly regulated at the transcriptional and post-translational levels, with their corresponding genes being regulated by transcription factors (TFs) interacting with gene promoters (Li et al. 2017). Acid invertases are capable of hydrolyzing fructose residues of disaccharides and -fructose-containing oligosaccharides, such as raffinose and stachyose (Sturm 1999). Additionally, CWINs are co-expressed along with hexose in the phloem plasma membrane, suggesting an important role in phloem apoplastic unloading (Braun et al. 2014). When sucrose is transported from the SEs into the apoplast, CWINs will cleave sucrose into glucose and fructose, which are then transported into sink cells by the hexose transporters on the plasma membrane (Roitsch and González 2004). Neutral/alkaline invertases (CIN) are localized in the cytoplasm and have a pH optima between 7-7.8 (Sturm 1999; Wan et al. 2018). In contrast to CWINs and VINs, CINs are highly unstable as they are not glycosylated (Ruan 2014). Although CINs have been less well characterized, these enzymes target sucrose and are considered critical for maintaining 6cytoplasmic sugar homeostasis, cellular functions, signalling and reproductive development (Li et al. 2017; Ruan 2014; Sturm 1999). 1.4.2 Sucrose synthase in higher plants Sucrose synthase reversibly catalyses the degradation of sucrose, yielding fructose and UDP-glucose as products (Koch 2004); the latter being a precursor for cellulose and callose biosynthesis (Joshi et al. 2004). There is evidence to suggest that sucrose synthase might be involved in determining the strength of sink tissues (Zrenner et al. 1995), and might also have a minor role in the biosynthesis of starch (Wang et al. 1993). Structurally, SuSy is a tetrameric enzyme with subunits of approximately 90kDa (Hardin and Huber 2004) and conserved glycosyltransferase domains (Zheng et al. 2011). Across plant species, SuSys are characterized by multigene families. A total of six isoforms have been found in Arabidopsis (Baud et al. 2004), seven in rice (Cho et al. 2011), 15 in poplar (An et al. 2014), eight in cotton (Zou et al. 2013), three in peas and maize (Barratt et al. 2001; Carlson et al. 2002), and five in grapes (Zhu et al. 2017). 1.4.2.1 Spatiotemporal localization of SuSy in Arabidopsis Several studies have successfully localized SuSy via immunolabelling and GUS staining in different species, including Arabidopsis (Barratt et al. 2009), maize (Nolte and Koch 1993; Hänggi and Fleming 2001), potato (Zrenner et al. 1995), and citrus (Nolte and Koch 1993). Sucrose synthase appears to be localized to companion cells, where it plays a role in phloem loading and unloading (Nolte and Koch 1993). The six isoforms of SuSy have been successfully localized in Arabidopsis (Yao et al. 2020). SuSy1 and SuSy4 were localized in the phloem companion cells, SuSy2 and SuSy3 were localized in the embryo of developing seeds, with the latter also found in the stomata guard cells, and SuSy5 and SuSy6 were localized in the sieve elements (Yao et al. 2020). There was no presence of SuSy isoforms in the xylem tissue (Yao et al. 2020), supporting the argument that, in Arabidopsis, SuSy does not play a significant role in cellulose biosynthesis (Barratt et al. 2009). This is further supported by the Arabidopsis sus123456 mutants, which show no measurable SUS activity, and exhibited WT-like ADP-glucose and UDP-glucose content (Fünfgeld et al. 2022; Wang et al. 2022). Additionally, there is evidence to suggest that the functional redundancy of the SuSy genes allows for cellulose and starch biosynthesis to proceed at normal rates when any of the six SuSy genes is silenced (Baroja-Fernández et al. 2012). Additionally, it has been suggested that 7SuSy is involved in the deposition of cellulose and callose as it is a cytoplasmic enzyme and often interacts with the plasma membrane (Amor et al. 1995). 1.4.2.2 Spatiotemporal localization of SuSy in Populus Although the SuSy family has been successfully characterized in A. thaliana, the multigene SuSy family of aspen (Populus) and other trees needs further investigation. Bioinformatics and molecular cloning originally revealed at least seven members of the poplar SuSy multigene family (Zhang et al. 2011). Most recently, updated genomic data from Populus trichocarpa has revealed 15 distinct members, making the poplar SuSy family the largest one to date amongst land plants (Tuskan et al. 2006; An et al. 2014). PtSUS1 and PtSUS2 seem to be the two best characterized members of this family. The protein sequences of PtSUS1 and PtSUS2 show high homology (80.9 – 85.7%) with SuSy1 and SuSy4 of Arabidopsis, tomato, and potato (Zhang et al. 2011). PtSUS1 and PtSUS2 seem to be preferentially expressed in mature xylem sink tissue, with medium expression levels in the roots, mature leaves and developing xylem (Zhang et al. 2011). Transcript profiling of PtSUS1-7 suggests that all family members are expressed in mature leaf at different levels, and they are also expressed preferentially in other organs. PtSUS3 has high expression in the shoot apex, PtSUS4 and PtSUS5 are expressed in the roots and have low expression in the cambium, xylem, and young leaves, PtSUS6 and PtSUS7 are confined to the mature source leaves, and are present at lower levels in the root and shoot apex (Zhang et al. 2011). Additionally, transcript levels of the remaining six members show that 15 genes are expressed in the roots, stems, leaves, floral buds, and vegetative buds at different levels (An et al. 2014). Although transcript profiling provides insight into the expression of this multigene family, there has not been a visual localization of the proteins in the tissue. 1.5 Promoters In DNA, promoter regions are located upstream of the coding strand of a gene. It is difficult to predict the exact length of a promoter, but for practical purposes the minimal sequence to be studied usually spans from 0.5Kb to 2Kb upstream of the transcriptional start site (TSS) (Pandiarajan and Grover 2018). They are essential for the transcription of DNA into RNA, as they provide a binding site for RNA polymerase and transcription factors (TFs), dictating the levels, time and location in which a gene is expressed. Promoters in both prokaryotes and 8eukaryotes are characterized by a lower stability, regardless of their nucleotide composition, when compared to the coding regions located downstream (Kanhere and Bansal 2005). It is hypothesized that within the promoter itself, DNA stability would be lower in the upstream regions, mostly due to the AT-rich content of the sequence. (Kanhere and Bansal 2005; Rajewska et al. 2012). Eukaryotic promoters are complex in the sense that they can span for several kilobases and can have regulatory elements far away from the TSS, making them difficult to fully characterize. 1.5.1 Promoter anatomy A promoter is defined as the non-coding region in the genome typically located upstream of the TSS of a gene, where relevant proteins bind to initiate transcription of that gene. Usually, promoters are characterized by containing several AT-rich regions, especially in their origin of replication, as AT-rich sequences are the most universally conserved structural elements in both eukaryotes and prokaryotes (Rajewska et al. 2012). AT-rich regions contain repeats of various lengths and are essential for DNA unwinding and replication (Rajewska et al. 2012). Most promoters can be subdivided into three parts: core, proximal, and distal regions. The core promoter is sometimes considered to be part of the proximal region and it contains the TSS, while the proximal and distal promoter regions contain several transcription regulators like enhancers and silencers (Abeel et al. 2008). The core promoter is considered to be the minimal DNA sequence required to activate transcription and determine its direction (Andersson and Sandelin 2020). It is located approximately 50bp upstream of the TSS, and contains essential transcriptional machinery such as the TATA box, the initiator, and the B-recognition element (Abeel et al. 2008; Hill et al. 2021). Although the core promoter is short in length, it contains several highly conserved elements that are essential for the recruitment and positioning of numerous proteins required to form the transcriptional multiprotein complex (Hill et al. 2021). Core promoters can be classified based on the presence or absence of their transcriptional elements, and their transcription initiation mechanism dictated by the interaction of such elements (Xu et al. 2016). The proximal promoter is adjacent to the core region and spans a few hundred base pairs. This region contains Transcription Factor Binding sites (TFBS) and is responsible for assembling RNA polymerase II at the correct position in order to start transcription; this is regulated by the TATA box in the core promoter and other TFs specific to RNA polymerase II (Nikolov and Burley 1997; Berk 1999; Rombauts et al. 2003; Pandiarajan and Grover 2018). 9It has also been determined that elements regulating TSS selection are mostly found in this region (Shahmuradov et al. 2003). However, due to the arbitrary size of the proximal region, determining its limits requires a technique known as ‘promoter bashing’ to test which subregions have a regulatory input (Andersson and Sandelin 2020). The distal promoter is usually located several thousands of base pairs away from TSS and contains regulatory elements related to spatiotemporal gene expression (Tjian and Maniatis 1994; Fessele et al. 2002; Rombauts et al. 2003). Enhancer elements tend to be more common within this region than silencers, yet both have the capability of being active in specific tissues or under certain environmental conditions (Biłas et al. 2016). In some cases, it is difficult to precisely identify where the proximal region ends and the distal region begins (Rombauts et al. 2003), as both of these regions contain multiple elements that participate in the regulation of transcription at several levels, including cell-, organ-, developmental- and environmental-factors (Shahmuradov et al. 2003). 1.5.2 Role in regulation Promoters have various levels of complexity. The infinite outcomes of gene regulation come from a combination of mechanisms that allow for diverse expression patterns with a number of regulatory elements available (Benfey and Chua 1990). DNA is constantly battling between packaging and accessibility. The dynamic competition occurs between nucleosomes packaging DNA into chromatin compactly, and ‘chromatin remodelers’ reconfiguring specific nucleosomes to provide access to important cis-regulatory sites (Asturias et al. 2003; Cairns 2009). Due to nucleosome phasing patterns in eukaryotic gene promoters, similar binding motifs may have repeats along multiple spacing groups to stabilize protein interactions spanning multiple helical turns in the DNA (O’Malley et al. 2016). In addition, there is evidence to suggest that regulatory elements of the distal promoter will sometimes target and activate core promoter nucleosome-remodeling factors in genes that are to be co-expressed (Nikolov and Burley 1997). The promoter region can bring different TFs together by assuming different three dimensional configurations to activate transcriptional machinery (Berk 1999; Rombauts et al. 2003) so that the interactive protein domains are aligned with the DNA helix and preferred nucleosome positions (O’Malley et al. 2016). Altering promoter architecture allows for different regulatory sites to be exposed under the appropriate conditions, and thus regulating gene expression (Cairns 2009). 10Additionally, promoters vary in their site of action, efficiency, and period of activity. Constitutive promoters are active in vivo at constant levels throughout all developmental stages, while tissue-specific promoters are active at a certain developmental stage or tissue. Some promoters also require activation from specific stimuli, upon which they bind to RNA polymerase and transcription factors to initiate transcription (Biłas et al. 2016). 1.5.2.1 Motifs Promoters play a very important role in transcriptional regulation. DNA-binding proteins, such as TFs, are recruited by specific nucleotide sequences in the promoter. These short sequences, known as motifs, are essential functional components of a promoter as they specify protein binding (Hudson and Quail 2003). Motifs can be identified by comparative analysis of known sequences of promoters without having prior knowledge of TFBSs. Also, co-regulated genes or genes within the same family that follow similar expression pathways tend to have conserved common motif sequences, allowing for techniques such as sequence alignment and other computational methods to be used for identification within a promoter sequence (Hudson and Quail 2003). Much effort has been devoted to identifying the protein recognition elements in promoters, and several techniques have been developed to identify TF in the genome and match them to existing and known TFBS in databases. Chromatin immunoprecipitation sequencing (ChIP-seq) allows for identification of TF in a genome-wide scale (Kheradpour and Kellis 2014). However, due to the demand for high quality antibodies and the difficulties related to rare or low expressed proteins, the resulting information on binding sites is limited to a few TFs, and the TFBS coverage has only been detailed in human and model organisms (Kidder et al. 2011; O’Malley et al. 2016). Interestingly, this approach has highlighted the complex behavior of TFs. Binding has been shown to be dynamic across cell types, often requiring the coordinated binding of cofactors or specific chromatin layouts for TF activity. Yet, it has been demonstrated that sometimes motifs are not essential for TF binding, and that binding will often occur in the absence of the motif recognition pattern (Kheradpour and Kellis 2014). In terms of databases, ReMap 2020 is the first large-scale integrated initiative, which aims to provide the largest catalogs of high quality regulatory regions, resulting from the integration of large-scale DNA-bindings assays of hundreds of TF and regulatory elements in the human and Arabidopsis genomes (Chèneby et al. 2020). It is expected that, in the future, the integration of the regulatory profiles of other species will expand this database. 11 1.5.2.2 Cis-elements Cis-elements are linear nucleotide regulatory sequences of non-coding DNA which produce another level of complexity when it comes to gene expression at all developmental stages (Biłas et al. 2016). The combination of different cis-elements can alter expression in a particular tissue at a certain time point, making it possible to have different expression patterns across a wide range of tissues (Benfey and Chua 1990). Cis-regulatory modules (CRMs) are made up of a combination of cis-regulatory elements (CREs), promoters, transcriptional regulators, and insulator elements. CRMs serve as sequence-specific TFBS and can determine the level of expression, time, and type of cell at which a gene is expressed (Schmitz et al. 2022). In fact, a gene’s expression profile can be largely determined from the combination of CREs present in the promoter under which the gene of interest is driven (Pandiarajan and Grover 2018). However, it remains quite challenging to predict which TF will bind to a particular CRE, mostly because this requires extensive understanding of cell types, TF expression patterns compared to the putative target genes, and accurately determining whether a CRE will be available for binding (Schmitz et al. 2022). Currently, research on this topic has been focused mostly on the discovery of cis-elements, but the long-term goal relies on having a better understanding of the functions and interactions of CREs and CRMs. In the last decade, several plant promoters have been found to not contain a TATA-box, and in such case, other cis-elements seem to play a role in transcription initiation (Mehrotra et al. 2011; Mejía-Guerra et al. 2015; Schmitz et al. 2022). Cis-elements have often been considered to be a type of regulatory element in promoters, however, as new data reveals the cis-regulome of plant genomes, the definition for promoters might become more exclusive to the core promoter region (Schmitz et al. 2022). 1.6 Research objectives Plant researchers have aimed to optimize gene expression for decades. The need to effectively manipulate and drive gene expression in plants for scientific and industrial purposes has shifted research focus on promoters. However, there is a lack of data regarding the characterization of the functional components that could potentially be driving spatiotemporal localization of proteins. The field of plant biotechnology is characterized for being extremely dynamic, but also quite demanding as implementation of novel methods largely depends on public 12acceptance and industrial economic benefits. A more comprehensive understanding of promoter functionality, as well as identifying fundamental regulatory elements or sequences could lead to viable promoter engineering strategies. The creation and implementation of synthetic promoters could give rise to consistent expression systems and highly predictable results that will achieve better crop efficiency and resource use for material production across numerous economic sectors. The primary objective of this research was to identify differences in the spatiotemporal localization of SuSy by focusing analysis on the native SuSy promoters of Arabidopsis thaliana and Populus trichocarpa. I set out to assess the putative contribution of the promoter regions to the functional role(s) of SuSy in plant growth and development. To better understand the role of the SuSy promoters in gene expression and protein localization, this study had two objectives: 1. To identify the essential motifs within the SuSy promoter sequences of A. thaliana and P. trichocarpa (Chapter 3). 