Science, Faculty of
Botany, Department of
DSpace
UBCV
Strelau, Matthew
Master of Science - MSc
University of British Columbia
The responses of plants to adverse environmental conditions require the adequate sensing of stress signals from the environment and their integration into coordinated cellular responses. These coordinated responses in plants often involve information exchanges between intracellular organelles that rely on vesicular trafficking and/or non-vesicular transport at membrane contact sites. This thesis focuses on the plant endoplasmic reticulum (ER) plasma membrane (PM) contact sites (EPCS), which are evolutionarily conserved nanostructures that facilitate exchanges of information between the cortical ER and the PM. The close association between membranes at EPCS is facilitated by physical tethering often mediated by lipid-transfer proteins. Members of one of these families, known as the plant synaptotagmins, are important elements conferring tolerance to biotic and abiotic stresses in Arabidopsis. Still, little is known about the regulatory mechanisms and environmental triggers controlling the expression and activity of the different synaptotagmins in plants. In this thesis, I use luciferase-based markers to perform systematic analyses of the SYNAPTOTAGMIN1 (SYT1) promoter activity and protein accumulation in Arabidopsis. My results show that SYT1 expression increases in response to salt-induced ionic and osmotic stresses, particularly when the stress generator contains Cl-. Remarkably, the amount of SYT1 protein does not change in response to salt stress indicating that SYT1 transcriptional and translational levels are uncoupled. By measuring the stability of SYT1 in response to NaCl, I determined that salt stress has an increased effect on the stability of the SYT1 protein and that its degradation is partially controlled by the 26S ubiquitin proteasome. I conducted an in silico analyses of gene expression for different members of the Arabidopsis synaptotagmin family and validated the results using SYT promoter - GUS fusions. My results show that while some Arabidopsis SYTs are ubiquitous, some others are expressed in specific tissues, and that their expression is environmentally and developmentally controlled. My results advance our understanding of the expression and regulation of individual SYTs in Arabidopsis and will facilitate the study of their specific contribution/s to the overall stress tolerance in plants.
ANALYSIS OF THE ARABIDOPSIS SYNAPTOTAGMINS GENE EXPRESSION AND PROTEIN ACCUMULATION DURING STRESS USING LUCIFERASE-BASED MARKERS by MATTHEW STRELAU B.Sc., Trinity Western University, 2017 A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (BOTANY) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2022 © Matthew Strelau, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: ANALYSIS OF THE ARABIDOPSIS SYNAPTOTAGMINS GENE EXPRESSION AND PROTEIN ACCUMULATION DURING STRESS USING LUCIFERASE-BASED MARKERS submitted by MATTHEW STRELAU in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in BOTANY Examining Committee: Abel Rosado, Associate Professor, Botany, UBC Supervisor George Haughn, Professor, Botany, UBC Supervisory Committee Member Lacey Samuels, Professor, Head of Botany, UBC Supervisory Committee Member Simone Castellarin, Associate Professor, Land and Food Systems, UBC Additional Examiner Additional Supervisory Committee Members: Geoffrey Wasteneys, Professor, Botany, UBC Supervisory Committee Member iii Abstract The responses of plants to adverse environmental conditions require the adequate sensing of stress signals from the environment and their integration into coordinated cellular responses. These coordinated responses in plants often involve information exchanges between intracellular organelles that rely on vesicular trafficking and/or non-vesicular transport at membrane contact sites. This thesis focuses on the plant endoplasmic reticulum (ER) plasma membrane (PM) contact sites (EPCS), which are evolutionarily conserved nanostructures that facilitate exchanges of information between the cortical ER and the PM. The close association between membranes at EPCS is facilitated by physical tethering often mediated by lipid-transfer proteins. Members of one of these families, known as the plant synaptotagmins, are important elements conferring tolerance to biotic and abiotic stresses in Arabidopsis. Still, little is known about the regulatory mechanisms and environmental triggers controlling the expression and activity of the different synaptotagmins in plants. In this thesis, I use luciferase-based markers to perform systematic analyses of the SYNAPTOTAGMIN1 (SYT1) promoter activity and protein accumulation in Arabidopsis. My results show that SYT1 expression increases in response to salt-induced ionic and osmotic stresses, particularly when the stress generator contains Cl-. Remarkably, the amount of SYT1 protein does not change in response to salt stress indicating that SYT1 transcriptional and translational levels are uncoupled. By measuring the stability of SYT1 in response to NaCl, I determined that salt stress has an increased effect on the stability of the SYT1 protein and that its degradation is partially controlled by the 26S ubiquitin proteasome. I conducted an in silico analyses of gene expression for different members of the Arabidopsis synaptotagmin family and validated the results using SYT promoter - GUS fusions. iv My results show that while some Arabidopsis SYTs are ubiquitous, some others are expressed in specific tissues, and that their expression is environmentally and developmentally controlled. My results advance our understanding of the expression and regulation of individual SYTs in Arabidopsis and will facilitate the study of their specific contribution/s to the overall stress tolerance in plants. v Lay Summary Plants endure changes to the environment, such as freezing temperatures, drought, flooding, and salt accumulation. The plant protein SYNAPTOTAGMIN1 (SYT1) has an important role in plant survival, especially when the soil becomes too salty due to fertilizers; salt tolerance decreases when SYT1 is absent from plants. Protein amounts are determined by the activity of the respective gene; to study the relationship between the SYT1 gene and the SYT1 protein, I measured when the gene is turned on and how much protein is made in different salts. I found that the SYT1 gene is turned on during most salt stresses, but the protein does not increase. I searched for changing gene expression for other SYTs and found that genetic expression varied within the members of the Arabidopsis SYT family. My results aid in our understanding of stress responsive genes and their relationship to protein amounts. vi Preface The transgenic pSYT1:LUC plant lines used in Chapter 3 and Chapter 4 were generated by Makoto Shirakawa. The pSYT1:SYT1-LUC transgenic lines used for Chapter 3 and 4 were generated by EunKyoung Lee. The generation of the complementation lines, SYT1-LUC/syt1-2 were generated by me under the guidance of EunKyoung Lee. Segregation of the luciferase lines were initiated by Elizabeth Samuels and completed by me. The IAA1-LUC line used in Chapter 4 was generously supplied by Judy Callis. The liquid treatments conducted in Chapter 3 were often completed with the help of Brenda Vila Nova Santana. Imaging and transfer of seedlings in Chapter 3 were completed with the help of Diana Vasileva and Erica Marandola. DNA work for Chapter 5 was organized by EunKyoung Lee and performed by me. The remaining experiments and transgenic lines were performed and generated by me. vii Table of Contents Abstract ...............................................................................................................................................................iii Lay Summary ...................................................................................................................................................... v Preface ................................................................................................................................................................. vi Table of Contents ............................................................................................................................................ vii List of Tables ....................................................................................................................................................... x List of Figures .................................................................................................................................................... xi List of Abbreviations.................................................................................................................................... xiii Acknowledgements ..................................................................................................................................... xvii Dedication ..................................................................................................................................................... xviii Chapter 1: Introduction .................................................................................................................................. 1 1.1 Soil salinization and plant stress ........................................................................................................................................... 1 1.2 Cellular Signalling in Response to Salt Stress .................................................................................................................. 2 1.3 Membrane contact sites and stress ...................................................................................................................................... 3 1.4 Endoplasmic reticulum-plasma membrane contact sites and associated tethers .......................................... 4 1.5 The Arabidopsis SYNAPTOTAGMIN1 (SYT1) EPCS tether. ....................................................................................... 7 1.6 Research Objectives .................................................................................................................................................................... 9 Chapter 2: Materials and Methods ........................................................................................................... 12 2.1 Plant Materials and Growth Conditions ........................................................................................................................... 12 2.2 Cloning ............................................................................................................................................................................................ 12 2.2.1 Transformation of E. coli ............................................................................................................................................... 12 2.2.2 Transformation of Agrobacterium ........................................................................................................................... 14 2.2.3 Transformation of Arabidopsis .................................................................................................................................. 15 viii 2.2.4 DNA and RNA Extraction .............................................................................................................................................. 15 2.3 Salt Sensitivity Tests ................................................................................................................................................................. 16 2.4 Chemical Treatment ................................................................................................................................................................. 16 2.4.1 Liquid Stress Treatment ................................................................................................................................................ 16 2.4.2 Cycloheximide Treatment ............................................................................................................................................ 16 2.5 Bioluminescence Assay ........................................................................................................................................................... 17 2.5.1 Image Acquisition ............................................................................................................................................................. 17 2.5.2 Image Quantification ....................................................................................................................................................... 18 2.6 Histochemical GUS staining ............................................................................................................................................. 18 2.7 Bioinformatic Analysis ............................................................................................................................................................ 19 2.8 Accession Numbers ................................................................................................................................................................... 20 Chapter 3: Study of the Correlation Between SYT1 Gene Activity and SYT1 Protein Amounts ............................................................................................................................................................ 21 3.1 Introduction ................................................................................................................................................................................. 21 3.2 Results ............................................................................................................................................................................................. 26 3.2.1 SYT1 expression throughout development .......................................................................................................... 26 3.2.2 Changes to the SYT1 promoter expression in response to abiotic stress................................................ 31 3.2.3 Changes in SYT1 protein levels in response to abiotic stress ....................................................................... 37 3.3 Discussion ..................................................................................................................................................................................... 44 Chapter 4: SYT1 Stability and Degradation .......................................................................................... 50 4.1 Introduction ................................................................................................................................................................................. 50 4.2 Results ............................................................................................................................................................................................. 53 4.2.1 Leaf disc samples accurately measure in vivo protein degradation .......................................................... 53 4.2.2 The stability of SYT1 increases in response to NaCl ......................................................................................... 55 4.2.3 SYT1 stability in response to proteasome inhibition ....................................................................................... 56 4.3 Discussion ..................................................................................................................................................................................... 58 ix Chapter 5: Comparative Gene Expression Analysis of the Arabidopsis Synaptotagmins .... 60 5.1 Introduction ................................................................................................................................................................................. 