2. To compare promoter activity of PtSUS1p in the vascular tissues of A. thaliana and P. trichocarpa using a glucuronidase (GUS) reporter driven by the same promoter (Chapter 4). The completion of these objectives relied heavily on the viability of successfully amplifying and manipulating the promoter sequences for Populus trichocarpa Sucrose Synthase 1 promoter (PtSUS1p) and Populus trichocarpa Sucrose Synthase 2 promoter (PtSUS2p). Unfortunately, the latter presented significant difficulties in the amplification and cloning process due significant AT-richness and repetitiveness, and therefore the results in this study are limited to the experiments and analysis of PtSUS1p. In Chapter 3, I address the first objective by focusing my bioinformatic analysis on the identification of several motif-sequences that have been previously characterized to be related to xylem cells and secondary cell wall deposition. My main focus was on comparing the homologous poplar and Arabidopsis promoter sequences for SuSy, but I expanded my focus to include a total of seven promoter sequences: PtSUS1, PtSUS2, AtSUS1, AtSUS4, PtCesA4, PtCesA7, PtCesA2. The secondary CesA promoters were included in the analysis to expand the scope of the motif analysis and comparison, given that research has shown SuSy to be associated to cellulose biosynthesis driven by the CesAs (Coleman et al. 2009; Payyavula et al. 2011; Gerber et al. 2014). Based on the bioinformatic analysis, I designed three promoter truncations that would allow me to refine which set of motifs or promoter region was 13influencing poplar SuSy’s expression in the xylem. Due to a frameshift in the GUS vector (explained in further detail in Chapter 4), it was not possible to obtain the truncation results. Yet, the bioinformatic data and truncations design remain viable for future experiments. In Chapter 4, I address my second objective by generating transgenic hybrid poplar trees and Arabidopsis, which were transformed with a GUS vector for promoter analysis of PtSUS1p. My main objective was to observe the GUS protein localization between xylem and phloem tissues of Arabidopsis and poplar. More specifically, I was interested in testing whether gene expression of GUS would be directed to the xylem of either plant when driven under the poplar promoter of PtSUS1. I set out to do this by creating transgenic lines and subsequently subjecting them to staining and imaging. Unfortunately, due to an intron sequencing error, a frameshift in the protein sequence exists, which created an early stop codon and prevented any GUS activity. For this reason, my results are limited to expression data obtained from quantitative real-time PCR (qRT-PCR) analysis on the hybrid poplar GUS lines grown in the greenhouse. Results show that PtSUS1 promoter is more active in the developing xylem than in the phloem tissue. Although no protein visualization was achieved, the expression analysis results are encouraging, as they show that PtSUS1p appears to have xylem-rich expression, and therefore has potential for tissue-specific gene expression. 14Chapter 2: Materials and Methods 2.1 Plant material and growth conditions 2.1.1 Hybrid poplar Hybrid poplar, P. alba x P. grandidentata: P39, were grown and maintained in vitro in Magenta boxes containing Woody Plant Media (WPM) growth medium. The cultures were grown at 23C under a long-day light cycle (16 hours light, 8 hours dark). The plants were subcultured using aseptic methods every 6 weeks, unless otherwise specified. 2.1.2 Arabidopsis thaliana Arabidopsis thaliana ecotype Columbia – 0 (Col-0) wild-type seeds were obtained from Dr. Lisanne de Vries. The seeds were surface sterilized via a 5-minute 70% ethanol wash/incubation, followed by a 15-minute 10% bleach wash/incubation step. The seeds were then washed 4 times with autoclaved distilled water, and finally resuspended in 0.1% Agar solution and left at 4C in darkness for 48 hours. The seeds were then germinated under constant light on plates containing half-strength Murashige & Skoog (MS) medium. Approximately 10 days post germination, seedlings were transferred to potting soil and moved to a growth chamber programmed for a long-day light cycle (16 hours light, 8 hours dark) at 21C. For the first week in soil, seedlings were kept under a clear plastic cover to ease transition. Then, covers were removed, and the plants were left to grow for another 3 weeks or until flowering. At this stage, plants designated for transformation were transformed using the floral dip method (Zhang et al. 2006). Mature plants from which flowers had been replaced by fully formed siliques, were left to dry inside the growth chamber for approximately 1 month. Seeds were then collected for screening and propagation. 2.2 Genomic DNA extraction (P. trichocarpa) Approximately 50-200 mg of fresh leaf tissue was ground in liquid nitrogen and placed in a microcentrifuge tube. 1.0mL of CTAB buffer (2% (w/v) CTAB [Sigma], 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, 1% PVP, 2- mercaptoethanol, pH 8.0) was immediately added to the tube and the samples were incubated at 65C for 30 minutes. After incubation, the samples were centrifuged at 13,000rpm for 10 minutes. The supernatant was then transferred to a new tube and 1 volume of chilled phenol:chloroform:isoamyl alcohol (IAA) was added and mixed 15gently by inversion. The samples were centrifuged at 13,000rpm for 10 minutes and the clear supernatant was collected, transferred to a new tube and half a volume of isopropanol was added and gently mixed by inversion to precipitate the DNA by centrifugation (13,000rpm for 10 minutes). The supernatant was discarded and the pellet was washed with 500uL of 70% ethanol, and then air-dried for approximately 30 minutes. Finally, the pellet was resuspended in 30uL of nanopure water and stored at -20C for later use. 2.3 Promoter amplification and isolation 2.3.1 Primer design Poplar promoter sequences were obtained from the Phytozome database (Joint Genome Institute, USA) of the organism Populus trichocarpa v3.0. The promoter sequences were specified as the 2000bp upstream from the TSS. Primers were designed using Primer3 software (Koressaar et al., 2018) and SnapGene software (from Insightful Science; available at snapgene.com). 2.3.2 PCR reaction and gel isolation of PtSUS1 promoter The native poplar promoter SuSy1 (Potri.018G063500) was amplified from extracted genomic Poplar DNA via PCR. The reaction was performed using 0.2µL High-Fidelity Phusion Polymerase (Thermo Fisher Scientific, USA) per 20µL reaction, as well as 0.8µL of forward and reverse sequence-specific primers (Appendix table A.1). The promoter was successfully amplified under the following thermocycler conditions: initial denaturation of 95C for 3 minutes, followed by 35 cycles of 95C for 30 seconds, 56C for 2 minutes, and 72C for 30 seconds, and a final extension time of 72C for 7 minutes. The amplified fragment was then isolated from a 1% Agarose Gel using Qiagen’s QG Buffer (QIAGEN) and DNA Isolation from Gel protocol, eluted in 30 µL of nanopure water and sent for sequencing. 2.4 Generation of constructs 2.4.1 GUS construct The sequence of the SuSy1 promoter was amplified from poplar genomic DNA employing High-Fidelity Phusion Polymerase (Thermo Fisher Scientific, USA) via PCR using the primers listed in the Appendix (Appendix table A.1). The SuSy1 promoter sequence was first cloned into pDONR221, and subsequently transferred to the GUS vector pKGWFS7 using Gateway™ technology (Invitrogen, USA). These plasmids were transformed into chemically competent 16OneShot TOP10 E. coli cells and sequenced. This construct was transformed into 6-week-old P39 hybrid poplar trees and wildtype Arabidopsis Col-0 plants using Agrobacterium tumefaciens (strain GV3103). 2.5 Preparation and transformation of competent cells 2.5.1 Escherichia coli Chemically competent One Shot™ TOP10 E. coli cells were obtained from Thermo Fisher Scientific (USA) and stored at -80C. The cells, already aliquoted at 50µL per tube, were briefly left to thaw on ice for 1 minute before adding 1µL of the Gateway™ BP or LR reaction, and then placed on ice for another 15 minutes. The cells were then heat-shocked at 42C for 90 seconds, and immediately placed back on ice for 3 minutes. Then, 450µL of Super Optimal broth with Catabolite repression (SOC) medium was added to the tube, and the cells were left to incubate at 37C for 1 hour with shaking at 200rpm. After incubation, 100uL of the culture was plated on solid Lysogeny Broth (LB) medium containing the corresponding antibiotic for selection (either 50µg/mL of Kanamycin or 50µg/mL of Spectinomycin) and left to incubate at 37C overnight. The following day, any colonies present were used to inoculate 5mL liquid LB cultures with selective antibiotics, which were also incubated overnight at 37C before performing a plasmid prep to screen the colonies and assess via a colony PCR if the transformation was successful before proceeding with further transformations. 2.5.2 Agrobacterium tumefaciens Starter cultures of A. tumefaciens EHA105 and GV3103 were grown in a 5mL liquid YEP culture containing 25 µg/mL Rifampicin and incubated in an orbital shaker set at 160rpm and 28C overnight. The next day, a 50mL culture of the same medium was inoculated using the 5mL liquid culture grown the night before and set to incubate in the orbital shaker at 200rpm and 28C until an OD600=0.5 to 1.0 was obtained. Cells were then transferred to a 50mL falcon tube, chilled on ice for 15 minutes, and then pelleted at 10,000g for 10 minutes at 4C. The supernatant was then discarded, and cells were resuspended in 5mL of chilled 20mM CaCl2, followed by subsequent centrifugation and resuspension in 1mL of ice-cold 20mM CaCl2. Cells were then distributed into 100µL aliquots into pre-chilled snap cap tubes and flash frozen in liquid nitrogen before being stored at -80C. Vectors were transformed into A. tumefaciens using the freeze-thaw method. Competent cell aliquots were taken from the -80C freezer and placed on ice, where 1 µg of 17plasmid DNA was immediately added to cells before thawing. Cells were then flash frozen by submersion into liquid nitrogen for 5 minutes, and subsequently thawed at room temperature for 10 minutes. Cells were then transferred to a 15mL falcon tube containing 2mL of liquid YEP medium with no antibiotics and incubated in an orbital shaker at 200rpm and 28C for 2-4 hours. Following this incubation period, cells were pelleted and resuspended in 1mL of liquid YEP medium and spread in aliquots (100µL and 200µL) onto solid YEP medium plates containing antibiotics appropriate for plasmid selection (50µg/mL of Rifampicin and either 50µg/mL of Kanamycin or 50µg/mL of Spectinomycin). Plates were incubated at 28C for 2-3 days to allow Agrobacterium colonies to form. Verification of transformation was performed via a colony PCR using gene-specific primers and sent for sequencing before proceeding with plant transformations. 2.6 Agrobacterium-mediated plant transformations 2.6.1 Hybrid poplar Transformation of hybrid poplar P39 was performed as described by Wilkerson et al. (2014). Transformed Agrobacterium from glycerol stock were grown in 5mL liquid YEP medium with 25µg/mL of Rifampicin and either 50µg/mL of Kanamycin or 50µg/mL of Spectinomycin at 28C and 200rpm for 48 hours. These starter cultures were then used to inoculate a 200mL culture of WPM media with 100µM acetosyringone. Cultures were left to grow overnight at 28C and 200rpm until they reached an OD600= 0.1-0.2. Approximately, 500 Leaf disks were harvested from six-week-old P39 leaf tissue and co-cultivated with transformed Agrobacterium tumefaciens (GV3103) containing the plant expression vector. Around 50 leaf disks were co-cultivated with 20mL aliquots of Agrobacterium per 50mL falcon tube, for a period of 30 minutes at 28C and 100rpm with the tubes placed horizontally in a gyratory shaker. Following co-cultivation, leaf disks were removed from the media and blot dried on sterile filter paper. Disks were carefully placed abaxial side up onto solid WPM plates containing 0.1µM 1-Naphthaleneacetic acid (NAA), 0.1µM 6-benzylaminopurine (BA), and 0.1µM thidiazuron (TDZ), and placed in dark growth conditions for two days to recover. On the third day, leaf disks were transferred onto solid WPM plates containing 0.1µM NAA, BA, and TDZ, as well as 500mg/L carbenicillin and 250mg/L cefotaxime to eliminate remaining Agrobacterium. Plates were once again incubated in the dark for 2 days and subsequently transferred to selective WPM media plates containing 0.1µM NAA, BA, and TDZ, as well as 500mg/L carbenicillin, 250mg/L cefotaxime and the appropriate antibiotic for transformant selection 18(25mg/L Kanamycin). Plates were incubated for 4 weeks under reduced light conditions to facilitate the development of shoots, roots, and/or callus. Leaf disks presenting any of these forms of growth were transferred to Magenta™ boxes with WPM-agar containing 0.01µM BA, 500mg/L carbenicillin, 250mg/L cefotaxime and the selective antibiotic (25mg/L Kanamycin) to encourage the putative transformants to grow shoots over a 4-week period under full light conditions. A single shoot from each successful disk was then transferred to Magenta™ boxes with WPM-agar containing 0.01µM NAA, 500mg/L carbenicillin, 250mg/L cefotaxime and 50mg/L Kanamycin to enable the development of roots. Two weeks after, any shoots displaying root growth were screened via a DNA extraction from leaf tissue and a PCR reaction to confirm the presence of the gene of interest (either GUS or GFP) as well as the presence of the promoter of interest. Confirmed transformants with well-established shoots and roots were excised and transferred to new Magenta™ boxes with antibiotic-free WPM-agar and 0.01µM NAA. A DNA screen was performed before moving the plants to the greenhouse to confirm the transformation. 2.6.2 Arabidopsis thaliana Transformation and growth of transgenic Arabidopsis thaliana was performed as described by Zhang et al. (2006). Starter cultures were prepared by inoculating 5ml of liquid YEP medium supplemented with 25µg/mL of Rifampicin and 50µg/mL of Spectinomycin with transformed A. tumefaciens. Cultures were grown at 28C and 200rpm for 2 days. On the third day, a 200mL liquid culture of YEP medium supplemented with 50µg/mL of Rifampicin and 50µg/mL of Spectinomycin was inoculated using 1-2mL of the starter culture and set to incubate 28C and 160rpm overnight. Once the cell cultures reached the stationary phase at OD600 = 1.5-2, cells were centrifuged at 4,000g for 1 hour at room temperature and then resuspended in 500mL of freshly made 5% sucrose solution. Sylgard 309 (Dow Corning, USA) was added to the cell mix to a final concentration of 0.02% and mixed well before the dipping. Inflorescences were submerged into the Agrobacterium cell suspension for approximately 30 seconds with gentle swirling. Once removed, excess solution was removed by allowing the excess liquid to drip off the plant for 3-5 seconds, and then the plants were placed horizontally inside an opaque plastic bag to maintain a humid environment for 24 hours. After this recovery period, plants were removed from the plastic bag and returned to a vertical position inside the growth chamber. These plants were dipped a second time, following the same protocol, 7 days after the first floral dip. Plants were then left to grow normally for 1 month, after which if siliques had fully 19formed, watering was withheld to allow to plants to senesce. These T1 seeds were then collected, sterilized, stratified, and plated onto solid half-strength MS media containing 50mg/L Kanamycin for the selection of successful transformants, which were then potted onto autoclaved soil and allowed to grow for 2-3 weeks before collecting tissue for a DNA extraction to confirm the transformation. 2.7 Preliminary DNA screening Approximately 50-200mg of fresh leaf tissue was placed into an individually labelled microcentrifuge tube and immediately ground with a sterile micro-pestle and suspended in 400µL of QD extraction buffer (200mM Tris HCl, 250mM NaCl, 25mM NaEDTA, 0.5% SDS). Samples were then centrifuged at 13,000g for 3 minutes. Carefully avoiding the pellet, 300µL of supernatant was transferred to a new microcentrifuge tube, followed by 300µL of isopropanol. After inverting the tubes 5 times and allowing for a 10 minutes incubation at room temperature, the samples were again pelleted at 13,000g for 5 minutes and the supernatant discarded. Then, 500µL of 70% ethanol was used to wash the remaining pellet by vortexing each sample for 3 seconds before placing the tubes in a centrifuge at 13,000g for 2 minutes, then discarding the supernatant. Tubes were left upside down for 40 minutes to 1 hour to allow all the remaining ethanol to evaporate and dry under ambient conditions. DNA was then resuspended in 100µL of nanopure water and stored in -20C until needed. 2.8 Microscopy 2.8.1 Histochemical GUS staining Fresh stem and leaf tissue was collected from both Arabidopsis and poplar transformants and placed in a microcentrifuge tube. Tissue was then fixed by immersing it in 90% cold acetone and left on ice for 20 minutes. Acetone was then removed and replaced with rinsing solution (0.1M NaPO4 [pH 7.0], 0.5mM ferricyanide, 0.5mM ferrocyanide). After 2 minutes, rinsing solution was removed from the tube and tissue was then immersed in 1mL of GUS staining solution (0.1M NaPO4 [pH 7.0], 0.1% Triton X-100 (w/v), 1mM ferricyanide, 1mM ferrocyanide, 1mM X-Gluc, 10mM EDTA [pH 8.0]). Samples were then vacuum infiltrated for 5-10 minutes, covered in aluminum foil, and left to incubate overnight at 37C. The next day, the staining solution was removed and 1mL of FAA fixative (50mL ethanol, 5mL acetic acid, 10mL of 37% formaldehyde, 35mL water) was added to the samples and left to incubate 20for 30 minutes. Afterwards, the remaining chlorophyll was removed from the tissue by immersing it in 95% ethanol for 2-6 hours or until chlorophyll was no longer evident. 