60 5.2 Results ............................................................................................................................................................................................. 63 5.2.1 Arabidopsis SYTs change expression throughout development ................................................................. 63 5.2.2 Most Arabidopsis SYTs do not change expression in response to abiotic stress ................................. 65 5.2.3 GUS Expression of the Arabidopsis SYTs are controlled by the respective upstream promoter regions .............................................................................................................................................................................................. 67 5.2.4 The Arabidopsis SYTs are grouped into four major clades ........................................................................... 71 5.3 Discussion ..................................................................................................................................................................................... 74 Chapter 6: Conclusions and Future Directions ................................................................................... 80 Bibliography .................................................................................................................................................... 82 Appendices ....................................................................................................................................................... 94 x List of Tables Table 2.1. Primers used for the amplification of promoter regions using gDNA in Arabidopsis thaliana (Col-0). ......................................................................................................................... 14 Table 2.2. Sequence size of the promoter region used for the SYT genes. ................................... 14 Table 3.1. Changes to pSYT1:LUC promoter activity and SYT1-LUC protein levels in response to abiotic stress-inducing chemicals. ...................................................................................... 44 Table 4.1. Summary of protein half-life and associated statistics in the presence of NaCl and MG132. ................................................................................................................................................................... 58 Table 5.1. The Arabidopsis family of synaptotagmins investigated in this thesis. ................... 64 Table 5.2. Frequency of the Arabidopsis SYT expression up-regulation and SYT expression downregulation in response to abiotic stress. ........................................................................................ 67 Table A1. Segregation analysis of GUS-expressing promoter lines the T2 generation. .......... 94 Table A2. Segregation analysis of pSYT1:LUC in Col-0 in the T2 generation. ............................ 94 Table A3. Tissue samples, in order from left to right of the x-axis in Figure 5.1 and the respective source for each datum. ............................................................................................................... 96 Table A4. Tissue samples and respective abiotic treatment in order from left to right of the x-axis in Figure 5.2. ............................................................................................................................................ 99 Table A5. Promoter binding sequences found in plant promoter databases, with the respective location in SYT upstream from the translational start site. ...................................... 100 Table A6. Predicted Motifs in the upstream promoter region of Arabidopsis NTMCs. ........ 100 xi List of Figures Figure 1. Protein domains and 3D structure of a canonical synaptotagmin in plants. .................... 7 Figure 3.1 Schematic summary of lipid transfer and associated proteins at the plasma membrane and endoplasmic reticulum in eukaryotes. .................................................................................... 25 Figure 3.2. pSYT1:LUC expression in the shoots and roots of Arabidopsis seedlings. ................ 27 Figure 3.3. Root elongation phenotypic complementation of the syt1 mutant with SYT1-LUC. 29 Figure 3.4. SYT1 protein levels in the shoots and roots of Arabidopsis seedlings. ..................... 30 Figure 3.5. pSYT1:LUC expression and SYT1-LUC protein levels in the shoot of adult Arabidopsis plants. ........................................................................................................................ 30 Figure 3.6. The change in pSYT1:LUC expression in response to salt stress. .............................. 32 Figure 3.7. The change in pSYT1:LUC expression in response to osmotic stress. ....................... 33 Figure 3.8. The change in pSYT1:LUC expression in response to Ca2+ channel disruption and chelation. ....................................................................................................................................... 35 Figure 3.9. The change in pSYT1:LUC expression in response to actin and microtubule disruption. ..................................................................................................................................... 36 Figure 3.10. The change in pSYT1:LUC expression in response to PI(4,5)P2 accumulation. ...... 37 Figure 3.11. The change in SYT1-LUC protein levels in response to salt stress. ........................ 38 Figure 3.12. The change in SYT1-LUC protein levels in response to osmotic stress. ................. 39 Figure 3.13. The change in SYT1-LUC protein levels in response to Ca2+ channel disruption and chelation. ....................................................................................................................................... 40 Figure 3.14. The change in SYT1-LUC protein levels in response to actin and microtubule disruption. ..................................................................................................................................... 41 Figure 3.15. The change in SYT1-LUC protein levels in response to PI(4,5)P2 accumulation. .. 42 Figure 4.1. The half-life of IAA1-LUC. ....................................................................................... 54 Figure 4.2. Changes to SYT1 protein stability in response to NaCl. ............................................ 56 Figure 4.3. Changes to SYT1 protein stability in response to the proteasome inhibitor MG132. 57 Figure 5.1. Gene expression of Arabidopsis SYT1-6 and CLB1 throughout development. .......... 65 xii Figure 5.2. The change in gene expression of Arabidopsis SYTs after exposure to abiotic stressors. ........................................................................................................................................ 67 Figure 5.3. Schematic diagram of gene structure for Arabidopsis SYT1-6/CLB1. ....................... 68 Figure 5.4. GUS expression of SYT1-6 and CLB1 one week after germination. .......................... 69 Figure 5.5. GUS expression of SYTs 1-6 and CLB1 in seedling roots. ......................................... 70 Figure 5.6. GUS stains in floral tissues......................................................................................... 71 Figure 5.7. The amino acid similarity between the Arabidopsis SYTs SMP domain. ................. 73 Figure 5.8. Simplified schematic of SYT expression in Arabidopsis seedlings. ........................... 79 Figure A1. Relationship between luciferase light intensity compared to the total mass of the seedlings for pSYT1:LUC. .......................................................................................................... 101 Figure A2. Relationship between luciferase light intensity compared to the total mass of the seedlings for SYT1-LUC. ........................................................................................................... 101 Figure A3. pSYT1:LUC expression in rosette leaves of 3 week old Arabidopsis plants. ........... 102 Figure A4. SYT1-LUC expression in rosette leaves of 3 week old Arabidopsis plants. ............ 