2.8.2 Sectioning and visualization Stem tissue was sectioned using the Leica VT1000S Vibratome. Stem cross sections were made by placing the piece of stem tissue into 3% low melting agarose gel and cutting it out as a block once solidified. This agarose block was then super-glued in a vertical position onto the vibratome stage. The vibratome was set at speed 8 (1mm/s) and frequency 7 (70Hz) for poplar stems and a speed of 9 (1.75 mm/s) and frequency 8 (80Hz) for Arabidopsis stems, to make 50µm cross sections. Thicker tissues, such as mature stems and roots harvested from the greenhouse lines, were sectioned using a manual Spencer AO860 sledge microtome (Spencer Lens Co., NY, USA), which was set to produce 60µm cross sections. Cross sections were then carefully mounted onto microscope glass slides using mounting media (50% Glycerol in 1X PBS) and then visualized using the Olympus BX53 microscope. 2.9 Expression analysis 2.9.1 Poplar greenhouse trial and harvest From the DNA analysis, six transgenic poplar lines were selected and sent to the South Campus Greenhouse alongside two wild-type controls. The plants were potted into 1-gallon pots filled with Potting mix Soil and irrigated by hand for 1 month. Due to inconsistencies in growth, the potted lines were transferred to the Main Campus Greenhouse at approximately the 6-week mark. Manual watering was switched to table-flooding. The trees were allowed to grow for 3 more months in these conditions before harvesting. Tissues for GUS assays were placed in 90% acetone, rinsed, and stored in distilled water and kept at 4 C until processed. The tissues collected for this purpose were source leaf, sink leaf, main root, top 5cm of the stem, and bottom 7cm of the stem. The tissues harvested for RNA extractions were the developing xylem, phloem, and main root; these were collected in aluminum foil pouches and placed directly in liquid nitrogen to prevent degradation and stored at -80C until needed. 212.9.2 RNA extraction Tissues collected for RNA extractions were kept frozen at all times by being immersed in liquid nitrogen and then ground using the GenoGrinder 2010 (SPEX SamplePrep, NJ, USA) set at 1500 rpm for 30 seconds. Xylem and phloem tissues were ground to a homogenous powder in 1 cycle, while root tissue required 6 applications to obtain a similar consistency. Ground tissues were placed in their respective pre-chilled screwcap tubes and stored at -80C until needed. For RNA extraction, 100mg of ground tissue was aliquoted into 2mL safe-cap RNAse-free tubes and resuspended in 0.75mL of Working Extraction Buffer (1M Tris-HCl [pH8.5], Lauryl Sulfate Lithium Salt, 8M LiCl, 0.5M Disodium Salt EDTA, Deoxycholic Sodium Salt, Tergitol NP-40(w/v), 1mM Aurintricarboxylic acid, 10mM dithiothreitol, 55mM Thiourea, 2% PVPP (w/v)) and vortexed. Samples were kept frozen in liquid nitrogen until all samples were processed. Samples were then placed at 37C with shaking until thawed, followed by centrifuging at 11,000g for 15 min at 4C, and transferring the supernatant to a new 2mL safe-cap RNAse-free tube. Due to poplar tissues containing significant levels of inherent polysaccharides, 25µL of 3.3M sodium acetate and 75µL of 100% ethanol were added to the tube, incubated on ice for 10min and centrifuged under the previous conditions. The supernatant was then transferred to a new 2mL tube where 85µL of 3.3M sodium acetate and 850µL of chilled isopropanol were added and mixed by inverting the tubes. The samples were kept frozen overnight at -80C. The following day, samples were thawed at room temperature and then centrifuged at 8,000rpm for 30 minutes at 4C. Supernatant was discarded, and pellet was resuspended over a period of 30 minutes with incubation on ice using 400µL of TE buffer (10mM Tris [pH8.0], 1mM EDTA [pH8.0]) and 400µL of 5M NaCl. Then, 200µL of 10% CTAB was added to the samples, mixed by vortexing, and set to incubate at 65C for 5 minutes. Samples were then extracted with 500µL of Chloroform:IAA 24:1, mixed for 20 seconds by vortexing, followed by centrifugation at 5,000g for 6 minutes at 4C. The aqueous phase was then transferred to a new tube and extracted once more following the same guidelines. Then, 150µL of 8M LiCl was added to the samples, mixed by vortexing, and then incubated at -30C overnight. The following day, samples were briefly thawed at room temperature before being centrifuged at 14,000g for 30 minutes at 4C. Supernatant was then discarded, and the pellet was carefully washed with 70% ethanol. Samples were then centrifuged at 14,000g for 10 minutes at 4C, after which the ethanol was discarded, and the pellet was set to dry for 10 minutes. Finally, pellet was carefully resuspended in 15µL of DEPC water and kept on ice for 30 minutes with periodic mixing until completely dissolved. 22 2.9.3 cDNA synthesis Contaminating DNA was removed from RNA samples using TURBO™ DNAse and following the manufacturer’s instructions (Thermo Fisher Scientific, USA). RNA purity and concentration was determined spectrophotometrically using a Nanodrop 1000 (Thermo Fisher Scientific, USA). RNA samples were then diluted to a concentration of 1µg using autoclaved nanopure water to be used as template for cDNA synthesis. Reactions were assembled in an RNAse free environment, and the following components were added to chilled RNAse free tubes: 4µL RT buffer, 1µL dNTPs, 1µL random primers, 1µg of cDNA, 1µL of OneScript Plus RTase (Applied Biological Materials, Richmond, BC), and nuclease free water up to a final volume of 20µL. Samples were then incubated at 55C for 50 minutes, and then stored at -80C. 2.9.4 qRT-PCR analysis Quantitative real-time PCR (qRT-PCR) was used to determine the relative expression levels of GUS gene in xylem and phloem tissues harvested from the poplar lines grown in the greenhouse. cDNA from the corresponding tissues was synthesized from RNA as detailed above. cDNA was then diluted 5x to a concentration of 200ng using nanopure water prior to setting up the individual reactions. Each reaction consisted of 10µL of BlasTaq 2X qPCR MasterMix (Applied Biological Materials, Richmond, BC), 0.5µL of qPCR forward and reverse primers (Appendix table A.2) at a concentration of 10µM, 1µL of diluted cDNA and nanopure water to a final volume of 20µL. All reactions were performed in triplicate. Reactions were run in a Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, USA) using the following cycling parameters: 1 cycle of 3 minutes at 95C, 40 cycles of 95C for 15 seconds, and 60C for 1 minute, followed by a melt curve cycle of 55C to 95C at 5C increments for 5 seconds to ensure amplification of 1 product. Target Ct values were normalized to the average expression of the housekeeping gene PtrEF1B, and in one case, values were normalized to the average expression of housekeeping gene PtrUBQ11. Relative fold expression levels were calculated through the double-delta Ct analysis (△△Ct), where △△Ct= △Cttarget sample - △Ctreference sample (Livak and Schmittgen 2001). 232.10 Bioinformatic analysis 2.10.1 Sequence analysis and motif identification Poplar promoter sequences were obtained from the Phytozome database (Joint Genome Institute, USA) under organism Populus trichocarpa v.3.0, and Arabidopsis promoter sequences were obtained from the TAIR database (Phoenix Bioinformatics Corporation, USA). Promoter sequences were specified as approximately 2000bp upstream from the translational codon, unless specified otherwise by the database resources. These sequences were imported to SnapGene (from Insightful Science; available at snapgene.com) for further analysis and labelling. Motifs were identified via two methods. First, promoter sequences were exported to the FIMO tool from The MEME suite (Grant, Bailey, & Stafford Noble, 2011) and compared against the six motif sequences of interest. Matches were annotated in the corresponding Snapgene file. Afterwards, the motif sequences were added to Snapgene to find any other possible matches within the sequence that might have been looked over by the MEME software and were highlighted and labelled accordingly. 2.10.3 Promoter truncations Three truncations were designed based on PtSUS1 promoter sequence and bioinformatic analysis using Snapgene (from Insightful Science; available at snapgene.com). Truncations were designed so that the promoter sequence would be truncated in a gradual manner from the 5’ end towards the 3’ end. The PCR reactions to amplify each of the designed truncations were performed using 0.2µL High-Fidelity Phusion Polymerase (Thermo Fisher Scientific, USA) per 20µL reaction, as well as 0.8µL of reverse primer #26 and the corresponding truncation-designed forward primer (Appendix table A.1). Each truncation was successfully amplified under the following thermocycler conditions: initial denaturation of 98C for 2 minutes, followed by 35 cycles of 98C for 20 seconds, 64C for 30 seconds, and 72C for 3 minutes, and a final extension time of 72C for 7 minutes. The amplified fragment was then isolated from a 1% Agarose Gel using Qiagen’s QG Buffer (QIAGEN) and DNA Isolation from Gel protocol, eluted in 30 µL of nanopure water and sent for sequencing. The isolated fragments were then used as templates in individual PCR reactions to add attB sites for cloning. The reactions were performed using 0.2µL High-Fidelity Phusion Polymerase (Thermo Fisher Scientific, USA) per 20µL reaction, as well as 0.8µL of the attB forward primers #132-134 and attB reverse primer #39 (Appendix table A.1). The thermocycler 24conditions were the following: initial denaturation of 98C for 1 minute and 30 seconds, followed by 35 cycles of 98C for 30 seconds, 50C for 30 seconds, and 65C for 4 minutes and 30 seconds, and a final extension time of 65C for 10 minutes. The incorporation of attB sites to the longest truncated fragment was not successful, however the other two truncated versions of the promoter were successfully cloned into pDONR221, and subsequently transferred to the GUS vector pKGWFS7 using Gateway™ technology (Invitrogen, USA). These plasmids were transformed into chemically competent OneShot TOP10 E. coli cells and sequenced. These constructs were transformed into wildtype Arabidopsis Col-0 plants using Agrobacterium tumefaciens (strain GV3103). 25Chapter 3: Bioinformatic analysis of xylem and secondary cell wall motifs identified in select promoters 3.1 Introduction Motifs are defined as small nucleotide sequences, usually ranging from 5 to 20 nucleotides long, that specify protein recruitment and binding (Hudson and Quail 2003; Rombauts et al. 2003). This is especially important in gene expression, as motifs are the essential functional components of promoters, necessary for the recruitment of transcription factors and other regulatory machinery (Hudson and Quail 2003). Research continues to direct efforts on developing techniques to identify motifs within promoter sequences and determine transcription factor specificity. Experimental techniques, such as CHIP-seq and protein binding microarrays have been successful at identifying transcription factor binding sites (TFBS), but have molecular limitations due to promoter complexity and are often very expensive (Kidder et al. 2011; Peng et al. 2016). In vitro mapping techniques offer a more inexpensive and scalable alternative, however, most of these methods employ synthetic DNA that lacks genomic properties known to impact TF binding (O’Malley et al. 2016). At present, computational analyses of promoter sequences seem to be the preferred option as motif and TFBS databases are constantly updated and refined. There are several bioinformatic software packages publicly available, such as MDscan, MEME, Bioinspector, and SCOPE, among others (Pandiarajan and Grover 2018). Most recently, ReMap 2020 offers the largest catalog of high-quality regulatory regions, however it is currently limited to the human and Arabidopsis genomes (Chèneby et al. 2020). Previously, TFBS and motifs could only be detected by databases when their sequences were previously known (Hudson and Quail 2003; Rombauts et al. 2003; Peng et al. 2016). In contrast to conventional motif matching approaches, DNA affinity purification sequencing (DAP-seq) is a low cost in vitro TF-DNA binding assay that can rapidly generate genome-wide binding site maps that specify binding locations and sequence motifs using genomic DNA (O’Malley et al. 2016). However, motif detection in promoters is not a straightforward process, as shorter sequences are statistically more likely to occur at random every few hundred base pairs, thus creating the additional challenge of discriminating “true” from “false” regulatory elements (Rombauts et al. 2003), often resulting in programs predicting several false positives (Rombauts et al. 2003; Peng et al. 2016). Finally, models used to detect potential motif matches are usually optimized for yeast or mammalian genomes, and require position weight matrices or consensus sequences which 26are not available for all organisms and thus limit the options for plant researchers (Hudson and Quail 2003; Abeel et al. 2008). 3.2 Selection of motifs of interest Previous studies show that SuSy 1 and SuSy 2 seem to be preferentially expressed in the mature xylem of the poplar family (Zhang et al. 2011), whereas in Arabidopsis there is no evidence of any SuSy isoforms present in the xylem tissue (Yao et al. 2020). In addition, there is evidence to suggest that SuSy is strongly associated to cellulose production in plants, as its catalytic reaction yields UDP-glucose, a precursor of cellulose biosynthesis, as one of its main products (Amor et al. 1995; Joshi et al. 2004; Koch 2004). Using this information, a total of six motifs were selected for this study from the available literature based on their putative roles in xylem development or cell wall formation (Appendix table B.1). A group of R2R3-type MYB TFs, comprising functionally redundant proteins MYB46 and MYB83, have been shown to activate transcription of secondary cell wall (SCW) biosynthesis genes, including lignin biosynthesis enzymes and SCW carbohydrate-active enzymes (CAZymes) in higher plants (Winzell et al. 2010). Plants have over 1000 CAZyme-encoding genes in their genome, and the ensuing enzymes have become subject of much research due to their involvement in the synthesis of plant cell walls, which produce two of the most abundant natural polymers on earth, cellulose and lignin. Moreover, understanding the complex pathways of cell wall biosynthesis eventually could aid in the development of tools to address cell wall recalcitrance (Cao et al. 2015). Additionally, CAZymes are essential to plants’ carbohydrate metabolism as they convert carbon in sugars into plethora of carbohydrates (Cao et al. 2015). Four CA-rich motifs from this category, labelled as CAm1- CAm4, were over-represented in xylem-specific SCW genes in Populus and Arabidopsis (Winzell et al. 2010). For this reason, motifs CAm1- CAm4 were selected as motifs of interest in my analysis. The NAC protein family is a very large and diverse group of plant transcription factors involved in plant senescence, SCW deposition, and stress responses. A core consensus DNA-binding sequence was identified for a small group of stress-induced NAC transcription factors with different binding affinities (Lindemose et al. 2014). This motif sequence has been identified to also be a core binding site for NAC transcription factors involved in SCW biosynthesis (Lindemose et al. 2014), and thus was selected for my analysis. 27Another important category of interest is the Vascular-related NAC Domain (VND) proteins VND3, VND7, and SND1 due to their vital role in xylem vessel formation and differentiation. More specifically, I was interested in the motif sequences that could bind to these transcription factors. There are two remarkably similar motif sequences known as the TERE (tracheary element-regulating cis-element) and SNBE-like (secondary-wall NAC binding element) motifs which have been identified as binding sites of NST (NAC secondary wall thickening factor) and VND NAC proteins (Negi et al. 2018; Olins et al. 2018; Tamura et al. 2019), therefore providing justification for inclusion as interesting targets in my analysis. In addition to these, it has been shown that VND7 is closely related to SCW deposition by interacting and activating several SCW-related target genes (Tamura et al. 2019), and therefore its motif sequence was also added to my analysis. Additionally, NAC Domain transcription factor SND1, also known as NAC Secondary wall Thickening promoting factor 3 (NST3) is involved in SCW biosynthesis in interfascicular and xylary fibers, and serves as a key differentiation regulator of Arabidopsis fiber cells (Ohtani et al. 2011; Takata et al. 2019; Tamura et al. 2019). Additionally, P. trichocarpa has 16 VNS genes, which can be subdivided into VND- and NST-type genes. These are expressed in developing xylem and have been found to play a role in woody cell differentiation in poplar (Akiyoshi et al. 2021). For these reasons, the motif sequence for SND1 was included in my analysis. 3.3 Scope and limitations The distal and proximal regions of promoters hold scientific interest largely because they contain several regulatory elements that can become accessible at different times and in different tissues, depending on the three-dimensional configuration of the promoter. (Rombauts et al. 2003; Cairns 2009). Pioneer TFs are capable of binding to nucleosomal DNA and recruiting histone modifying enzymes and chromatin remodeling complexes that will open the chromatin structure, allowing TFs to bind to accessible binding sites in the DNA and fully activating CRMs (Schmitz et al. 2022). For the purposes of this project, 3D configuration was not considered as a factor in spatiotemporal gene regulation, and motifs were identified solely on the promoter sequences obtained from available databases. Although this type of bioinformatic study is informative to a degree, the sole presence of the motifs does not necessarily dictate that the TF is being recruited by the promoter motif at a given point in time. Additionally, there may be false positive matches for motifs of interest that were identified by the programs employed (Rombauts et al. 2003; Peng et al. 2016). It is also important to note 28that this analysis is limited to only the six chosen motifs and their known sequences, and, it is more than likely that other motifs and regulatory elements not identified in this study are also interacting and taking part in the spatiotemporal gene regulation process. The aim of this bioinformatic analysis was to compare the motif distribution in the selected promoters and to assess whether the few motifs identified in the proximal and distal promoter regions have a significant influence on spatiotemporal gene expression. Furthermore, this analysis intended to find motif events, whether individual or combined, that could potentially explain the difference in the SuSy gene expression in the vascular tissues of Arabidopsis and hybrid poplar. 3.4 Results 3.4.1 Analysis of the poplar SuSy1 and SuSy2 promoters Analysis of the motifs identified in the two primary xylem-localized poplar SuSy promoters and their distribution along the promoter sequences reveal similarities that could potentially be responsible for gene expression to the xylem (Figure 3.1). At the distal end of both sequences, two instances of the SNBE-like motif were identified in close proximity to one another. These motifs are located 123bp away from each other in the PtSUS1 promoter and only 23bp away in the PtSUS2 promoter. Similarly, there were two SND1 motifs near one another in the proximal region of both SuSy promoters. Also, both promoters contain one TERE motif, yet, in PtSUS1 it is present in the proximal region, while in PtSUS2 it is in the distal region. Other motifs were identified several times throughout the sequence, such as the CAm 2-4 motifs and the stress-inducible NAC TF motif. However, these do not seem to present themselves in a specific pattern. Additionally, the VND7 motif was only identified in the PtSUS1 promoter, and there were no close matches to the motif sequence in the PtSUS2 promoter. 29 Figure 3.1: Motif comparison of PtSUS1 and PtSUS2 promoters. Scale bar as shown. 303.4.2 Analysis of the Arabidopsis SuSy1 and SuSy4 promoters Motif analysis of the SUS1 and SUS4 promoters in A. thaliana offered a few differences to the motifs found in the SUS1 promoter of P. trichocarpa (Figure 3.2). As previously mentioned, the PtSUS1 promoter has two occurrences of the SNBE-like motif in the distal region, close to the 5’ end, whereas the Arabidopsis homologs lack this pairing. Only one SNBE-like motif was identified in the promoter sequence of AtSUS1, and AtSUS4 does not have an SNBE-like motif at all. The AtSUS4 promoter also lacks the TERE and VND7 motifs, while its ortholog contains two of each of these. One similarity between the Arabidopsis and poplar promoters is the presence of two SND1 motifs: one located in the distal region and another copy closer to the 3’ end. In addition, there seem to be several inducible NAC TF motifs scattered throughout the sequences of these promoters, however, there is no clear pattern to their occurrence. 31 Figure 3.2: Motif comparison between AtSUS1, AtSUS4 and PtSUS1 promoters. Scale bars as shown. 323.4.3 Analysis of the poplar CesA4, CesA7 and CesA2 promoters The three poplar CesA promoters were specifically considered because they are homologs to the Arabidopsis SCW CesAs. Given that a strong association between the SuSy and the CesA proteins in cellulose biosynthesis has been suggested, motif comparison between these promoter sequences might provide more insight into the possible role of motifs in gene expression (Figure 3.3). Motif analysis of the three SCW CesA promoters showed that none of them matched the distal positioning of the pair of SNBE-like motifs identified in PtSUS1, however, the PtCesA2 promoter does contain two SNBE-like motifs, although they are located over 900bp away from each other and seem to be part of the proximal promoter region. PtCesA4 has one copy of the SNBE-like motif located close to the core promoter region, and PtCesA7 lacks this motif altogether. PtCesA7 promoter has three SND1 motifs, and their location is similar to that of the SND1 motifs in the PtSUS1 promoter: one copy in the distal region towards the 5’ end, and two copies relatively close to each other near the 3’ end. This pattern is not present in either PtCesA4 and PtCesA2, however, they have one and two SND1 motifs, respectively. In addition, both PtCesA2 and PtCesA7 promoters have a VND7 motif located approximately in the middle of their sequence, which is also the case for PtSUS1 promoter. PtCesA7 also has another VND7 motif towards the 5’ end, in the distal region. The PtCesA4 promoter does not contain any VND7 motifs, but it has two TERE motifs within 105bp of each other in the distal region of its sequence, and although PtCesA2 only has one TERE motif, it is located approximately in the same region of the promoter sequence as its paralog. The overall distribution of the inducible NAC TF motifs and the CAm 1-4 motifs does not show a clear pattern when comparing the promoters. 33 Figure 3.3: Motif comparison between P. trichocarpa’s CesA 4, 7, and 2 promoters and the SuSy1 promoter. Scale bars as shown. 343.5 Discussion 3.5.1 Core promoter Eukaryotic promoters play an essential role in gene expression due to their many functional components, such as motifs and cis-elements, but also due to the presence of the transcriptional machinery in their core regions (Benfey and Chua 1990; Abeel et al. 2008; Pandiarajan and Grover 2018). The core region is approximately 35-50bp in length and contains highly conserved elements that orchestrate the recruitment and positioning of transcriptional machinery, such as RNA Polymerase II, among other proteins, that form part of the transcriptional multiprotein complex (Goodrich, 2001). However, for this study, the core regions of the selected promoters were not of major interest as they do not contain motifs that could potentially impact spatiotemporal gene expression. 3.5.2 Proximal promoter The proximal promoter contains several TFBS, several of which are associated to RNA polymerase II, but in some cases it is also a site of motif clusters and other regulatory elements that mediate tissue-specific gene expression (Winzell et al. 2010). The promoter region is adjacent to the core promoter and expands for a few hundred base pairs, however, it is difficult to determine where the proximal region ends and the distal region begins (Rombauts et al. 2003). For the purposes of this study, I will refer to the proximal promoter as the 700 bp adjacent to the core region (50bp from TSS). When comparing the proximal region of poplar SuSy1 and SuSy2 promoters, it is apparent that there is conservation of a few motifs in this region. Both sequences have two copies of the SND1 motif, located 204bp away from each other in the PtSUS1 promoter and 194bp away from each other in the PtSUS2 promoter. This similarity could be an indication that this pair of motifs are conserved sequences among the SuSy multigene family, however, other promoters from this family would have to be analyzed before reaching such a conclusion. SND1, also known as NAC012 and NST3, is a motif that has been identified as being involved in SCW biosynthesis in interfascicular and xylary fibers in stems (Ohtani et al. 2011; Takata et al. 2019; Tamura et al. 2019). Due to this motif’s involvement in SCW thickening and essential role in fiber development (Eckert et al. 2019), it was anticipated that a similar pattern would be apparent in the PtCesA promoters. However, of the three CesA promoters analyzed, only PtCesA7 contained two SND1 motifs in its proximal promoter region, similar to the pattern found in the two SuSy promoters. PtCesA2 also had two occurrences of the SND1 motif, however, one of these is found further upstream in the distal region at a much larger 35distance from the first SND1 event compared to the distances seen in poplar CesA7, SUS1, and SUS2 promoters. It could be that this double-SND1 event, present in both poplar SuSy promoters and not in the Arabidopsis homologs, could be affecting the spatiotemporal localization of the SuSy proteins and driving expression to the xylary fibers in the xylem. Thus, this motif pattern could be a potential candidate region for further observation. Even though the double-SND1 pattern was not present in all the proximal regions of the PtCesA promoters, other NAC and NST TFs have been found to be redundant to SND1, and were not necessarily identified in this analysis (Eckert et al. 2019). For instance, we see at least one instance of the stress inducible-NAC motif across all seven promoters in their proximal regions. Although it would be hard to determine the level of conservation or influence of this motif across all promoters from this analysis, this stress-inducible motif has been previously linked to SCW deposition (Lindemose et al. 2014), and therefore could be acting redundantly to the double-SND1 motif events in the xylary fibers in poplar, but also across other tissues where SND1 is not active and SCW is still being deposited. However, because there is no clear pattern and no discernable differences between the poplar and Arabidopsis SuSy promoters with respect to this motif, it may not be considered a global candidate for spatiotemporal regulation. Additionally, one TERE motif was identified in the proximal region of the PtSUS1 and AtSUS1 promoters. Given the phylogenetic similarity between Arabidopsis and poplar SuSy genes, it would not be unexpected to find motifs, such as TERE, to be conserved between homologous promoters (Zhang et al. 2011). Then again, because this motif is found in both species, it is unlikely that the TERE motif in this particular case is the driver of distinct spatiotemporal localization in the vascular tissues. Finally, the proximal region of all these promoters, except for AtSUS4, seem to contain one or more CAm motifs. This is not unexpected, especially for poplar, because CAZymes are involved in highly recalcitrant plant cell wall synthesis and carbohydrate synthesis pathways (Cao et al. 2015). Several of these promoters have more CAm motifs spread throughout their distal region as well; yet these motif events seem to occur in no specific pattern across the promoters analyzed. Additionally, in the two SuSy promoters and PtCesA 7 and 2 promoters, CAm motifs seem to occur in tight clusters. For this reason, it would be difficult to accurately identify the role of the four individual CAm motif sequences detected in this study successfully. 363.5.3 Distal promoter The distal promoter region is located the farthest away from the TSS site, and can span for several thousands of base pairs (Rombauts et al. 2003; Abeel et al. 2008). Research has previously shown that several regulatory elements are contained in this region and that they directly affect spatiotemporal gene expression (Tjian and Maniatis 1994; Fessele et al. 2002; Rombauts et al. 2003). Additionally, it has been suggested that activated transcription in vivo requires the three promoter regions to be accessible by RNA Polymerase II transcriptional activators, and that distal enhancer elements can sometimes target nucleosome-remodeling factors in core promoters of other genes to be co-expressed (Nikolov and Burley 1997; Schmitz et al. 2022). For the purposes of this study, I have delineated the distal region of the promoter to be anything immediately upstream of the proximal promoter limit to approximately 2000bp site upstream of the TSS. It is possible that the promoter sequences in my analysis are shorter or longer than what I have specified as it is unpredictable how far the distal region extends (Rombauts et al. 2003). When comparing the distal regions of both SuSy promoters, there was a noticeable difference in the quantity of motif events present in each. PtSUS1 had a total of six motif events, and among these, no CAm or TERE motifs were not found. On the other hand, PtSUS2 has a total of 13 motif events, among which there is a clear cluster consisting of six or seven CAm and stress inducible NAC motifs. Although the quantity of motifs found in PtSUS2 were almost double that of PtSUS1, the VND7 and SND1 motifs are missing from PtSUS2’s distal promoter. For this reason, I did not consider any of these missing motifs as promising candidates responsible for the spatiotemporal localization of the SuSy proteins to the xylem in poplar. The motif event that was most attractive was the pair of SNBE-like motifs, located relatively close to one another towards the 5’ end of the distal region in both poplar SuSy promoters (Fig.3.1). In addition, when comparing the poplar SuSy promoters to their orthologs in Arabidopsis, the latter promoters do not have an analogous motif event. AtSUS1 contains one SNBE-like motif in its distal region, but a second copy was not found; while AtSUS4 did not contain any SNBE-like motif sequence (Fig.3.2). Moreover, the presence of these SNBE-like motifs in the distal region of the poplar SuSy promoters may direct spatiotemporal regulation of the SuSy proteins in xylem, as previous research supports that SNBE-like motifs have been previously linked to xylem-specific regulatory regions (Negi et al. 2018). SNBE-like motifs are common cis-elements associated with the regulation of genes related to the secondary wall, including VND, NST, and MYB genes (Negi et al. 2018). By this reasoning, 37it is plausible that the pair of SNBE-like motifs identified in the promoters’ distal region are binding to their corresponding TFs and regulating others such as SND1, VND7, and CAm 1-4, which motifs were also found in the promoter sequence. Furthermore, there might be other motif sequences, which were not chosen for this analysis, that contribute to TFs from these families binding to DNA and directing spatiotemporal regulation in the xylem. Nonetheless, due to its absence from the Arabidopsis SuSy promoters, its xylem-specific regulatory role for other TFs, and its distal location within the poplar SuSy promoters’ sequences, the double SNBE-like motif event was selected as a strong candidate for further observation. 3.6 Promoter truncations design To test the importance of some of the motifs identified in the bioinformatic analysis and their impact on gene expression and protein localization, promoter truncations were designed for the PtSUS1 promoter’s sequence. Targeting motifs individually, whether for complete removal or sequence modification, would have been a costly and lengthy challenge, so a broader truncation strategy was implemented. The objective behind this was to refine which region of the promoter, when absent, would impact gene expression in the vascular tissues of the plant. Notably, promoters present several challenges for molecular work due to their repetitive and highly AT-rich sequences, which can result in limitations when designing the truncation strategy in this study. Additionally, the motifs identified in my bioinformatic analysis are certainly not the only motifs present in the promoter, so unquestionably, other regulatory sites might have also been affected at the sites of truncation. Truncations were designed to gradually remove parts of the regulatory regions of the promoter in a 5’ to 3’ direction. The main reason to keep the 3’ end completely unaltered was to conserve the essential transcriptional machinery, like the TATA box, the initiator, and other required elements (Abeel et al. 2008; Xu et al. 2016; Pandiarajan and Grover 2018). Three truncations were specifically designed: one at the 5’ end of the distal region, targeting the double SNBE-like motif event; another truncation aiming to remove the remaining motifs in the distal region and leaving the few in the proximal region intact, and finally, a third truncation which would remove most of the regulatory regions, leaving only the core promoter (Fig. 3.4). I will refer to these truncations as the distal truncation (DPT), proximal truncation (PPT), and core truncation (CPT) respectively. 38 Figure 3.4: Truncation design for PtSUS1 promoter based on the bioinformatic analysis of select motifs. Scale bar as shown. 39 The DPT aimed to remove the pair of SNBE-like motifs present at the 5’end of the distal region of PtSUS1 promoter. As previously mentioned, this motif event is a strong candidate for spatiotemporal regulation due to its location in the poplar SuSy promoters, its absence from the Arabidopsis homologs, and its role in xylem-specific regulation. My hypothesis for this truncation was that gene expression would be affected to some level, and the GUS reporter signal would perhaps be visible in tissues other than the xylem. This hypothesis was based on the fact that most of the promoter would still be intact, and therefore most of the regulatory regions would likely remain unchanged. Thus, transcription and translation should still occur at similar levels compared to the complete promoter. If the localization of GUS, when driven under the promoter PtSUS1 – DPT, shows anything other than xylem-specific localization, it would be interesting to explore further the role of the double SNBE-like motifs through a more closely-targeted approach. The PPT aimed to remove some of the other motifs present in the distal region including four inducible NAC motifs, one SND1-binding motif, and the only VND7-binding motif identified in the promoter. This strategy would leave the truncated promoter with a few more than half of the original motifs identified in the PtSUS1 promoter. Visually, I would have expected the GUS signal to still be detected in the plant, but localization would be over wide range of tissues. This hypothesis was guided by the fact that most of the regulatory regions associated with spatiotemporal localization are generally found in the distal region, which in this truncation strategy would be removed, but all essential transcriptional machinery in the core promoter and the binding site for RNA Polymerase II in the proximal promoter would remain in place (Abeel et al. 2008; Pandiarajan and Grover 2018). Finally, the CPT was designed such that all identified motifs in the proximal and distal promoter regions were removed, leaving a 297bp promoter. This truncation would technically retain the core promoter and its transcriptional components, but would result in a promoter without most of the regulatory regions. It would be difficult to predict the functionality of the ensuing promoter. With most of the TFBS removed, the TFs required to bind and create the transcriptional complex would not be able to come together and regulate the transcription of the gene (Cairns 2009). Therefore, I hypothesized that plants transformed with the CPT would show little to no GUS staining. 