102 Figure A5. The DNA nucleotide similarity between the Arabidopsis SYTs promoter region. ... 103 xiii List of Abbreviations BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid CCD Charge Coupled Device cER Cortical Endoplasmic Reticulum CHX Cycloheximide CLB1 CALCIUM-DEPENDENT LIPID-BINDING PROTEIN DAG Days After Germination EGTA Ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid EPCS Endoplasmic Reticulum-Plasma Membrane Contact Sites ER Endoplasmic Reticulum E-Syt Extended-Synaptotagmin FRAP Fluorescence Recovery After Photobleaching GFP Green Fluorescent Protein GTF General Transcription Factor GUS β-glucuronidase HKT High-affinity Potassium Transporter IAA1 INDOLE-3 ACETIC ACID INDUCIBLE 1 IP3 Inositol Triphosphate LatB Latrunculin B LPA Lyso-Phosphatidic Acid LUC Luciferase MAPPER Membrane-attached Peripheral ER xiv MCS Membrane Contact Sites MEGA Molecular Evolutionary Genetics Analysis mRNA Messenger RNA MG132 Carbobenzoxy-Leu-Leu-leucinal MT Microtubule MUSCLE Multiple Sequence Comparison by Log-Expectation NET3C NETWORKED 3C NHX N+/H+ EXCHANGER 1 NSCC Non-selective Cation Channels NSF N-ethylmaleimide sensitive fusion NTMC N-Terminal Transmembrane C2 Domain Proteins ORP OSBP-Related Protein OSBP Oxysterol-Binding Protein PA Phosphatidic Acid PCC Pearson Correlation Coefficient PCR Polymerase Chain Reaction PI Phosphatidylinositol PI4KA Phosphatidylinositol 4-Kinase Alpha PI4P Phosphatidylinositol 4-Phosphate PI(4,5)P2 Phosphatidylinositol 4,5-Bisphosphate PIP5K Phosphatidylinositol 4-Phosphate 5 Kinase PLACE Plant Cis-acting Regulatory DNA Elements PLC Phospholipase C xv PLD Phospholipase D PM Plasma Membrane PS Phosphatidylserine Pol II RNA Polymerase II pSYT1:LUC proSYT1:LUCIFERASE REE Rare earth elements ROS Reactive Oxygen Species r-protein Ribosomal-protein SCaBP8 SOS3-like Calcium Binding Protein 8 S-EPCS SYT1 ER-PM Contact Sites SHM1 SERINE HYDROXYMETHYLTRANSFERASE siRNA Small interfering RNA SMP Synaptotagmin-like mitochondrial lipid-binding domain SNAP Soluble NSF Attachment SNARE SNAP Receptor SOS Salt Overly Sensitive SYT Synaptotagmin SYT1 SYNAPTOTAGMIN1 SYT1-LUC proSYT1:SYT1-LUCIFERASE Tcb Tricalbin TraVA Transcriptome Variation Analysis tRNA Transfer RNA TULIP Tubular Lipid-Binding xvi U-73122 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] -1H-pyrrole-2,5-dione USB16 Ubiquitin-specific-protease 16 VAMP Vesicle-Associated Membrane Protein VAP Vesicle-Associated Membrane Protein-Associated Protein V-EPCS VAP27 ER-PM Contact Sites X-gluc 5-bromo-4-chloro-3-indoyl- β -D-glucuronic acid xvii Acknowledgements My greatest acknowledgement goes to Abel, for guiding me throughout my projects, assistance on this jarring journey through a global pandemic, supporting me with advice alongside repeatedly failed experiments, and for helping me grow as a scientist. Thank you to my family for providing the emotional and social support that distracted me from the utmost stressful and strained moments. Thank you, Kyle, for the academic advice, loving support, the reminder to relax, while also the critical accountability that enforced me to finish my work. I would also like to thank my committee members, George Haughn, Lacey Samuels, and Geoffrey Wasteneys for their time sacrificed towards advising my project and for the unique viewpoint that each member provided. Additionally, I would like to thank my advisor, Keith Adams. The company of my colleagues was a tremendous help, and I thank EunKyoung, Brenda, Ana, Gill, and the dedicated undergraduates. All the technical skills I acquired can be attributed to the help I received from Eun. She played an instrumental role in my growth in the lab and provided a tremendous support that I cannot put into words. Brenda was my biggest supporter within the first year of my degree and I hope to visit Brazil to see her. Ana has been my closest friend throughout my degree; through laughter, tears, and sweat (and sometimes blood), our bond has pushed us through the hardest moments. I would like to thank the Department of Botany, UBC, and acknowledge that UBC Vancouver is located on the traditional, ancestral, and unceded territory of the Musqueam people. xviii Dedication I would like to dedicate this thesis to my family and friends, with a special mention to my grandmother and my late “Gramps” (Wally Strelau, 1933-2020) and Omi (Erika Norgaard, 1936-2020). 1 Chapter 1: Introduction 1.1 Soil salinization and plant stress Plants are sessile and therefore incapable of rapidly changing their environment to avoid environmental stressors. As such, plants must simultaneously withstand multiple abiotic factors (e.g., low/high temperature, humidity, soil quality) and biotic factors (e.g., pathogens and herbivory) that stunt their growth and lower their survival rates. In agriculture, a common and unfavourable condition faced by crop plants is soil salinization. This adverse condition is often generated by the overuse of fertilizers, pesticides, and agricultural run-off that increases the concentration of toxic ions in the agricultural soils (Bouwer, 2003). Salinization affects more than 800 million hectares worldwide, and salt-induced land degradation is estimated to cost the global agriculture industry over 30 billion CAD annually (Munns & Tester, 2008; Qadir et al., 2014). Soil salinization is detrimental for plants as it triggers osmotic and ionic stress. Osmotic stress influences the osmoregulation of cells, and ionic stress induces the accumulation of toxic ions, which reduces plant fitness (Botella et al., 2007). Additionally, salinization is often present with additional stressors such as drought and cold, in a process known as multifactorial stress combination (Zandalinas et al., 2021). Understanding the complex plant responses to salinization and multifactorial stress combination is becoming an important focus of research, especially as the threats of climate change become pervasive (Zandalinas et al., 2021). This knowledge is essential for developing salt-tolerant varieties that improve the productivity of agriculture (Wang et al., 2003; Munns et al., 2012). 2 1.2 Cellular Signalling in Response to Salt Stress Plants exposed to high salt concentrations must sense the presence of the stressor and mount a coordinated response to survive. This response often involves three primary activities: (1) prevention or alleviation of damage, (2) re-establishment of cellular homeostasis, and (3) growth resumption (Zhang et al., 2022). The cellular responses to salt stress rely on sensor proteins that perceive salt-induced transient elevation of free cytosolic calcium ions ([Ca2+]Cyt) and trigger cascades that induce the activation or inactivation of specific stress-inducible signalling pathways (Davletova et al., 2005; Swarbreck et al., 2013; Zhu, 2016). These signalling pathways modulate different cellular responses to NaCl stress (Botella et al., 2007). In non-stressed conditions, nutritional Na+ uptake occurs through high-affinity potassium transporters (HKT) (Ji et al., 2013). As [Na+] increases in soil, Na+ enters the symplastic pathway through non-selective cation channels (NSCCs), and generates toxic [Na+] in the cytosol. As a result, plant cells activate safeguard mechanisms that reduce Na+ entry, compartmentalize cytosolic Na+ in the vacuole, and export cytosolic Na+ outside the cell to avoid further accumulation of Na+ in the cytosol (Ji et al., 2013). These processes are often coordinated by chemical signals, protein complex formation, and transporter activation (Zhu, 2016). Classical forward and reverse genetic screens using the model plant Arabidopsis thaliana have been essential to elucidate molecular mechanisms involved in the salt stress response in plants, such as the salt overly sensitive (SOS) signalling pathway. In Arabidopsis, salt stress triggers a rapid spike in cytosolic Ca2+ concentration which is recognized by Ca2+ sensors, specifically the calcium-binding protein SALT OVERLY SENSITIVE 3 (SOS3) in roots and the SOS3-LIKE CALCIUM BINDING PROTEIN 8 (SCaBP8) in shoots (Zhu, 2000). The binding of Ca2+ to these sensors stimulates their interaction and the subsequent formation of complexes 3 with the serine/threonine-protein kinase SOS2 (Halfter et al., 2000). The SOS2-SOS3 and SOS2-SCaBP8 complexes then re-localize the cytosolic SOS2 to the plasma membrane (PM), where the phosphorylation and activation of the Na+/H+ antiporter SOS1 occurs (Quan et al., 2007). The SOS1 Na+/H+ antiporter exports Na+ from the cytosol, reducing the [Na+]cyt to non-toxic levels and thus facilitating the Na+ homeostasis in the cell (Oh et al., 2009). 1.3 Membrane contact sites and stress In addition to the SOS pathway, plants have acquired alternative mechanisms to tolerate Na+ toxicity and withstand unfavourable conditions. These cellular responses often require the coordinated activity of intracellular membrane-bound organelles that relay signals and macromolecules across lipid membranes (Prinz, 2014). During NaCl stress, different organelles establish vesicular and non-vesicular forms of communication that bypass the organelle compartmentalization to integrate stress signals and coordinate the cellular responses. Examples of vesicular mechanisms activated in response to NaCl stress include the activation of bulk-flow endocytosis (Baral et al., 2015; Zwiewka et al., 2015) and the activation of the secretory pathway in which vesicles containing the SNARE PROTEIN SYNTAXIN OF PLANTS 61 (SYP61) are thought to transport the drought-sensitive aquaporin PIP2;7 from the trans-Golgi network (TGN) to the PM (Drakakaki et al., 2011; Besserer et al., 2012; Zhang et al., 2016; see Wang et al., 2019 for a comprehensive list of endomembrane trafficked proteins that participate in abiotic stress responses). Another mechanism that is believed to be active in response to NaCl stress is the non-vesicular transfer of information between the endoplasmic reticulum (ER) and associated organelles through membrane contact sites (MCS) (Pérez-Sancho et al., 2016). MCS are evolutionarily conserved nanostructures often established between the ER and closely associated 4 (3-80 nm) organelle membranes. These small intermembrane distances enable specific information exchange between organelles without the need for secretory vesicles (Stefan et al., 2013). MCSs can also be defined based on additional characteristics such as membrane composition, tethering mechanisms, and protein content that provide functional specificity (Pérez-Sancho et al., 2016). MCS establishment and function are facilitated by specialized proteins that prevent membrane fusions, regulate the bidirectional transport of molecules (e.g. lipids), and control the positioning of enzymes in trans (Scorrano et al., 2019). Based on their specific activities, MCS-resident proteins can be classified into four categories: structural proteins that facilitate membrane tethering, functional proteins such as lipid-transfer proteins, sorter proteins that define the lipidome and proteome at MCSs, and regulatory proteins that influence MCS protein activity (Scorrano et al., 2019). The collection of these proteins and membrane structural features confers specificity to the membrane contact site function. 1.4 Endoplasmic reticulum-plasma membrane contact sites and associated tethers A large proportion of the ER membranes in plants are found at the cell cortex and can potentially establish MCS association with the plasma membrane (Bayer et al., 2017). These ER-PM contact sites (EPCSs) are facilitated by tether proteins that stabilize the two membranes at a distance ranging from 3 nm at plasmodesmata EPCSs to an arbitrarily defined distance of 30 nm (Bayer et al., 2017), which is the estimated distance between the cortical ER (cER) and the PM that a single protein tether can span (McFarlane et al., 2017; Michaud & Jouhet, 2019; Rosado & Bayer, 2021). In eukaryotes, similar EPCS tethering components have been associated with different functions, altogether making the study of EPCS organism-dependent (Tilsner et al., 2016). For example, the vesicle-associated membrane protein (VAMP)-associated protein (VAP) 5 tether contains an ER-anchored transmembrane domain and a lipid-binding domain with organism-dependent functions (Kagiwada & Hashimoto, 2007). Animal VAPs were first identified within soluble N-ethylmaleimide Sensitive Fusion (NSF) Attachment (SNAP) Receptor (SNARE) protein complexes aiding in vesicle trafficking from the ER (Skehel et al., 1995). VAPs in yeast form ER-PM tethering complexes with the lipid transfer protein Oxysterol-Binding Homology (Osh) proteins, and among their functions, control the phosphatidylinositol-4-phosphate (PI4P) levels at the PM and the regulation of the phosphatase activity at EPCSs (Stefan et al., 2011). In plants, co-immunoprecipitation and co-localization analysis have demonstrated that VAPs (VAP27-1 and VAP27-3) localize to EPCSs and interact with the actin-binding protein NETWORKED 3C (NET3C) (Wang et al., 2014, 2016). Plant VAPs have a role in endocytosis (Stefano et al., 2018), ER-cytoskeleton interactions (Wang et al., 2016), and might be involved in the transfer of lipids through interactions with oxysterol-binding proteins (Saravanan et al., 2009; Wang et al., 2017). This diversity within various protein interactions and roles illustrates the complexity of functions carried out by similar EPCS components in different organisms. Another family of EPCS components is the synaptotagmin (SYT) tethering proteins. The SYT structure contains an N-terminal transmembrane domain, a variable length linker, a synaptotagmin-like-mitochondrial-lipid binding domain (SMP), and two distinct tandem C-terminal calcium-binding domains called C2A and C2B (Figure 1) (Craxton, 2004). The mammalian Extended Synaptotagmins (E-Syts) are anchored to the ER via a hairpin sequence to form homo- and heteromeric complexes that facilitate contact with the PM (Giordano et al., 2013). Binding to the PM is mediated by C2 domains and requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM (Giordano et al., 2013). The SMP domain functions to 6 transfer lipids between membranes, such as glycerophospholipids in the mammalian E-Syt2 (Argyraki et al., 2018). The Tricalbins (Tcb) are the yeast orthologs of the plant SYTs and form tethering complexes at EPCSs (Creutz et al., 2004). Plant SYTs were initially described as homologs of mammalian synaptotagmins involved in neurotransmitter release, but further studies demonstrated that plant SYTs are more closely related to the mammalian Extended-Synaptotagmins involved in non-vesicular lipid transfer (Schapire et al., 2008; Yamazaki et al., 2008; Pérez-Sancho et al., 2015, 2016). Accordingly, a recent publication demonstrated that the Arabidopsis SYT1 and SYT3 have a functional role in regulating the diacylglycerol homeostasis between the cER and the PM during abiotic stress (Ruiz-Lopez et al., 2021). The Arabidopsis SYT family was identified through a sequence homology search and comparison of ten sequenced genomes (Craxton, 2004). SYTs belong to a superfamily of N-terminal transmembrane C2 domain proteins (NTMC) that contains five SYTs with two C2 domains (SYT1-5), a sixth SYT with three C2 domains (SYT6), and the seventh tether with one C2 domain, CLB1 (Pérez-Sancho et al., 2016; E. K. Lee et al., 2020; Ishikawa et al., 2020). 7 Figure 1. Protein domains and 3D structure of a canonical synaptotagmin in plants. A) Schematic diagram of the unifying features of synaptotagmins. TM, transmembrane domain; SMP, synaptotagmin-like mitochondrial-lipid binding domain; C2A/C2B, Ca2+- and lipid-binding domain. B) Predicted protein structure of SYNAPTOTAGMIN in Arabidopsis. Blue, SMP domain; pink, C2A/C2B domain. SYT1 protein model was generated through homology modelling using Phyre2 with >99% confidence per domain. 