403.7 Protein localization in truncated promoter transformants Although the techniques and strategies employed were appropriate for the imaging portion of this experiment, an unexpected sequencing error caused a frameshift mutation in the translated sequence. This resulted in a shortened GUS protein, which could not produce the signature blue staining required to observe protein localization under the microscope. This issue is discussed further in Chapter 4. 41Chapter 4: Investigating the role of PtSUS1 promoter in spatiotemporal expression 4.1 Introduction To date, most of the research focusing on SuSy genes has specifically examined their expression levels and protein localization, while little research has been conducted to better understand the role of their promoters in spatiotemporal localization. The difference in SuSy protein localization in Arabidopsis and poplar offered the perfect opportunity to analyze the divergences of their respective promoters, and test how the promoters were driving expression in different tissues. To address this question, I created a GUS construct and fused it with the PtSUS1 promoter to drive expression, and subsequently used it to generate transgenic hybrid poplar and A. thaliana plants. The purpose of this experiment was to visually confirm the localization of the GUS protein when driven under the same promoter in different species. Transgenic lines in both species were created at roughly the same time, via two different Agrobacterium-mediated transformation protocols to maximize efficiency in each species. In both cases, transgenic lines were confirmed via two methods: selective antibiotic growth and DNA screening. Select transgenic poplar lines were sent to the greenhouse to grow for a longer period of time to produce mature vascular tissues for harvesting, and to promote independent sucrose production in the plant instead of relying on the sucrose found inherently in tissue culture media. In contrast, transgenic Arabidopsis were grown in bulk and selected for until the T1 generation, at which point seeds were collected and kept in dry storage until used for growth and tissue harvest for GUS staining. Due to difficulties in GUS staining visualization, an expression analysis was conducted on the developing xylem, phloem, and root tissue collected from the greenhouse-grown poplar transgenic trees. Although expression work was not originally considered for this study, these tissues were flash-frozen at harvesting to preserve RNA. The goal behind the expression analysis was to have a better understanding, at a molecular level, of the issues preventing GUS visualization, and to obtain data that may indicate whether gene expression in the xylem differed from that in the phloem, when driven under the PtSUS1 promoter. 424.2 Expression analysis results To generate plants that would facilitate protein localization when observed under a microscope, a construct (PtSUS1p::GUS) was created via Gateway cloning and subsequently used for Agrobacterium-mediated plant transformations. Hybrid poplar P39 and Arabidopsis thaliana Col-0 were transformed with the GUS construct, grown, and harvested for staining. None of the transgenic lines produced any observable phenotype. Therefore, an expression analysis of the developing xylem, phloem, and root tissues was conducted to verify whether transcription of the GUS gene was occurring. The expression analysis had to be limited to the greenhouse-grown transgenic poplar lines, as trees provide ample individual tissues that could be harvested and stored for unique analyses. qRT-PCR was employed to investigate expression analysis of each tissue, for which four primer sets were used: one pair for a housekeeping gene (PtEF1B or PtUBQ11), one pair for the GFP gene present in the construct, and two pairs of GUS-specific primers to test whether the GUS transcript was being produced at the expected size (Appendix table A.2). GFP expression levels were negligible, thus confirming that the GFP gene present in the vector does not function (as suggested by Dr. Mathias Schuetz, personal communication). For this reason, all GFP-related values were not considered further in the data analysis. For the analysis, the double-delta method was used (Livak and Schmittgen 2001), and all expression values were normalized relative to the housekeeping gene employed. Samples from the same tissue type were run together to compare all transgenic lines to the P39 WT controls, and to assess whether any of the transgenic lines displayed differences in transcript abundance. Gene expression in phloem was measured and normalized relative to PtrUBQ11. None of the lines showed any significant difference in GUS expression levels when compared to the P39 WT controls (Figure 4.1). Moreover, due to very low expression levels, the standard error is large compared to those from other tissues. 43 Figure 4.1: Fold change of GUS expression levels in the phloem tissue of greenhouse-grown transgenic PtSUS1p::GUS poplar lines. Expression normalized to PtrUBQ11. Error bars represent standard error of the mean (n=3). Expression data from the root tissue was normalized relative to PtrEF1B. A significant increase in GUS expression was detected in lines 55 and 64 compared to the WT lines (Figure 4.2). Additionally, standard error for this set of data was smaller compared to the standard error from the phloem data set. Thus, the root tissue expression data mean is a more accurate reflection of the population mean. From this data set, line 55 seems to be a possible high-expressing line, followed by line 64. -1.0-0.50.00.51.01.52.02.53.03.54.0WT-1 WT-2 Line 10 Line 55 Line 64 Line 76 Line 78 Line 89Fold Gene Expression (2^-△△Ct)Fold Change of GUS relative to UBQ11 in Phloem 44 Figure 4.2: Fold change of GUS expression levels in the root tissue of greenhouse-grown transgenic PtSUS1p::GUS poplar lines. Expression normalized to PtrEF1B. Significance is shown as *= 𝝆 < 𝟎. 𝟎𝟓 and ** = 𝝆 < 𝟎. 𝟎𝟏 compared to P39 WT GUS expression levels. Error bars represent standard error of the mean (n=3). Interestingly, when examining the vascular tissues significant differences in the expression levels of GUS were apparent when driven under the PtSUS1 promoter (Figure 4.3). It is evident from the data that expression levels of GUS in the xylem tissue are significantly higher than those in the phloem in all but one of the poplar transgenic lines. In order, the top three highest expressing lines appear to be lines 89, 76, and 55, followed closely by lines 78 and 64. Line 10 showed no significant differences in GUS expression levels compared to the WT controls. ***-1.00.01.02.03.04.05.06.07.08.0WT-1 WT-2 Line 10 Line 55 Line 64 Line 76 Line 78 Line 89Fold Gene Expression (2^-△△Ct)Fold Change of GUS relative to EF1B in Roots 45 Figure 4.3: Fold change of GUS expression levels in the xylem and phloem tissues of greenhouse-grown transgenic PtSUS1p::GUS poplar lines. Expression normalized to PtrEF1B. Significance is shown as *= 𝝆 < 𝟎. 𝟎𝟓 and ** = 𝝆 < 𝟎. 𝟎𝟏 compared to P39 WT GUS expression levels. Error bars represent standard deviation for the △Ct value. GUS expression levels of three high expressing PtSUS1p::GUS transgenic lines, as well as both WT controls, were compared to three independent greenhouse-grown P39 WT trees to verify that there was no contamination, or cross-over of any kind between my transgenic lines and my controls. The independent controls were grown by another student for approximately six months, but under similar environmental and growth conditions. I will refer to these lines as “external controls”. Expression data for this set was obtained with the two pairs of GUS-specific primers to verify transcript length and results were normalized to PtrEF1B (Figure 4.4). Expression values for each primer pair per line are very similar to each other, indicating that the transcription of GUS is occurring at full length. Additionally, the data shows that line 55 is once again the highest expressing line of those tested, demonstrating a significant increase in GUS expression compared to all control trees. Line 64 also showed significant levels of GUS expression compared to the control group. Surprisingly, line 78, although previously marked as a high GUS expressing line in Figure 4.3, was not significantly different to the *********-2.00.02.04.06.08.010.012.014.0WT-1 WT-2 Line 10 Line 55 Line 64 Line 76 Line 78 Line 89Fold Gene Expression (2^-△△Ct)Xylem Phloem 46external control group (Figure 4.4). This contradiction in the expression results could be due to improper technique or degradation of the cDNA sample. Figure 4.4: Fold change of GUS expression levels in the xylem tissue of select greenhouse-grown high expressing transgenic PtSUS1p::GUS poplar lines and external P39 controls. Expression normalized to PtrEF1B. Significance is shown as *= 𝝆 < 𝟎. 𝟎𝟓 and ** = 𝝆 < 𝟎. 𝟎𝟏 compared to the GUS expression levels of a comprehensive P39 WT group consisting of the three external controls and the other two internal control lines. Error bars represent standard deviation for the △Ct value. 4.3 Discussion It was previously shown that PtSUS1 and PtSUS2 are preferentially expressed in the mature xylem sink tissue, and medium levels of expression have also been found in the roots, mature leaves, and developing xylem of poplar trees (Zhang et al. 2011). However, the RNA sequencing data from which this information was obtained referenced only the native transcript levels, and did not explore the role or strength of the native promoters. Research has shown that promoters play an essential role in gene expression and regulation (Benfey and Chua 1990; Rombauts et al. 2003; Cairns 2009; Biłas et al. 2016), yet, the extent of promoters analysis remains minor compared to the wide range of genes that have been studied over time. Plant biotechnology aims to optimize gene expression in favour of increasing resource efficiency, and for this reason, it is essential that more promoters are thoroughly analyzed. Here I have -5.00.05.010.015.020.025.030.0Ext.Control 1Ext.Control 2Ext.Control 3 WT-1 WT-2 Line 55 Line 64 Line 78Fold Gene Expression (2^-△△Ct)GUS (5' end) GUS (3' end) 47demonstrated that the PtSUS1 promoter may provide a means for driving gene expression in the xylem tissue. Although the generated GUS lines failed to produce a visible GUS phenotype, the expression analysis data show significant differences in transcript levels across different tissues harvested from verified transgenic lines. Figure 4.3 demonstrates a clear difference in the expression levels of the GUS transcript being produced in the developing xylem compared to phloem tissue of the same transgenic poplar lines. From the six randomly selected transgenic lines, five of them showed significantly higher expression in the xylem than in the phloem (Figure 4.3). Line 10 demonstrated WT-levels of expression across all tissues, and this may be due to the expression cassette being inserted in a non-expressing site in the genome. However, lines 55 and 64 consistently displayed significant expression differences to the WT control lines in the root and xylem tissues (Figure 4.2-4.4). However, in the phloem, these high expressing lines displayed expression levels similar to WT (Figure 4.1). In fact, none of the transgenic lines had significant differences in the phloem expression of GUS compared to WT. These differences in expression across tissues match the previously known RNA sequencing data (Zhang et al. 2011), and in addition, also provide better insight into the promoter’s role in protein localization. The expression analysis results are interesting because the data provides information directly related to the promoter’s function. In the transgenic poplar trees, the PtSUS1 promoter is native to the species, but the GUS gene being expressed is not. Previous work demonstrated that the protein PtSUS1 localized mainly to the mature and developing xylem (Zhang et al. 2011), and we see the same pattern occurring in the current transgenic lines with GUS. Although the GUS protein was not formed correctly in my trees, the transcript levels detected in the expression analysis are evidence of this tissue-specific expression. Since GUS is not native to poplar and is not being co-expressed with another gene, there is no reason that it would be preferentially expressed in any of the tissues, other than from the expression being driven by the PtSUS1 promoter. In other words, in this study, PtSUS1p was solely responsible for the difference in expression of GUS between the xylem and phloem tissues. This is of particular interest when it comes to manipulating gene expression, as suggests this promoter and not the protein or gene is responsible for spatiotemporal gene expression of sucrose synthase. This also creates the opportunity to introduce PtSUS1p into other species or drive the specific expression of other genes of interest to xylem tissue. While the expression analysis data provided evidence to support that the promoter is the driving the spatiotemporal gene expression, questions regarding the role of promoter 48architecture elements, such as several of the motifs identified in Chapter 3, remain unanswered. The importance of this information might be key to fully understand promoter activity (Nikolov and Burley 1997; Berk 1999; Rombauts et al. 2003). More remarkably, it could aid in designing promoter architecture by manipulating regulatory regions and elements of interest, and creating synthetic promoters that will optimize crop efficiency and resource use across many economic sectors (Mehrotra et al. 2011; Pandiarajan and Grover 2018). 4.4 Background troubleshooting The data obtained in this study does not reflect the amount of work and learning that took place throughout this degree. Although the expression analysis data provided some insight into the tissue-specific expression of genes driven by one of the poplar SuSy promoters, there are still many questions left to be answered. I am certain that some of these could have been resolved by the truncation and visualization experiments that were originally designed for this work. However, there were several challenges along the way that resulted in these experiments being unsuccessful. However, it remains extremely important to consider each failure as a learning opportunity to correct the course of progress and implement new and improved strategies. The complexity of this project came mostly from the novelty of dealing with promoters instead of genes. Promoters are rapidly becoming the focus of scientific research as more information on their importance and effect on gene expression becomes available. The challenges encountered throughout this study were not entirely unexpected. This is because polymerase-assisted amplification, cloning, and altering promoter sequences are not optimized towards promoter characteristics, but rather standardized for genes. Molecular protocols are always in constant change, adjusting and recalibrating according to new data, new specimens, or new problems. For this reason, I find it essential to report the background troubleshooting that took place throughout this study in hopes that my challenges can provide a better insight into the intricacies of working with promoters. 4.4.1 GUS staining and imaging While the transgenic poplar lines were growing in the greenhouse, GUS assay trials were conducted on several positive control samples to test and optimize the staining protocol. All positive controls were A. thaliana transgenics (Appendix table C.1) grown from T3 seeds on selective media for two weeks, and then grown on soil for approximately another six weeks. Plant tissue was harvested for staining from two-week-old seedlings, as well as from more 49mature tissues at the four- and six-week marks to test incubation times, solution penetration, and intensity of stain. Samples were then visualized and photographed under the microscope to confirm GUS staining. Successful staining of two-week-old seedlings (Figure 4.5 - 4.6), as well as four- and six-week-old stem tissue and leaves (Figure 4.7 - 4.8) confirmed that the protocol, as well as the appropriateness of the reaction solutions, would yield the signature blue stain where GUS was expressed. Figure 4.5: Two-week old positive control RPL4A::GUS seedling. GUS activity in shown in (A) whole seedling, (B) hypocotyl and early root, (C) first true leaves and cotyledon. Seeds provided by Dr. Abel Rosado. Scale bar as shown. 50 Figure 4.6: Two-week old positive control PrABCB11::GUS seedling. GUS activity in shown in (A) top section of seedling, (B) hypocotyl, (C) early primary root. Seeds provided by Dr. Lacey Samuels. Scale bar as shown. 