1.5 The Arabidopsis SYNAPTOTAGMIN1 (SYT1) EPCS tether. One plant synaptotagmin, SYNAPTOTAGMIN1 (SYT1), is highly expressed and ubiquitous in Arabidopsis (Zimmermann et al., 2014; Pérez-Sancho et al., 2015; Lee et al., 2019). The Arabidopsis SYT1 was first established as a synaptotagmin-like protein after plasma membrane fraction purification and characterization after cold acclimation (Kawamura & Uemura, 2003; Alexandersson et al., 2004). SYT1 expression was present in all tissues, with the strongest expression observed in the vascular tissue and elongation zone of roots (Schapire et al., 2008). This was determined using a β-glucuronidase reporter for the SYT1 cis-regulatory sequence, as well as gene expression measured through microarray experiments (Winter et al., 8 2007; Schapire et al., 2008). SYT1 was found to be co-localized at the PM and the ER, using the PM-localized PIN2-GFP and the ER marker HDEL-GFP (Schapire et al., 2008; Yamazaki et al., 2008; Pérez-Sancho et al., 2015). Additionally, SYT1 subcellular localization was confirmed by co-localization with an artificial marker that fuses the mammalian membrane-attached peripheral ER domain (MAPPER) with GFP and is stabilized when the intermembrane distance between the ER and the PM is less than 25 nm (Lee et al., 2019). The SYT1 function as a tethering protein was inferred from biochemical assays that confirmed Ca2+- and phospholipid-dependent membrane binding through the C2 domains (Schapire et al., 2008; Pérez-Sancho et al., 2015). The syt1-2 loss-of-function T-DNA mutant also showed growth hypersensitivity in response to NaCl stress indicated by a reduction in fresh weight and root length compared to the wild type (Schapire et al., 2008). SYT1-labelled EPCSs expand in response to ionic stress in a cytoskeleton-independent manner. However, chemical treatments and analyses using Fluorescence Recovery After Photobleaching (FRAP) of SYT1 determined that the establishment of SYT1-EPCSs is cytoskeleton-dependent (Lee et al., 2019). Apart from its role conferring NaCl tolerance, SYT1 is involved in freezing tolerance, interacts with viral movement proteins at plasmodesmata, confers tissue and cellular mechanical stability, and regulates the plant immune responses to powdery mildew (Yamazaki et al., 2008; Levy et al., 2015; Pérez-Sancho et al., 2015; Kim et al., 2016; Ishikawa et al., 2018). The diversity of roles for SYT1 highlights the importance of EPCS function for plants, and the need to advance our understanding of the expression patterns and functions of additional members of the SYT family in Arabidopsis. 9 1.6 Research Objectives Synaptotagmin-like structured proteins are found in the genomes of all phyla (Craxton, 2004), and ample synaptotagmin research has been conducted in metazoans (Saheki & De Camilli, 2017). In plants, the expression pattern and function of the Arabidopsis SYT1 tether has been previously reported (Schapire et al., 2008; Yamazaki et al., 2008; Pérez-Sancho et al., 2015), but there is limited information on how SYT1 and additional SYT family members are regulated. This thesis analyzes the transcriptional and translational regulation of SYT1 and addresses questions regarding the distribution and putative function of additional members of the SYT family. This thesis addresses three research questions that guide the objectives described below: 1. Is SYT1 transcription and translation differentially regulated by NaCl stress? 2. Is SYT1 stability regulated by NaCl stress? 3. Are different SYTs responding to specific stresses in different tissues? Objective One: Compare the activity of the SYT1 promoter and the SYT1 protein levels in control and stress conditions (Chapter 3). This objective analyzes whether SYT1 gene expression correlates with the amount of SYT1 protein present in seedlings. I hypothesized that SYT1 protein accumulation throughout development and in response to abiotic stress is primarily controlled by SYT1 gene expression. To determine whether changes in SYT1 expression correlate with SYT1 protein accumulation, I generated stable pSYT1:LUC and pSYT1:SYT1-LUC transgenic reporter lines. I selected lines that displayed high luminescence and contained a homozygous single insertion, 10 and I used them to measure changes in SYT1 promoter activity and SYT1 protein accumulation throughout early development and in response to various stress-inducing chemical treatments. Objective Two: Establish whether the stability of the SYT1 protein changes in response to stress and identify mechanisms involved in SYT1 degradation (Chapter 4). The results from Objective One showed that SYT1 promoter activity increased, but SYT1 protein accumulation remained constant in response to salt stress. Based on this observation, I hypothesized that SYT1 protein accumulation is not strictly coupled to gene transcription and predicted that SYT1 protein stability would decrease in response to salt stress. I specifically explored whether its degradation was mediated by the 26S proteasome. I determined the stability of the SYT1 protein by examining the changes in the luminescence of the pSYT1:SYT1-LUC line in response to the protein synthesis inhibitor cycloheximide (Kao et al., 2015; Gilkerson et al., 2016). I also evaluated whether SYT1 degradation occurs through the 26S proteasome pathway by examining the changes in the luminescence of the pSYT1:SYT1-LUC line using the 26S proteasome inhibitor MG132 (Vannini et al., 2014). Objective Three: Establish the gene expression patterns for the different members of the Arabidopsis SYT family in response to developmental and environmental cues (Chapter 5). This objective addresses two interconnected questions: (1) do different SYTs display tissue expression specificity, and (2) is the expression of the different SYTs responsive to environmental stress? I hypothesized that the members of Arabidopsis SYT family would be differentially expressed throughout development, and that their individual expression patterns could change in response to specific environmental stresses. To identify tissues and 11 developmental conditions that could be important for the function of the different SYTs, I performed in silico gene expression profiles on SYT1-6 and the SYT-related CLB1. Additionally, I performed an in silico analysis of the changes in gene expression of SYT1-6/CLB1 in response to selected abiotic stresses. To validate the in silico analyses, I measured in vivo gene expression using pSYT:GUS reporter lines for SYT1-6 and CLB1. 12 Chapter 2: Materials and Methods 2.1 Plant Materials and Growth Conditions Arabidopsis thaliana (ecotype Columbia; A. thaliana Col-0) seeds were surface sterilized with a 4:1 solution of 87.5% EtOH and 30% H202 for 60 seconds or by incubation in chlorine gas for two hours within a sealed container (Lindsey et al., 2017). Previously published plant lines used include IAA1-LUC provided by Dr. Judy Callis (Gilkerson et al., 2016) and syt1-2 (SAIL_775_A08) provided by the ABRC (Schapire et al., 2008). Seeds were sown on half-strength Linsmaier and Skoog (LS, Caisson Labs, Smithfield UT, USA) medium with 0.8% agar. Seedlings were grown from 3 to 14 days for seedling development experiments and eight days for chemical treatment in a plant growth chamber (Percival Scientific, Perry Iowa, USA) under a 16-hr light/8-h dark cycle at a constant 22°C. Plants grown beyond 14 days were transferred to soil (Sunshine mix #4; Sun Gro Horticulture Canada) in a 22°C 16-hr light/8-h dark cycle photoperiod. 2.2 Cloning 2.2.1 Transformation of E. coli The promoter sequences for the SYT genes used in this thesis are approximately 1500 bp directly upstream from the start codon (ATG) of each respective gene. The SYT1 promoter region was unsuccessfully cloned, rather, the established plant line pSYT1:GUS line was used as a replacement (Schapire et al., 2008). All promoter regions were PCR amplified from Arabidopsis gDNA using Phusion polymerase (Thermo Fisher, Ottawa ON, Canada), and purified using the Monarch Gel Extraction Kit (New England Biolabs, Whitby ON, Canada) or 13 EZ-10 Spin Column PCR Products Purification Kit (Bio Basic Inc., Markham ON, Canada). Each DNA fragment was then cloned into pENTR/D-TOPO (attL1/attL2) (Thermo Fisher, Ottawa ON, Canada). This vector was used for the transformation of high efficiency chemically competent DH10β E. coli (New England Biolabs, Whitby ON, Canada) using heat shock (Inoue et al., 1990). LR Clonase™ II Plus (Life Technologies) was used to recombine the pSYT-containing pENTR vector into pBGWFS7/GUS-GFP (attR1/attR2). Each recombined construct was then transformed into chemically competent DH5ɑ E. coli (Fisher Scientific, Ottawa ON, Canada) using heat shock. Vector-positive colonies were selected on 50 µg/ml kanamycin or 100 µg/ml spectinomycin Lysogenic Broth (LB) agar plates (Thermo Fisher, Ottawa ON, Canada). The validity of the promoter was confirmed through Sanger Sequencing (Psomagen, Rockville MD, USA). Genomic and promoter sequence for SYT1 was amplified from extracted DNA using the high fidelity Phusion polymerase (New England Biolabs). The luciferase coding sequence was cloned by Makoto Shirakawa under the expression of the amplified native SYT1 promoter (pSYT1:LUC). To clone pSYT1:SYT1-LUC, the promoter sequence was cloned into pENTRTM5'-TOPO (attL4/attR1), the SYT1 coding sequence was cloned into pENTRTM'/D-TOPO (attL1/attL2), and the luciferase coding sequence was cloned into pENTRTM3'-TOPO (attR2/attL3) by EunKyoung Lee using Multisite Gateway®; recombination into pDESTTMR4-R3 (attR4/attR3) was completed using LR ClonaseTM II Plus (Life Technologies). 14 Table 2.1. Primers used for the amplification of promoter regions using gDNA in Arabidopsis thaliana (Col-0). Primer Name Sequence pSYT2 fw 1396 bp fragment CACCTGTACTGTCTAGTGTCTGTG pSYT2 rv up to ATG GTCTCATCAACTCAAATCTTCTGACAAG pSYT3 fw 1754 bp fragment CACCAACGCACTGTATCTCTC pSYT3 rv up to ATG GTTTTTGTTTTGTTTTTCCCGATGAAAATC pSYT4 fw 1416 bp fragment CACCATAAAGCTACTTGTTGAAACAG pSYT4 rv up to ATG TTTTGTTCACACTTCTCGAAGGAC pSYT5 fw 1660 bp fragment CACCAGAAGGGCATGAAGAAAG pSYT5 rv up to ATG ATTTAATCGCCGGATTTAGAAAAATTGC pSYT6 fw 1629 bp fragment CACCATTCAGCGTGATGGTTTGG pSYT6 rv up to ATG CTCTTCGATTCGCCTTCAAGCTTC pCLB1 fw 1564 bp fragment CACCAACGCTTCCGTAAATTCTTG pCLB1 rv up to ATG TTTTCCTGCAACCGAAACAAAGATTAC Table 2.2. Sequence size of the promoter region used for the SYT genes. Region Sequence size upstream of ATG pSYT2 1396 bp pSYT3 1754 bp pSYT4 1416 bp pSYT5 1660 bp pSYT6 1629 bp pCLB1 1564 bp 2.2.2 Transformation of Agrobacterium The SYT2/3/4/5/6/CLB1 promoter-driven GUS-GFP expression vectors were transformed into Agrobacterium tumefaciens (strain GV3101) by electroporation (Miki et al., 1993). Vector- 15 positive colonies were selected on 25 µg/ml rifampicin and 100 µg/ml spectinomycin LB agar plates. The presence of the plasmid was verified using insert-specific PCR amplification. 2.2.3 Transformation of Arabidopsis The plasmid constructs were introduced into Arabidopsis wild type Col-0 and syt1-2 using floral dip (Clough & Bent, 1998). Transgenic lines were segregated on half-strength LS media containing either 25 µg/mL hygromycin or 15 µg/mL glufosinate-ammonium (Sigma-Aldrich Canada Co., Oakville ON, Canada). 2.2.4 DNA and RNA Extraction For the confirmation of T-DNA insertion SAIL_775_A08, one 2-week old syt1-2 rosette leaf was stamped onto a Whatman Flinders Technology Associates (FTA) card (Sigma-Aldrich Canada Co., Oakville ON, Canada) and dried at room temperature for 30 minutes. One FTA paper disc was incubated in an extraction buffer (10 mM Tris, pH 7.5, 2 mM EDTA, 0.1% Tween20) for 30 minutes and washed with TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA). The hole-punched FTA disc was used as the template in PCR reaction tubes. DNeasy Miniprep Kit (Qiagen, Montreal QC, Canada) and Monarch Total RNA Miniprep kit (New England Biolabs, Whitby ON, Canada) were used for Arabidopsis DNA and RNA extraction respectively. Plasmid DNA was extracted from E. coli colonies using the EZ-10 Spin Column Plasmid DNA Minipreps Kit (Bio Basic Canada Inc., Markham ON, Canada) following the manufacturer's protocol. DNA was purified using the Monarch DNA Gel Extraction Kit (New England Biolabs, Whitby ON, Canada) and the EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic Canada Inc., Markham ON, Canada). 16 2.3 Salt Sensitivity Tests Seeds of the wild-type Col-0, T-DNA mutant syt1-2, and pSYT1:SYT1-LUC were sown on half-strength LS media and stratified for three days. Seedlings at three days after germination were transferred to 1/10 LS solid media or 1/10 LS solid media supplemented with 150 mM NaCl and grown vertically. Seedlings were imaged seven days later using an EPSON Perfection V2000 scanner. Seedling root growth was quantified using FIJI imaging software (Schneider et al., 2012). Statistical differences in root length were determined using a Student’s T-Test (p<0.05) in Microsoft Excel. Graphs were generated in GraphPad Prism 8 (https://www.graphpad.com, version 8.3.1). 2.4 Chemical Treatment 2.4.1 Liquid Stress Treatment The 8-day-old seedlings were incubated in liquid 1/10 LS media containing mock, 150 mM NaCl, 100 mM KCl, 50 mM MgCl2, 50 mM Na2SO4, 50 mM K2SO4, 200 mM Mannitol, 200 mM sorbitol, or 50 µM U-73122 for 16-hr, or in 25 µM Latrunculin B (LatB) for 2-hr. All reagents, excluding LatB (Abcam, Cambridge UK) were purchased from Sigma (Sigma-Aldrich Canada Co., Oakville ON, Canada). 2.4.2 Cycloheximide Treatment Fresh rosette leaf discs (5 mm diameter) from 6-week grown plants were hole-punched and placed within a white opaque 96-well microplate, following a modified protocol outlined by Gilkerson et al. (2016). The leaf discs were incubated in each well (1 mM luciferin, and/or 200 µM cycloheximide, and/or 100 µM MG132) (Sigma-Aldrich Canada Co., Oakville ON, Canada) 17 from 0-16 hours and were measured in a Synergy 2 Multi-Mode Microplate Reader (BioTek, Vermont, USA). Leaf discs were incubated in wells containing solution (a combination of luciferase, DMSO, MG132, or NaCl) for two hours before the addition of CHX. An initial reading was measured prior to CHX and was immediately placed into the reader for the time series after the CHX. Wells were quantified using BioTek Gen5 data analysis software and exported as an excel file. Data was imported to GraphPad Prism 8. The half-life and graphs were generated using best-fit Nonlinear regression analysis, then further calculated using One phase decay analysis. In the case of unfit curves, the Two-phase decay analysis was used. The differences between datasets were compared using the two-way ANOVA (or mixed model) analysis. The confirmation and change in fluorescence were qualitatively imaged under a charge-coupled device (CCD). 2.5 Bioluminescence Assay 2.5.1 Image Acquisition Activation of the firefly luciferase gene was characterized by measuring the bioluminescence using in vivo image capture. Aliquots of D-luciferin sodium salt (Cedarlane Goldbio, Burlington ON, Canada) in water were stored at -20°C and thawed before incubation. Seedlings were incubated with supplementary 1mM D-luciferin at the initiation of respective treatments. Subsequent to chemical treatment, seedlings were transferred to a 1/10 LS containing 0.8% agar and the respective treatment concentration (e.g., seedlings incubated in liquid 50 mM MgCl2 were transferred to 50 mM MgCl2 0.8% agar). Seedlings were incubated in the absence of light for 30 minutes and then imaged using a top-mounted ultra-sensitive CCD (Princeton Instruments, Trenton NJ, USA; PIXIS 1300b) within a dark chamber. When imaging the shoot 18 and root seedling growth and development, Petri dishes containing 64 seedlings were sprayed with 1 ml of 1 mM D-luciferin approximately 24 hours before imaging, as well as incubated in the dark and sprayed 30 minutes before imaging. The CCD is connected to a 50 mm lens with Kenko Auto Extension Tubes (Amplis Foto Inc., Markham, ON Canada) and imaged using high gain, 500 MHz analog to a digital conversation, and 300-second exposure settings. Raw images were processed through Princeton Instruments LightField imaging software (Princeton Instruments, Trenton NJ, USA). Three images were taken per sample, averaged, and exported as a TIFF file. 2.5.2 Image Quantification Images were quantified by measuring the raw mean luminescence intensity of seedling hypocotyl and cotyledons using FIJI imaging software (Schindelin et al., 2012). The average background intensity surrounding each plate was subtracted from each seedling measurement in Microsoft Excel (https://www.office.com). The student’s T-test and graphing were completed in GraphPad Prism 8. 