51 Figure 4.7: Four- and six-week old positive controls PrABCB11::GUS stems and leaf. Panels A-C show GUS staining in stem longitudinal sections at various magnifications, GUS activity present in the xylem parenchyma. Lines grown from seeds obtained from Dr. Lacey Samuels. Scale bar as shown. Figure 4.8: Stem cross-sections from six-week old positive controls for GUS assays. GUS activity indicated by arrows where coloured staining is light. GUS controls (A) 4CL::GUS, (B) PrABCB11::GUS, and (C) RPL4A::GUS were grown from seeds obtained from Dr. Abel Rosado and Dr. Lacey Samuels. Scale bar = 100µm. The same verified protocol was then implemented for the GUS assays of the tissues harvested from the transgenic poplar lines grown in the greenhouse, and a few transgenic T1 Arabidopsis lines. None of the PtSUS1p::GUS lines produced an observable GUS phenotype in any of the tissues visualized, as shown by the similarity between the transgenic lines and the WT controls (Figures 4.9-4.11). In some transgenic lines the presence of GUS artifacts were 52observed, however, these were labelled as such due to a lack of consistency across sections of the same line, as well as no visible patterning across the tissue (Figure 4.12). Figure 4.9: Stem cross-sections from the top 7cm of the main stem of the PtSUS1p::GUS greenhouse-grown lines after GUS assay. (A) P39 control for comparison. (B-G) No GUS activity visible in any of the transgenic lines. Scale as shown for P39 in panel A, scale bar in panel G applies to all transgenic lines (panels B-G). Figure 4.10: Stem cross-sections from the bottom 5cm of the main stem of the PtSUS1p::GUS greenhouse-grown lines after GUS assay. (A) P39 control for comparison. (B-G) No GUS activity visible in any of the transgenic lines. Scale bar= 500µm. 53 Figure 4.11: Root cross-sections from the main root of the PtSUS1p::GUS greenhouse-grown lines after GUS assay. (A) P39 control for comparison. (B-D, F) No GUS activity visible in any of these transgenic lines. (E) Faint blue artifact present. Scale bar= 200µm. Figure 4.12: GUS artifacts present in stem and root tissue from PtSUS1p::GUS greenhouse-grown lines. (A) Line 89 presented an artifact of approximately 25.4µm in length in one of its bottom stem cross sections. Artifact was characterized by dark staining and clear shape in one of the xylem vessel walls. (B) Line 76 presented an artifact of approximately 106.2µm in length in one of its main root cross sections. Artifact was light in colour and had an undefined shape. Scale bars as shown. 54Overall, it was surprising to not find a single line or tissue section that showed successful GUS staining, as all the transgenic lines had been confirmed prior to harvest via DNA screenings. This irregularity encouraged me to incorporate expression analysis to this project to better understand why the PtSUS1p::GUS poplar lines were not producing a visible GUS phenotype as expected. The expression data shows that the transcript for GUS was being produced and at full length in the tissues that I originally expected to see a positive visual indication. 4.4.2 Promoter and intron amplification Considering the expression analysis demonstrated active full-length transcription of the GUS transcript, I reanalyzed the promoter sequence and examined the open reading frames (ORFs) of the final construct using SnapGene. It was then apparent that the promoter sequence that I cloned into the GUS expression vector contains a fragment of the first intron of the PtSUS1 gene sequence. The intron sequence was 453bp long and sits immediately after the 135bp-long 5’UTR and a few base pairs prior to the start codon of the eGFP gene of the expression vector. The presence of the intron was not surprising, as I had intentionally designed my reverse primer to anneal to a sequence downstream of the TSS. The challenge of amplifying these particular set of promoters lied in their characteristic AT-richness and high level of repetitiveness in their sequence. Most of the commercial polymerases I could find seem to be either optimized for an evenly distributed AT-GC sequence or in some cases for GC-rich sequences, which are characteristic of gene sequences. AT-rich regions, like those in PtSUS1p and PtSUS2p, tend to have a low thermodynamic stability (Rajewska et al. 2012), and in many cases this will interfere with amplification via PCR, especially with non-proofreading polymerases, as AT-rich sequences have lower melting temperatures than GC-rich DNA due to A-T pairs forming only two hydrogen bonds (New England BioLabs, 2023). Therefore, to improve polymerase binding to my promoter sequences, I was advised to design a reverse primer that would anneal to a more GC-rich sequence located downstream of the TSS. This strategy allowed me to amplify the PtSUS1p as a complete fragment and to proceed with the cloning process, as by including the intron in the sequence to be amplified I boosted GC richness of the fragment from 24% GC to 27% GC. And although that 3% difference does not seem significant, it proved to be a vast improvement when it came to PCR amplification. 554.4.3 Frameshift mutation Although creating a reverse primer at an annealing site downstream of the TSS proved successful in amplifying PtSUS1p in a single fragment, I did not take into consideration that in order to maintain translational accuracy I would have to modify my amplified promoter fragment further to remove the incomplete intron. I assumed that the intron’s presence would not impact transcription, as the start codon for eGFP and GUS would remain intact. Additionally, I considered that the intron would be spliced out of the transcript in post-transcriptional modification and would therefore not interfere in translation. In retrospect, I now know that this did not happen because the intron fragment that was cloned does not contain the specific 3’ splicing site needed for this process. Upon further analysis, I discovered that the intron fragment also contains an ATG codon in one of the possible ORFs, which would result in a +2 frameshift of the overall reading frame (Appendix figure C.1), and this would generate an early stop codon at the beginning of the GUS transcript. In other words, the presence of the intron fragment would affect the GUS protein by creating a nonsense mutation in the codon sequence (Appendix figure C.2). This would explain why although my transgenic lines produced full-length transcript of GUS, but did not display a visible phenotype after staining. Unfortunately, the only way to verify this hypothesis as the cause behind my non-phenotypic GUS lines would be to conduct protein analysis experiments such as western blotting, electron microscopy, x-ray diffraction, among others. 4.4.4 Arabidopsis experiments The promoter swapping experiment in Arabidopsis was conducted with the purpose of visualizing via GUS assays whether the poplar SuSy promoter would drive protein localization to the xylem instead of the phloem. The truncation experiments, on the other hand, aimed to test the effect and importance of motif events that could potentially drive protein localization to the xylem as described in Chapter 3. Both of these experiments involved GUS assays, and therefore, Arabidopsis WT Col-0 plants were transformed with the respective GUS constructs. However, all the constructs were created using the amplified PtSUS1 promoter fragment containing the intron. The experiments carried out in Arabidopsis presented a wide array of problems and difficulties in troubleshooting for several reasons. First, all plant transformations were conducted at around the same time. At that stage, there was no indication that the GUS 56construct would not result in the expected visual phenotype until the GUS assays for the poplar lines were carried out. Additionally, transgenic Arabidopsis lines were selected only until the T1 generation and not all the way to T3 due to a time constraint. This increased the assumption of any false negatives, as the variability among transgenic lines was quite large in the T1 generation. It would have not been unusual for positive transgenic lines in the T1 generation to display little to no staining, mostly due to the variation in insertion sites and heterozygous genotypes. Another problem encountered in the truncation experiments was that none of the constructs could be visualized, as all truncations were designed to truncate the 5’ end of the promoter, while keeping the 3’ end intact. With the intron existing downstream of the promoter’s 3’end, it was unintentionally conserved in all three truncation constructs. The decision behind this truncation design was based on the importance of conserving the transcriptional machinery present in the core promoter region, such as the TATA box, the initiator, and the B-recognition element (Abeel et al. 2008; Pandiarajan and Grover 2018). This was to ensure any expression differences came from the truncation of the motifs present in the regulatory regions and not due to a lack of essential transcriptional components. As mentioned, the same PtSUS1p::GUS construct was used to transform the Arabidopsis plants, and served as the basis for all the truncation constructs. The nonsense mutation caused by the intron fragment in these constructs also affected the outcome of these transformations. Thus, although the plants were transformed successfully, there was no visible GUS phenotype after staining. The other problem specific to Arabidopsis, was that due to their very small and delicate anatomy, isolating and harvesting the vascular tissues to then perform an expression analysis via qRT-PCR similar to that conducted for the poplar tissues was not possible. 57Chapter 5: Conclusion 5.1 Thesis summary This project originally aimed to identify motifs in the regulatory regions of native SuSy promoters of Arabidopsis thaliana and Populus trichocarpa, that could potentially influence the tissue-specific differences inherently observed in protein localization of the SuSy proteins in these two species. Additionally, I also aimed to compare GUS reporter protein localization in the vascular tissues of these two plant species when gene expression was driven by the same promoter. In Chapter 3, I analyzed and compared the sequences of seven promoters: PtSUS1p, PtSUS2p, AtSUS1p, AtSUS4p, PtCesA2p, PtCesA4p, and PtCesA7p. This bioinformatic analysis aimed to identify essential motifs within the sequences that could potentially drive protein localization to the xylem. A total of six SCW- and xylem-related motifs, whose sequences had been identified in previous research, were selected for motif mapping in the promoter sequences. The analysis showed that most of the motifs of interest were identified in the proximal and distal promoter regions, which was not unexpected, as these are the main regulatory regions (Rombauts et al. 2003). Although a few motif patterns were identified in the proximal region, none of the motif events showed a consistent configuration in the two poplar SuSy promoters that might uniquely suggest xylem-specific localization. On the distal region, 2 SNBE-like motifs were identified within close proximity to one another in the promoter sequences of both poplar SuSy promoters. This double-motif event became of major interest for further research as it has been previously shown that regulatory elements in the distal promoter region can directly affect spatiotemporal gene expression (Tjian and Maniatis 1994; Fessele et al. 2002; Rombauts et al. 2003). Additionally, these motifs were not found in a similar arrangement in either of the Arabidopsis SuSy promoters, nor in the poplar secondary CesA promoters analyzed. For these reasons, the double-SNBE motif event was considered as a strong candidate for the truncation experiments that were to follow. The truncation experiments were designed to test which promoter region might affect protein localization the most, and thus refine the putative list of motifs could be driving tissue-specific expression. Three truncations were designed based on the bioinformatic analysis conducted in Chapter 3, whereby sections of the promoter were gradually removed from the regulatory region in a 5’ to 3’ direction. Unfortunately, although the constructs for these 58truncations were created, there was an error in cloning, and as such none of the transgenic lines generated displayed a visual phenotype that could be analyzed to determine if these truncations were truly successful. In Chapter 4, via expression analysis I demonstrated that PtSUS1p could successfully drive GUS expression preferentially to the developing xylem, as well as in the root tissues. The poplar transgenic lines analyzed, showed significantly higher transcript abundance in the xylem tissues compared to associated phloem tissue. Additionally, none of the transgenic lines showed significantly different values for GUS expression in the phloem from those in the WT controls. The results from this analysis are interesting as the data concurs with previous profiling studies (Zhang et al. 2011), but also supports the original hypothesis that the promoter PtSUS1p is possibly the main regulator in driving tissue-specific expression to the xylem, in poplar trees. This finding provides opportunity to further evaluate PtSUS1p as a candidate for future endeavors that require a xylem-specific promoter to intentionally drive protein localization to this tissue. Similar to the truncation experiments, I initially intended to show tissue-specific expression driven by PtSUS1p via GUS visualization in both hybrid poplar and Arabidopsis thaliana. Unfortunately, due to the presence of an incomplete intron in the promoter sequence cloned, visualization of GUS was not possible, which prompted the need to complete the ensuing expression analysis experiment. Although the data obtained provided valuable insight into the tissue-specific expression driven by PtSUS1p, several questions remain unanswered. Nonetheless, I firmly believe that troubleshooting remains one of the most indispensable skills a scientist can have, as it is indisputable that all scientific research will present its own challenges in the pursuit of knowledge. 5.2 Relevance It has been a long-standing goal in molecular biology and plant biotechnology to develop tools and strategies that allow precise regulation of gene expression. The need to manipulate and enhance gene activity has brought much interest to promoter research. However, the use of wild-type promoters presents several challenges when it comes to actual implementation, reliability, and consistency of results (Venter and Botha 2010). For this reason, the development of synthetic promoters is of upmost importance. The main objective behind synthetic promoters is to enhance the expression of the target gene and increase the promoter’s strength and accuracy, while also reducing any undesirable leaky expression (Mehrotra et al. 592011; Pandey et al. 2018). The implementation of synthetic promoters to rescue phenotypes or direct gene expression to specific tissues has proven successful in many studies (De Meester et al. 2018). Nevertheless, synthetic promoter elements come from naturally occurring promoter sequences that have shown desirable characteristics that are linked to functional domains. These domains or elements can be swapped or recreated for integration into synthetic promoters (Mehrotra et al. 2011; Pandey et al. 2018; Pandiarajan and Grover 2018). Alternatively, other strategies involve modifying the native architecture of the core promoter elements, along with the proximal or distal promoter regions to manipulate specific regulatory elements (Cairns 2009; Venter and Botha 2010; Pandey et al. 2018). The research and development of synthetic promoters is quickly becoming a priority in genetic engineering. The research questions posed for this project aimed to contribute to the general pool of knowledge of promoter regulatory elements that could potentially be used to drive protein localization to the xylem tissue of plants. Despite many questions remaining unanswered, the results of this study partially support the hypothesis that PtSUS1p could act as a spatiotemporal promoter controlling gene expression within the xylem. While the specific regulatory elements require further research, this project has alluded to a few potential candidates for xylem-specific gene expression that could prove useful in the future. 5.3 Future research Promoter research is quickly becoming a major topic of interest for plant biologists and geneticists. However, there are limitations in the protocols, techniques, and materials that have been optimized for gene-related work thus far. As more promoter-specific resources become available, the difficulties of troubleshooting promoter-based experiments should be resolved, and future efforts may be less challenging during the actual cloning and manipulation. Although the results from this thesis propose a valuable promoter candidate for xylem-driven gene expression, many other research avenues remain open for further exploration. 5.3.1 Intron removal and evaluation of influence on gene expression Although my constructs failed to produce a GUS signal due to a frameshift mutation caused by an incomplete intron in the sequence, visual localization of the poplar SuSy proteins has yet to be conducted successfully. Additionally, the promoter truncation experiments, and their corresponding visualization are also pending. 60Obtaining the GUS signals from these constructs could be accomplished by either removing the intron from the amplified promoter or ensuring that it will be spliced in post-transcriptional modification prior to translation. The former option may present more challenges, as amplifying the promoter sequence without the intron could require finely tuned PCR optimization and high-fidelity polymerases that anneal tightly to AT-rich sequences. Post-amplification intron removal might be a better suited option, contingent on the availability of a restriction enzyme that will not affect the reading frame or the 5’UTR sequence of the transcript, which contains essential binding sites for the protein complexes of RNA polymerase and can enhance the expression of the transgene (Pandey et al. 2018). On the other hand, the latter option might be easier and would guarantee that the reading frame for GUS translation is maintained. For this, the extension of the intron amplification towards its native 3’ splice site (SS) or the incorporation of a synthetic 3’ SS via modified primers could guarantee their recognition by small nuclear RNA (snRNA) proteins, resulting in proper intron splicing (Shaul 2017). There is evidence to suggest that introns can influence transcript abundance, as well as affect the rate and efficiency of transcription (Shaul 2017). This phenomenon has been referred to as intron-mediated enhancement (IME). The exact mechanisms remain noticeably unknown, and despite decades of research focused on this topic, little progress has been made to better understand the factors involved in such mechanisms, as well as the relationship between introns and promoters (Gallegos and Rose 2015). Although for my constructs the first SuSy intron was mistakenly included in PtSUS1p’s amplification, it might be interesting to investigate whether this first intron, or any of the others, contribute to mRNA stability, rate or efficiency of transcription, and nuclear export. 5.3.2 Promoter swapping Arabidopsis and poplar SuSy homologs The second objective of this study aimed to investigate whether the poplar SuSy promoters PtSUS1 and PtSUS2 could drive protein localization to the xylem in Arabidopsis. Although, this could not be verified due to the non-phenotypic transgenic plants produced (no GUS expression). A promoter swap between the Arabidopsis and poplar homologous promoters and the use of a GUS construct, like the one in this study, or the implementation of a fluorescent tag for live-cell imaging, could yield further information on the influence of this set of SuSy promoters on spatial localization. This data would not only be valuable to fill knowledge gaps 61in the poplar SuSy multigene family, but might also expose strong promoters that could potentially be used in future tissue-specific expression studies. 5.3.3 Comparing SuSy and secondary CesA expression levels in vascular tissues The bioinformatic analysis and data presented in Chapter 3, identified the motifs of interest in the three poplar secondary CesA promoters. These promoters were included in the analysis due to their role in cellulose production and SCW deposition in mature xylem tissues, specifically xylem tracheary elements, which are essential for proper water transportation, and supportive xylary fibers (Schuetz et al. 2013). Additionally, there is some evidence to support an association between SuSy and the CesAs in cellulose biosynthesis (Coleman et al. 2009; Payyavula et al. 2011; Gerber et al. 2014). However, the poplar secondary CesAs served mainly for promoter sequence comparison and were not a focal point in the study. The Arabidopsis secondary CesA promoters have been previously used in other studies to target gene expression to the tracheary elements and fibers in the xylem tissue, specifically in cells undergoing SCW deposition (Smith et al. 2013). Measuring the vascular expression levels via qRT-PCR of the poplar and Arabidopsis secondary CesAs, and then comparing them to the corresponding SuSy expression might provide further insight into the strength, reliability, and expression pattern of each of these promoters. In other words, this expression data could hint at which promoters, if any, could be better suited to drive gene expression in specific vascular tissues in future transgenic plants. Alternatively, an experiment where the poplar SCW CesAs and SuSy are co-expressed could also provide insight into the interaction and regulation of these proteins. In addition, this data could also indicate a possible correlation in the SuSy and CesA expression of Arabidopsis and poplar that could potentially provide more information as to the degree of association between these families of proteins during cellulose biosynthesis. 62References Abeel, T., Saeys, Y., Bonnet, E., Rouzé, P., and Van De Peer, Y. 2008. Generic eukaryotic core promoter prediction using structural features of DNA. Genome Res. 18(2): 310–323. doi:10.1101/gr.6991408. Akiyoshi, N., Ihara, A., Matsumoto, T., Takebayashi, A., Hiroyama, R., Kikuchi, J., Demura, T., and Ohtani, M. 2021. Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties. Plant Cell Physiol. 62(12): 1963–1974. doi:10.1093/pcp/pcab102. Amor, Y., Haigler, C.H., Johnson, S., Wainscott, M., and Delmer, D.P. 1995. A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proc. Natl. Acad. Sci. U. S. A. 92(20): 9353–9357. doi:10.1073/pnas.92.20.9353. An, X., Chen, Z., Wang, J., Ye, M., Ji, L., Wang, J., Liao, W., and Ma, H. 2014. Identification and characterization of the Populus sucrose synthase gene family. Gene 539(1): 58–67. Elsevier B.V. doi:10.1016/j.gene.2014.01.062. Anderson, C.T., Carroll, A., Akhmetova, L., and Somerville, C. 2010. Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots. Plant Physiol. 152(2): 787–796. doi:10.1104/pp.109.150128. Andersson, R., and Sandelin, A. 2020. Determinants of enhancer and promoter activities of regulatory elements. Nat. Rev. Genet. 21(February): 71–87. Available from https://doi.org/10.1038/ s41576-019-0173-8. Aoki, N., Hirose, T., Scofield, G.N., Whitfeld, P.R., and Furbank, R.T. 2003. The sucrose transporter gene family in rice. Plant Cell Physiol. 44(3): 223–232. doi:10.1093/pcp/pcg030. 63Asturias, F.J., Ezeokonkwo, C., Kornberg, R.D., and Lorch, Y. 2003. Electron Microscopic Analysis of the RSC Chromatin Remodeling Complex. Methods Enzymol. 376(21): 48–62. doi:10.1016/S0076-6879(03)76004-2. Baroja-Fernández, E., Muñoz, F.J., Li, J., Bahaji, A., Almagro, G., Montero, M., Etxeberria, E., Hidalgo, M., Sesma, M.T., and Pozueta-Romero, J. 2012. Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production. Proc. Natl. Acad. Sci. U. S. A. 109(1): 321–326. doi:10.1073/pnas.1117099109. Barratt, D.H.P., Barber, L., Kruger, N.J., Smith, A.M., Wang, T.L., and Martin, C. 2001. Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiol. 127(2): 655–664. doi:10.1104/pp.010297. Barratt, D.H.P., Derbyshire, P., Findlay, K., Pike, M., Wellner, N., Lunn, J., Feil, R., Simpson, C., Maule, A.J., and Smith, A.M. 2009. Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proc. Natl. Acad. Sci. U. S. A. 106(31): 13124–13129. doi:10.1073/pnas.0900689106. Baud, S., Vaultier, M.N., and Rochat, C. 2004. Structure and expression profile of the sucrose synthase multigene family in Arabidopsis. J. Exp. Bot. 55(396): 397–409. doi:10.1093/jxb/erh047. Baud, S., Wuillème, S., Lemoine, R., Kronenberger, J., Caboche, M., Lepiniec, L., and Rochat, C. 2005. The AtSUC5 sucrose transporter specifically expressed in the endosperm is involved in early seed development in Arabidopsis. Plant J. 43(6): 824–836. doi:10.1111/j.1365-313X.2005.02496.x. Bauer, S., Vasu, P., Persson, S., Mort, A.J., and Somerville, C.R. 2006. Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls. Proc. Natl. Acad. Sci. U. S. A. 103(30): 11417–11422. 64doi:10.1073/pnas.0604632103. Van Bel, A.J.E. 2003. The phloem, a miracle of ingenuity. Plant, Cell Environ. 26(1): 125–149. doi:10.1046/j.1365-3040.2003.00963.x. Benfey, P.N., and Chua, N.H. 1990. The cauliflower mosaic virus 35S promoter: Combinatorial regulation of transcription in plants. Science (80-. ). 250(4983): 959–966. doi:10.1126/science.250.4983.959. Berk, A.J. 1999. Activation of RNA polymerase II transcription. Curr. Opin. Cell Biol. 11(3): 330–335. doi:10.1016/S0955-0674(99)80045-3. Biłas, R., Szafran, K., Hnatuszko-Konka, K., and Kononowicz, A.K. 2016. Cis-regulatory elements used to control gene expression in plants. Plant Cell. Tissue Organ Cult. 127(2): 269–287. Springer Netherlands. doi:10.1007/s11240-016-1057-7. Braun, D.M., Wang, L., and Ruan, Y.L. 2014. Understanding and manipulating sucrose phloem loading, unloading, metabolism, and signalling to enhance crop yield and food security. J. Exp. Bot. 65(7): 1713–1735. doi:10.1093/jxb/ert416. Burn, J.E., Hocart, C.H., Birch, R.J., Cork, A.C., and Williamson, R.E. 2002. Functional Analysis of the Cellulose Synthase Genes. Plant Physiol 129(June): 797–807. doi:10.1104/pp.010931.1. Cairns, B.R. 2009. The logic of chromatin architecture and remodelling at promoters. Nature 461(7261): 193–198. doi:10.1038/nature08450. Cao, H., Ekstrom, A., and Yin, Y. 2015. Plant Carbohydrate Active Enzyme (CAZyme) Repertoires: A Comparative Study. In Advances in the Understanding of Biological Sciences Using Next Generation Sequencing (NGS) Approaches. Edited by G. Sablok, S. Kumar, S. Ueno, J. Kuo, and C. Varotto. Springer International Publishing, Cham. pp. 115–134. doi:10.1007/978-3-319-17157-9_8. Carlson, S.J., Chourey, P.S., Helentjaris, T., and Datta, R. 2002. Gene expression studies on 65developing kernels of maize sucrose synthase (SuSy) mutants show evidence for a third SuSy gene. Plant Mol. Biol. 49(1): 15–29. doi:10.1023/A:1014457901992. Chèneby, J., Ménétrier, Z., Mestdagh, M., Rosnet, T., Douida, A., Rhalloussi, W., Bergon, A., Lopez, F., and Ballester, B. 2020. ReMap 2020: A database of regulatory regions from an integrative analysis of Human and Arabidopsis DNA-binding sequencing experiments. Nucleic Acids Res. 48(D1): D180–D188. doi:10.1093/nar/gkz945. Cho, J. Il, Kim, H.B., Kim, C.Y., Hahn, T.R., and Jeon, J.S. 2011. Identification and characterization of the duplicate rice sucrose synthase genes OsSUS5 and OsSUS7 which are associated with the plasma membrane. Mol. Cells 31(6): 553–561. doi:10.1007/s10059-011-1038-y. Coleman, H.D., Yan, J., and Mansfield, S.D. 2009. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure. Proc. Natl. Acad. Sci. U. S. A. 106(31): 13118–13123. doi:10.1073/pnas.0900188106. Cronshaw, J. 1981. Phloem Structure and Function. Annu. Rev. Plant Physiol. 32: 465–484. Desprez, T., Juraniec, M., Crowell, E.F., Jouy, H., Pochylova, Z., Parcy, F., Höfte, H., Gonneau, M., and Vernhettes, S. 2007. Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 104(39): 15572–15577. doi:10.1073/pnas.0706569104. Doblin, M.S., Kurek, I., Jacob-Wilk, D., and Delmer, D.P. 2002. Cellulose biosynthesis in plants: From genes to rosettes. Plant Cell Physiol. 43(12): 1407–1420. doi:10.1093/pcp/pcf164. Eckert, C., Sharmin, S., Kogel, A., Yu, D., Kins, L., Strijkstra, G.J., and Polle, A. 2019. What makes the wood? Exploring the molecular mechanisms of xylem acclimation in hardwoods to an ever-changing environment. Forests 10(4). doi:10.3390/f10040358. Egli, B., Kölling, K., Köhler, C., Zeeman, S.C., and Streb, S. 2010. Loss of cytosolic 66phosphoglucomutase compromises gametophyte development in arabidopsis. Plant Physiol. 154(4): 1659–1671. doi:10.1104/pp.110.165027. Fessele, S., Maier, H., Zischek, C., Nelson, P.J., and Werner, T. 2002. Regulatory context is a crucial part of gene function. Trends Genet. 18(2): 60–63. doi:10.1016/S0168-9525(02)02591-X. Fünfgeld, M.M.F.F., Wang, W., Ishihara, H., Arrivault, S., Feil, R., Smith, A.M., Stitt, M., Lunn, J.E., and Niittylä, T. 2022. Sucrose synthases are not involved in starch synthesis in Arabidopsis leaves. Nat. Plants 8(5): 574–582. Springer US. doi:10.1038/s41477-022-01140-y. Gallegos, J.E., and Rose, A.B. 2015. The enduring mystery of intron-mediated enhancement. Plant Sci. 237: 8–15. Elsevier Ireland Ltd. doi:10.1016/j.plantsci.2015.04.017. Gerber, L., Zhang, B., Roach, M., Rende, U., Gorzsás, A., Kumar, M., Burgert, I., Niittylä, T., and Sundberg, B. 2014. Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers. New Phytol. 203(4): 1220–1230. doi:10.1111/nph.12888. Hänggi, E., and Fleming, A.J. 2001. Sucrose synthase expression pattern in young maize leaves: Implications for phloem transport. Planta 214(2): 326–329. doi:10.1007/s00425-001-0680-6. Hardin, S.C., and Huber, S.C. 2004. Proteasome activity and the post-translational control of sucrose synthase stability in maize leaves. Plant Physiol. Biochem. 42(3): 197–208. doi:10.1016/j.plaphy.2003.12.004. Hill, M.S., Vande Zande, P., and Wittkopp, P.J. 2021. Molecular and evolutionary processes generating variation in gene expression. Nat. Rev. Genet. 22(4): 203–215. doi:10.1038/s41576-020-00304-w. Huber, S.C., and Akazawa, T. 1986. A Novel Sucrose Synthase Pathway for Sucrose 67Degradation in Cultured Sycamore Cells. Plant Physiol. 81(4): 1008–1013. doi:10.1104/pp.81.4.1008. Hudson, M.E., and Quail, P.H. 2003. Identification of Promoter Motifs Involved in the Network of Phytochrome A-Regulated Gene Expression by Combined Analysis of Genomic Sequence and Microarray Data. Plant Physiol. 133(4): 1605–1616. doi:10.1104/pp.103.030437. Johansson, H., Sterky, F., Amini, B., Lundeberg, J., and Kleczkowski, L.A. 2002. Molecular cloning and characterization of a cDNA encoding poplar UDP-glucose dehydrogenase, a key gene of hemicellulose/pectin formation. Biochim. Biophys. Acta - Gene Struct. Expr. 1576(1–2): 53–58. doi:10.1016/S0167-4781(02)00292-0. Joshi, C.P., Bhandari, S., Ranjan, P., Kalluri, U.C., Liang, X., Fujino, T., and Samuga, A. 2004. Genomics of cellulose biosynthesis in poplars. New Phytol. 164(1): 53–61. doi:10.1111/j.1469-8137.2004.01155.x. Kanhere, A., and Bansal, M. 2005. Structural properties of promoters: Similarities and differences between prokaryotes and eukaryotes. Nucleic Acids Res. 33(10): 3165–3175. doi:10.1093/nar/gki627. Kheradpour, P., and Kellis, M. 2014. Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments. Nucleic Acids Res. 42(5): 2976–2987. doi:10.1093/nar/gkt1249. Kidder, B.L., Hu, G., and Zhao, K. 2011. ChIP-Seq: technical considerations for obtaining high-quality data. Nat. Immunol. Volume 12(Number 10): 918–922. Kleczkowski, L.A. 1994. Glucose Activation and Metabolism Through in Plants Udp-Glucose. Phytochemistry 37(6): 1507–1515. Kleczkowski, L.A., Kunz, S., and Wilczynska, M. 2010. Mechanisms of UDP-glucose synthesis in plants. CRC. Crit. Rev. Plant Sci. 29(4): 191–203. 68doi:10.1080/07352689.2010.483578. Koch, K. 2004. Sucrose metabolism: Regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr. Opin. Plant Biol. 7(3): 235–246. doi:10.1016/j.pbi.2004.03.014. Kubicki, J.D., Yang, H., Sawada, D., O’Neill, H., Oehme, D., and Cosgrove, D. 2018. The Shape of Native Plant Cellulose Microfibrils. Sci. Rep. 8(1): 4–11. doi:10.1038/s41598-018-32211-w. Kühn, C., and Grof, C.P.L. 2010. Sucrose transporters of higher plants. Curr. Opin. Plant Biol. 13(3): 287–297. doi:10.1016/j.pbi.2010.02.001. Li, J., Wu, L., Foster, R., and Ruan, Y.L. 2017. Molecular regulation of sucrose catabolism and sugar transport for development, defence and phloem function. J. Integr. Plant Biol. 59(5): 322–335. doi:10.1111/jipb.12539. Lindemose, S., Jensen, M.K., De Velde, J. Van, O’Shea, C., Heyndrickx, K.S., Workman, C.T., Vandepoele, K., Skriver, K., and Masi, F. De. 2014. A DNA-binding-site landscape and regulatory network analysis for NAC transcription factors in Arabidopsis thaliana. Nucleic Acids Res. 42(12): 7681–7693. doi:10.1093/nar/gku502. Livak, K.J., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25(4): 402–408. doi:10.1006/meth.2001.1262. Mahboubi, A., Ratke, C., Gorzsás, A., Kumar, M., Mellerowicz, E.J., and Niittylä, T. 2013. Aspen SUCROSE TRANSPORTER3 allocates carbon into wood fibers. Plant Physiol. 163(4): 1729–1740. doi:10.1104/pp.113.227603. De Meester, B., De Vries, L., Özparpucu, M., Gierlinger, N., Corneillie, S., Pallidis, A., Goeminne, G., Morreel, K., De Bruyne, M., De Rycke, R., Vanholme, R., and Boerjana, W. 2018. Vessel-specific reintroduction of CINNAMOYL-COA REDUCTASE1 69(CCR1) in Dwarfed ccr1 mutants restores vessel and Xylary fiber integrity and increases biomass. Plant Physiol. 176(1): 611–633. doi:10.1104/pp.17.01462. Mehrotra, R., Gupta, G., Sethi, R., Bhalothia, P., Kumar, N., and Mehrotra, S. 2011. Designer promoter: An artwork of cis engineering. Plant Mol. Biol. 75(6): 527–536. doi:10.1007/s11103-011-9755-3. Mejía-Guerra, M.K., Li, W., Galeano, N.F., Vidal, M., Gray, J., Doseff, A.I., and Grotewold, E. 2015. Core promoter plasticity between maize tissues and genotypes contrasts with predominance of sharp transcription initiation sitesopen. Plant Cell 27(12): 3309–3320. doi:10.1105/tpc.15.00630. Meyer, S., Melzer, M., Truernit, E., Hümmer, C., Besenbeck, R., Stadler, R., and Sauer, N. 2000. AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer. Plant J. 24(6): 869–882. doi:10.1046/j.1365-313X.2000.00934.x. Milne, R.J., Reinders, A., Ward, J.M., Offler, C.E., Patrick, J.W., and Grof, C.P.L. 2017. Contribution of sucrose transporters to phloem unloading within Sorghum bicolor stem internodes. Plant Signal. Behav. 12(5): 5–8. Taylor & Francis. doi:10.1080/15592324.2017.1319030. Nakano, Y., Yamaguchi, M., Endo, H., Rejab, N.A., and Ohtani, M. 2015. NAC-MYB-based transcriptional regulation of secondary cell wall biosynthesis in land plants. Front. Plant Sci. 6(MAY): 1–18. doi:10.3389/fpls.2015.00288. Negi, S., Tak, H., and Ganapathi, T.R. 2018. Xylem specific activation of 5’ upstream regulatory region of two NAC transcription factors (MusaVND6 and MusaVND7) in banana is regulated by SNBE-like sites. PLoS One 13(2): 1–23. doi:10.1371/journal.pone.0192852. Nikolov, D.B., and Burley, S.K. 1997. RNA polymerase II transcription initiation: A 70structural view. Proc. Natl. Acad. Sci. U. S. A. 94(1): 15–22. doi:10.1073/pnas.94.1.15. Nixon, B.T., Mansouri, K., Singh, A., Du, J., Davis, J.K., Lee, J.G., Slabaugh, E., Vandavasi, V.G., O’Neill, H., Roberts, E.M., Roberts, A.W., Yingling, Y.G., and Haigler, C.H. 2016. Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex. Sci. Rep. 6(March): 1–14. Nature Publishing Group. doi:10.1038/srep28696. Nolte, K.D., and Koch, K.E. 1993. Companion-cell specific localization of sucrose synthase in zones of phloem loading and unloading. Plant Physiol. 101(3): 899–905. doi:10.1104/pp.101.3.899. O’Malley, R.C., Huang, S.S.C., Song, L., Lewsey, M.G., Bartlett, A., Nery, J.R., Galli, M., Gallavotti, A., and Ecker, J.R. 2016. Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape. Cell 165(5): 1280–1292. Elsevier Inc. doi:10.1016/j.cell.2016.04.038. O’Sullivan, A.C. 1997. Cellulose: the structure slowly unravels. Cellulose 4(3): 173–207. Available from http://link.springer.com/article/10.1023/A:1018431705579. Ohtani, M., Nishikubo, N., Xu, B., Yamaguchi, M., Mitsuda, N., Goué, N., Shi, F., Ohme-Takagi, M., and Demura, T. 2011. A NAC domain protein family contributing to the regulation of wood formation in poplar. Plant J. 67(3): 499–512. doi:10.1111/j.1365-313X.2011.04614.x. Olins, J.R., Lin, L., Lee, S.J., Trabucco, G.M., Mackinnon, K.J.M., and Hazen, S.P. 2018. Secondary wall regulating NACs differentially bind at the promoter at a CELLULOSE SYNTHASE A4 Cis-eQTL. Front. Plant Sci. 9(December): 1–12. doi:10.3389/fpls.2018.01895. Oparka, K.J., and Turgeon, R. 1999. Sieve elements and companion cells - Traffic control centers of the phloem. Plant Cell 11(4): 739–750. doi:10.1105/tpc.11.4.739. 71Pandey, B., Prakash, P., Verma, P.C., and Srivastava, R. 2018. Regulated gene expression by synthetic modulation of the promoter architecture in plants. In Current Developments in Biotechnology and Bioengineering: Synthetic Biology, Cell Engineering and Bioprocessing Technologies. Elsevier B.V. doi:10.1016/B978-0-444-64085-7.00010-1. Pandiarajan, R., and Grover, A. 2018. In vivo promoter engineering in plants: Are we ready? Plant Sci. 277(May): 132–138. doi:10.1016/j.plantsci.2018.10.011. Payyavula, R.S., Tay, K.H.C., Tsai, C.J., and Harding, S.A. 2011. The sucrose transporter family in populus: The importance of a tonoplast PtaSUT4 to biomass and carbon partitioning. Plant J. 65(5): 757–770. doi:10.1111/j.1365-313X.2010.04463.x. Peng, F.Y., Hu, Z., and Yang, R.C. 2016. Bioinformatic prediction of transcription factor binding sites at promoter regions of genes for photoperiod and vernalization responses in model and temperate cereal plants. BMC Genomics 17(1): 1–16. BMC Genomics. doi:10.1186/s12864-016-2916-7. Pommerrenig, B., Popko, J., Heilmann, M., Schulmeister, S., Dietel, K., Schmitt, B., Stadler, R., Feussner, I., and Sauer, N. 2013. SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation. Plant J. 73(3): 392–404. doi:10.1111/tpj.12037. Poudel, K., Luo, X., Chen, L., Jing, D., Xia, X., Tang, L., Li, H., and Cao, S. 2020. Identification of the SUT gene family in pomegranate (Punica granatum l.) and functional analysis of pgl0145810.1. Int. J. Mol. Sci. 21(18): 1–14. doi:10.3390/ijms21186608. Purushotham, P., Ho, R., and Zimmer, J. 2020. Architecture of a catalytically active homotrimeric plant cellulose synthase complex. Science (80-. ). 369(6507): 1089–1094. doi:10.1126/science.abb2978. Rajewska, M., Wegrzyn, K., and Konieczny, I. 2012. AT-rich region and repeated sequences 72- the essential elements of replication origins of bacterial replicons. FEMS Microbiol. Rev. 36(2): 408–434. doi:10.1111/j.1574-6976.2011.00300.x. Riesmeier, J.W., Willmitzer, L., and Frommer, W.B. 1994. Evidence for an essential role of the sucrose transporter in phloem loading and assimilate partitioning. EMBO J. 13(1): 1–7. doi:10.1002/j.1460-2075.1994.tb06229.x. Roitsch, T., and González, M.C. 2004. Function and regulation of plant invertases: Sweet sensations. Trends Plant Sci. 9(12): 606–613. doi:10.1016/j.tplants.2004.10.009. Rombauts, S., Florquin, K., Lescot, M., Marchal, K., Rouzé, P., and van de Peer, Y. 2003. Computational approaches to identify promoters and cis-regulatory elements in plant genomes. Plant Physiol. 132(3): 1162–76. doi:10.1104/pp.102.017715.nomes. Rottmann, T.M., Fritz, C., Lauter, A., Schneider, S., Fischer, C., Danzberger, N., Dietrich, P., Sauer, N., and Stadler, R. 2018. Protoplast-esculin assay as a new method to assay plant sucrose transporters: Characterization of AtSUC6 and AtSUC7 sucrose uptake activity in arabidopsis Col-0 ecotype. Front. Plant Sci. 9(April): 1–22. doi:10.3389/fpls.2018.00430. Ruan, Y.-L. 2014. Sucrose Metabolism: Gateway to Diverse Carbon Use and Sugar Signaling. Annu. Rev. Plant Biol. 65(1): 33–67. doi:10.1146/annurev-arplant-050213-040251. Schmitz, R.J., Grotewold, E., and Stam, M. 2022. Cis-regulatory sequences in plants: Their importance, discovery, and future challenges. Plant Cell 34(2): 718–741. doi:10.1093/plcell/koab281. Schuetz, M., Smith, R., and Ellis, B. 2013. Xylem tissue specification, patterning, and differentiation mechanisms. J. Exp. Bot. 64(1): 11–31. Shahmuradov, I.A., Gammerman, A.J., Hancock, J.M., Bramley, P.M., and Solovyev, V. V. 2003. PlantProm: A database of plant promoter sequences. Nucleic Acids Res. 31(1): 73114–117. doi:10.1093/nar/gkg041. Shaul, O. 2017. How introns enhance gene expression. Int. J. Biochem. Cell Biol. 91(July): 145–155. Elsevier. doi:10.1016/j.biocel.2017.06.016. Sivitz, A.B., Reinders, A., Johnson, M.E., Krentz, A.D., Grof, C.P.L., Perroux, J.M., and Ward, J.M. 2007. Arabidopsis sucrose transporter AtSUC9. High-affinity transport activity, intragenic control of expression, and early flowering mutant phenotype. Plant Physiol. 143(1): 188–198. doi:10.1104/pp.106.089003. Slewinski, T.L., and Braun, D.M. 2010. Current perspectives on the regulation of whole-plant carbohydrate partitioning. Plant Sci. 178(4): 341–349. Elsevier Ireland Ltd. doi:10.1016/j.plantsci.2010.01.010. Slewinski, T.L., Garg, A., Johal, G.S., and Braun, D.M. 2010. Maize SUT1 functions in phloem loading. Plant Signal. Behav. 5(6): 687–690. doi:10.4161/psb.5.6.11575. Smith, R.A., Schuetz, M., Roach, M., Mansfield, S.D., Ellis, B., and Samuels, L. 2013. Neighboring parenchyma cells contribute to Arabidopsis xylem lignification, while lignification of interfascicular fibers is cell autonomous. Plant Cell 25(10): 3988–3999. doi:10.1105/tpc.113.117176. Srivastava, A.C., Ganesan, S., Ismail, I.O., and Ayre, B.G. 2008. Functional characterization of the Arabidopsis AtSUC2 sucrose/H+ symporter by tissue-specific complementation reveals an essential role in phloem loading but not in long-distance transport. Plant Physiol. 148(1): 200–211. doi:10.1104/pp.108.124776. Stadler, R., Truernit, E., Gahrtz, M., and Sauer, N. 1999. The AtSUC1 sucrose carrier may represent the osmotic driving force for anther dehiscence and pollen tube growth in Arabidopsis. Plant J. 19(3): 269–278. doi:10.1046/j.1365-313X.1999.00527.x. Stork, J., Harris, D., Griffiths, J., Williams, B., Beisson, F., Li-Beisson, Y., Mendu, V., Haughn, G., and DeBolt, S. 2010. CELLULOSE SYNTHASE9 serves a nonredundant 74role in secondary cell wall synthesis in arabidopsis epidermal testa cells. Plant Physiol. 153(2): 580–589. doi:10.1104/pp.110.154062. Sturm, A. 1999. Update on Biochemistry Invertases . Primary Structures , Functions , and Roles in Plant Development and Sucrose Partitioning Some Common Molecular Features but Differ. Plant Physiol. 121(September): 1–7. Sturm, A., and Chrispeels, M.J. 1990. CDNA cloning of carrot extracellular β-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell 2(11): 1107–1119. doi:10.1105/tpc.2.11.1107. Sullivan, S., Ralet, M.C., Berger, A., Diatloff, E., Bischoff, V., Gonneau, M., Marion-Poll, A., and North, H.M. 2011. CESA5 is required for the synthesis of cellulose with a role in structuring the adherent mucilage of Arabidopsis seeds. Plant Physiol. 156(4): 1725–1739. doi:10.1104/pp.111.179077. Takata, N., Awano, T., Nakata, M.T., Sano, Y., Sakamoto, S., Mitsuda, N., and Taniguchi, T. 2019. Populus NST/SND orthologs are key regulators of secondary cell wall formation in wood fibers, phloem fibers and xylem ray parenchyma cells. Tree Physiol. 39(4): 514–525. doi:10.1093/treephys/tpz004. Tamura, T., Endo, H., Suzuki, A., Sato, Y., Kato, K., Ohtani, M., Yamaguchi, M., and Demura, T. 2019. Affinity-based high-resolution analysis of DNA binding by VASCULAR-RELATED NAC-DOMAIN7 via fluorescence correlation spectroscopy. Plant J. 100(2): 298–313. doi:10.1111/tpj.14443. Taylor, N.G., Howells, R.M., Huttly, A.K., Vickers, K., and Turner, S.R. 2003. Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc. Natl. Acad. Sci. U. S. A. 100(3): 1450–1455. doi:10.1073/pnas.0337628100. Tjian, R., and Maniatis, T. 1994. Transcriptional activation: A complex puzzle with few easy pieces. Cell 77(1): 5–8. doi:10.1016/0092-8674(94)90227-5. 75Tuskan, G.A., DiFazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., Putnam, N., Ralph, S., Rombauts, S., Salamov, A., Schein, J., Sterck, L., Aerts, A., Bhalerao, R.R., Bhalerao, R.P., Blaudez, D., Boerjan, W., Brun, A., Brunner, A., Busov, V., Campbell, M., Carlson, J., Chalot, M., Chapman, J., Chen, G.-L., Cooper, D., Coutinho, P.M., Couturier, J., Covert, S., Cronk, Q., Cunningham, R., Davis, J., Degroeve, S., Dejardin, A., DePamphilis, C., Detter, J., Dirks, B., Dubchak, I., Duplessis, S., Ehlting, J., Ellis, B., Gendler, K., Goodstein, D., Gribskov, M., Grimwood, J., Groover, A., Gunter, L., Hamberger, B., Heinze, B., Helariutta, Y., Henrissat, B., Holligan, D., Holt, R., Huang, W., Islam-Faridi, N., Jones, S., Jones-Rhoades, M., Jorgensen, R., Joshi, C., Kangasjarvi, J., Karlsson, J., Kelleher, C., Kirkpatrick, R., Kirst, M., Kohler, A., Kalluri, U., Larimer, F., Leebens-Mack, J., Leple, J.-C., Locascio, P., Lou, Y., Lucas, S., Martin, F., Montanini, B., Napoli, C., Nelson, D.R., Nelson, C., Nieminen, K., Nilsson, O., Pereda, V., Peter, G., Phillipe, R., Pilate, G., Poliakov, A., Razumovskaya, J., Richardson, P., Rinaldi, C., Ritland, K., Rouze, P., Ryaboy, D., Schmutz, J., Schrader, J., Segerman, B., Shin, H., Siddiqui, A., Sterky, F., Terry, A., Tsai, C.-J., Uberbacher, E., Unneberg, P., Vahala, J., Wall, K., Wessler, S., Yang, G., Yin, T., Douglas, C., Marra, M., Sandberg, G., Van de Peer, Y., and Rokhsar, D. 2006. The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray). Science (80-. ). 313: 1596–1604. Venter, M., and Botha, F.C. 2010. Plant developmental biology. Plant Dev. Biol. 2: 393–414. doi:10.1007/978-3-642-04670-4. Verbančič, J., Lunn, J.E., Stitt, M., and Persson, S. 2018. Carbon Supply and the Regulation of Cell Wall Synthesis. Mol. Plant 11(1): 75–94. doi:10.1016/j.molp.2017.10.004. Wan, H., Wu, L., Yang, Y., Zhou, G., and Ruan, Y.L. 2018. Evolution of Sucrose Metabolism: The Dichotomy of Invertases and Beyond. Trends Plant Sci. 23(2): 163–177. Elsevier Ltd. doi:10.1016/j.tplants.2017.11.001. 76Wang, F., Sanz, A., Brenner, M.L., and Smith, A. 1993. Sucrose synthase, starch accumulation, and tomato fruit sink strength. Plant Physiol. 101(1): 321–327. doi:10.1104/pp.101.1.321. Wang, W., Viljamaa, S., Hodek, O., Moritz, T., and Niittylä, T. 2022. Sucrose synthase activity is not required for cellulose biosynthesis in Arabidopsis. Plant J. 110(5): 1493–1497. doi:10.1111/tpj.15752. Watanabe, Y., Meents, M.J., McDonnell, L.M., Barkwill, S., Sampathkumar, A., Cartwright, H.N., Demura, T., Ehrhardt, D.W., Samuels, A.., and Mansfield, S.. 2015. Visualization of cellulose synthases in Arabidopsis secondary cell walls. Plant Sci. 350(6257): 198–204. Wilkerson, C.G., Mansfield, S.D., Lu, F., Withers, S., Park, J.Y., Karlen, S.D., Gonzales-Vigil, E., Padmakshan, D., Unda, F., Rencoret, J., and Ralph, J. 2014. Monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone. Science (80-. ). 344(6179): 90–93. doi:10.1126/science.1250161. Willats, W.G.T., McCartney, L., Mackie, W., and Knox, J.P. 2001. Toward optimal ordering of prediction tasks. Plant Mol. Biol. 47: 9–27. doi:10.1137/1.9781611972795.76. Wind, J., Smeekens, S., and Hanson, J. 2010. Sucrose: Metabolite and signaling molecule. Phytochemistry 71(14–15): 1610–1614. Elsevier Ltd. doi:10.1016/j.phytochem.2010.07.007. Winzell, A., Aspeborg, H., Wang, Y., and Ezcurra, I. 2010. Conserved CA-rich motifs in gene promoters of Pt×tMYB021-responsive secondary cell wall carbohydrate-active enzymes in Populus. Biochem. Biophys. Res. Commun. 394(3): 848–853. Elsevier Inc. doi:10.1016/j.bbrc.2010.03.101. Wohlert, M., Benselfelt, T., Wågberg, L., Furó, I., Berglund, L.A., and Wohlert, J. 2022. Cellulose and the role of hydrogen bonds: not in charge of everything. Cellulose 29(1): 771–23. doi:10.1007/s10570-021-04325-4. Xu, M., Gonzalez-Hurtado, E., and Martinez, E. 2016. Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription. Biochim. Biophys. Acta - Gene Regul. Mech. 1859(4): 553–563. Elsevier B.V. doi:10.1016/j.bbagrm.2016.01.005. Yao, D., Gonzales-Vigil, E., and Mansfield, S.D. 2020. Arabidopsis sucrose synthase localization indicates a primary role in sucrose translocation in phloem. J. Exp. Bot. 71(6): 1858–1869. doi:10.1093/jxb/erz539. Zhang, C., Han, L., Slewinski, T.L., Sun, J., Zhang, J., Wang, Z.Y., and Turgeon, R. 2014. Symplastic phloem loading in poplar. Plant Physiol. 166(1): 306–313. doi:10.1104/pp.114.245845. Zhang, D., Xu, B., Yang, X., Zhang, Z., and Li, B. 2011. The sucrose synthase gene family in Populus: Structure, expression, and evolution. Tree Genet. Genomes 7(3): 443–456. doi:10.1007/s11295-010-0346-2. Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W., and Chua, N.H. 2006. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1(2): 641–646. doi:10.1038/nprot.2006.97. Zheng, Y., Anderson, S., Zhang, Y., and Garavito, R.M. 2011. The structure of sucrose synthase-1 from arabidopsis thaliana and its functional implications. J. Biol. Chem. 286(41): 36108–36118. doi:10.1074/jbc.M111.275974. Zhong, R., and Ye, Z.H. 2007. Regulation of cell wall biosynthesis. Curr. Opin. Plant Biol. 10(6): 564–572. doi:10.1016/j.pbi.2007.09.001. Zhu, X., Wang, M., Li, X., Jiu, S., Wang, C., and Fang, J. 2017. Genome-wide analysis of the Sucrose synthase gene family in grape (Vitis vinifera): Structure, evolution, and expression profiles. Genes (Basel). 8(4). doi:10.3390/genes8040111. 78Zou, C., Lu, C., Shang, H., Jing, X., Cheng, H., Zhang, Y., and Song, G. 2013. Genome-Wide analysis of the sus gene family in cotton. J. Integr. Plant Biol. 55(7): 643–653. doi:10.1111/jipb.12068. Zrenner, R., Salanoubat, M., Willmitzer, L., and Sonnewald, U. 1995. Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant J. 7(1): 97–107. doi:https://doi.org/10.1046/j.1365-313X.1995.07010097.x. 79 Appendices Appendix A: Additional Materials and Methods Appendix Table A.1. PCR primers employed in this study No. Name Use description Sequence (5'-3') 26 PtSUS1p_RV26 Amplification of complete PtSUS1 promoter GCAGTAGGCGGGTTCTATCA 35 PtSUS1p _FW35 GCCTAGCAACACTTGACCTC 38 PtSUS1p _FW38_attB1 attB site addition to PtSUS1p GGGGACAAGTTTGTACAAAAAAGCAGG CTTCGCCTAGCAACACTTGACCTC 39 PtSUS1p RV39_attB2 GGGGACCACTTTGTACAAGAAAGCTGGG TGGCAGTAGGCGGGTTCTATCA 32 PtSUS2p _FW32 Amplification second half of PtSUS2p CGCATAGCAGCACAACT 75 PtSUS2p _RV75 GTCCCTTTCACAAAACCGCT 89 PtSUS2p _FW89 Amplification first half of PtSUS2p CTCCAGGACACCAATGTAG 97 PtSUS2p _RV97 GACTGGTAGATGGATGGATG 110 GUS screening_110FW GUS screening CACCGAAGTTCATGCCAG 111 GUS screening_111RV GACGGCCTGTGGGCATTCAG 120 Pt.SUS1p_DP.trunc_120FW FW primer truncation 1 (DT) GTAAATGACCTGTGTTGCGA TATTAAATATC 121 Pt.SUS1p_PX.trunc_121FW FW primer truncation 2 (PT) CTTGTCGGTGCATGATGCCAC 122 Pt.SUS1p_CP.trunc_122FW FW primer truncation 3 (CT) CAAATTAAGAGAAAAGAGTGGTG 132 PtSUS1p _FW132_attB1 attB site addition to truncation 1 (DT) GGGGACAAGTTTGTACAAAAAAGCAGGC TTCGACCTGTGTTGCGATATTAAATATC 133 PtSUS1p _FW133_attB1 attB site addition to truncation 2 (PT) GGGGACAAGTTTGTACAAAAAAGCAGGC TTCCTTGTCGGTGCATGATGCCAC 134 PtSUS1p _FW134_attB1 attB site addition to truncation 3 (CT) GGGGACAAGTTTGTACAAAAAAGCAGGCT TCCAAATTAAGAGAAAAGAGTGGTG 80Appendix Table A.2. qRT-PCR primers employed in this study Name Sequence (5'-3') EF1B_FW_Pt GGCATTAAGTTTTGTCGGTCTG EF1B_RV_Pt GCGGTTCATCATTTCATCTGG UBQ11_FW_Pt GTTGATTTTTGCTGGGAAGC UBQ11_RV_Pt GATCTTGGCCTTCACGTTGT qPCR_SUS1intron_FW CCCAGCATCCTTTTCGCTTGCTT qPCR_SUS1intron_RV GGCAGTAGGCGGGTTCTATCATAG qPCR_GUS_FW GCACTCAATGTACACCGACATG qPCR_GUS_RV CGGCGAAATTCCATACCTGTTCAC qPCR_GUS.2_FW GTCGCGCAAGACTGTAACCACG qPCR_GUS.2_RV GCTAGTGCCTTGTCCAGTTGC qPCR_eGFP_FW GATCACTCTCGGCATGGACGAG qPCR_eGFP_RV CGATCCAGACTGAATGCCCACA Appendix B: Additional motif information Appendix Table B.1. Motifs identified in this study Motif Name Sequence (5' - 3') Description CAm1 CAGCAA MYB46-like TFs (target SCW biosynthesis genes, xylem-specific) CAm2 CCACCA CAm3 ACCAAC CAm4 CACCAA Inducible NAC TFs CGT[A/G] Core binding site for NAC TFs in SCW biosynthesis SNBE-like [T/A]NN[C/T][T/C/G]TNNNNNNNA[A/C] GN[A/C/T][A/T] Binding site for VNDs and SND1 TFs (xylem vessels) TERE CTTNAAAGCNA Binding site for VNDs and NST NAC proteins VND7 CTTNNCTTNNAAGNNAAG Closely related to SCW deposition, xylem vessel formation and differentiation SND1 T[G/A]CGT Involved in SCW biosynthesis in interfascicular and xylary fibers 81Appendix C: GUS staining controls and additional troubleshooting information Appendix Table C.1. GUS positive controls employed in this study Construct name Background Expected GUS activity Obtained from At4CL::GUS Landsberg Xylem tissues of the root and shoot Dr. Abel Rosado RPL4A::GUS Col-0 Correlated to free Auxin distribution (leaves, root tip, root vasculature, anthers, stigma, etc.) Dr. Abel Rosado PrABCB11::GUS Col-0 Phloem, xylem parenchyma, cortex, and epidermis Dr. Lacey Samuels Appendix Figure C.1. Vector map of the PtSUS1p::GUS construct (background pKGWFS7) displaying the two main ORFs (thin yellow arrows). ORF-1 shows the complete transcriptional length where transcription would start at the true start codon in the eGFP sequence, which would result in a viable GUS protein. ORF-2 shows a +2 frameshift, creating an earlier start codon within the intron sequence, as well as an early stop codon which would result in a nonsense mutation in the GUS protein sequence. 82 Appendix Figure C.2. Amino acid sequence comparison of the resulting protein translated from ORF-1 and ORF-2. (a) ORF-1 results in an 845 amino acid (aa) functional protein, capable of producing the signature blue GUS stain after treatment. (b) Due to the +2 frameshift in the cloned promoter sequence, ORF-2 results in a 332 aa non-functional protein.
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Comparing the sucrose synthase promoters in Arabidopsis thaliana and hybrid poplar
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