2.6 Histochemical GUS staining One-week seedlings and six-week flowers were removed from -GUS containing plants and placed in 90% acetone on ice. After preparation, the samples incubated at room temperature in the acetone for 20 minutes. The samples were washed with buffer solution without X-Gluc (H2O, phosphate buffer, 10% Triton-X, K3[Fe(CN)6], K4[Fe(CN)6]). The tissue samples were then incubated in buffer solution with X-Gluc, vacuum-infiltrated for 15-20 minutes on ice, then incubated at 37°C for 24 hours. Tissues were fixed in an EtOH series and stored in 70% EtOH. 19 2.7 Bioinformatic Analysis The genomic sequence and promoter region for SYT1, SYT2, SYT3, SYT4, SYT5, SYT6, and CLB1 were acquired from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org). Amino acid and nucleotide sequences for NTMC proteins (SYTs 1-6, CLB1) were aligned using MUltiple Sequence Comparison by Log-Expectation (MUSCLE) within MEGA X (Kumar et al., 2018). Protein domains were determined using Pfam (Mistry et al., 2021). Pairwise alignments and graphics were generated using Jalview (Waterhouse et al., 2009). Phylogenetic trees were computed using maximum-likelihood analysis, with 1000 bootstrap replicates. The Arabidopsis eFP Browser (Winter et al., 2007) was used to compare the expression of SYT genes (Klepikova et al., 2015, 2016; Kasianov et al., 2017). The topmost 50 co-expressed genes were individually compiled using the Pearson Correlation Coefficient (PCC) from Expression Angler for each SYT gene during general development, pollen germination, abiotic stress, chemical stress, within root tissue, and stigma and ovaries (Toufighi et al., 2005). Significantly enriched cis-regulatory elements for each co-expression network were identified using the Plant Cis-acting Regulatory DNA Elements (PLACE) database, Arabidopsis-specific PLACE database, and JASPAR database (1000 bp upstream from the translational start site, 3.0 distance between two non-parametric bootstrap distributions [(0<z<10)], 0.35 functional depth [(IC-ICmin)/(ICmax-ICmin)], >50% of all genes must contain the motif before being considered significant, forward strand only) (http://bar.utoronto.ca/cistome/cgi-bin/BAR_Cistome.cgi). Over-represented motifs that were 7 nucleotides long were predicted using the MEME suite accessed using Cistome using the same statistical parameters (1000 bp upstream, 3.0 z, 0.35 d, >50% genes, forward strand) (Austin et al., 2016). 20 2.8 Accession Numbers Nucleotide sequence data and gene information can be found on The Arabidopsis Information Resource using the locus accession numbers; SYT1 (AT2G20990), SYT2 (AT1G20080), SYT3 (AT5G04220), SYT4 (AT5G11100), SYT5 (AT1G05500), SYT6 (AT3G18370), and CLB1 (AT3G61050). 21 Chapter 3: Study of the Correlation Between SYT1 Gene Activity and SYT1 Protein Amounts 3.1 Introduction 3.1.1 The relationship between gene expression and protein synthesis The temporal and spatial expression of protein-encoding genes is fundamentally important for cell productivity and function. In many cases, the response to developmental cues and environmental stimuli is controlled by complex gene expression networks that tightly regulate the temporal and spatial distribution of target proteins. Therefore, examining the temporal and spatial relationship between gene expression and protein accumulation is an important step to understand how developmental cues and stress signals regulate the plant adaptive responses at the cellular level. Gene expression starts in the nucleus where a transcriptional complex formed by multiple cofactors, general transcription factors, and RNA polymerase II binds to a highly conserved promoter region known as the TATA-box. This transcriptional complex synthesizes pre-messenger RNA (pre-mRNA), and it is regulated by transcription factors that increase (enhancers) or decrease (repressors) the activity of the RNA polymerase II complex in response to the prevailing environmental and developmental conditions (Young, 1991). Numerous modifications occur in the nuclear pre-mRNA before maturation and export, such as the excision of introns in spliceosomes, 5’-capping, 3’ cleavage, and polyadenylation (Luehrsen et al., 1994; Clancy & Brown, 2008). Once matured, the resulting messenger RNA (mRNA) from protein-encoded genes is exported outside the nucleus, recognized by ribosomes and translated. 22 The correlations between gene expression, protein accumulation, and protein activity can vary because the promoter activity, mRNA transcription, mRNA processing, protein synthesis, and protein activity can be independently regulated in response to endogenous signals (e.g. secondary messengers) and environmental cues. 3.1.2 Proposed regulators of SYT1 gene expression, protein accumulation, and protein activity at the cellular level. 3.1.2.1 Cytosolic Ca2+ Signatures The activation of genes in response to environmental triggers is often mediated by Ca2+ -dependent signalling cascades. Ca2+ signal transduction generally involves four steps, (1) a transient or sustained increase in free cytosolic Ca2+, (2) the detection of these changes by sensor proteins, (3) a signalling pathway activated by the sensor proteins, and (4) the removal of excess cytosolic Ca2+ facilitated by Ca2+ uniporters, antiporters, and Ca2+-ATPases (Edel et al., 2017). The Ca2+ signals are decoded using signatures, whereby local changes in Ca2+ concentration of specific duration determine the specificity of the response. The activity of the Arabidopsis SYT tethers is believed to be regulated by Ca2+ signatures as SYTs are localized at the cortical ER and PM interface where local changes in cytosolic Ca2+ concentration are likely to occur (Pérez-Sancho et al., 2015; Ishikawa et al., 2018), and the in vitro lipid-binding activity of the SYT1 and SYT3 calcium-binding domains depends on the Ca2+ concentration (Schapire et al., 2008; Ruiz-Lopez et al., 2021). Specifically, biochemical studies have shown that the phospholipid-binding activity of the SYT1 (or SYT3) C2A domain is Ca2+- 23 dependent, but the phospholipid binding activity of the SYT1 (or SYT3) C2B domain is Ca2+-independent. Changes in Ca2+ concentration also influence the phenotypes observed in syt1 loss-of-function mutants. For example, the reduction in root elongation of syt1-2 is intensified under conditions where the extracellular Ca2+ concentration is low (Schapire et al., 2008), and reducing extracellular Ca2+ concentration also reduces the freezing tolerance of the syt1-2 mutant (Yamazaki et al., 2008). Against the notion that SYT1 activity is directly controlled by [Ca2+]cyt signatures is the fact that the [Ca2+]cyt signature in response to NaCl stress occurs within minutes, but the cellular responses of SYT1-labelled EPCS (S-EPCS) to salt stress require several hours (Kader & Lindberg, 2010; Lee et al., 2019). Hence, SYTs might be regulated by [Ca2+]cyt signatures but still require the slow activation of additional components (e.g. phospholipid synthesis) (Lee et al., 2019). The link between Ca2+ and SYT1-EPCS tethering activity is also illustrated by extracellular Ca2+ chelation assays and chemical treatments with non-selective Ca2+-channel blockers. These experiments showed that altering Ca2+ homeostasis changes the number of SYT1- and SYT5-labelled EPCSs and reduced the cortical ER reticule size (Ishikawa et al., 2018; Lee et al., 2020). 3.1.2.2 Cytoskeletal Elements Cytoskeletal elements are essential for the organization and stability of the cortical ER and the ER-PM contact sites (Wang et al., 2014). For example, the movement of the cER is dependent on the actin cytoskeleton, and the formation of EPCS anchors relies on interactions of the EPCS tether VAP27 and the actin-associated protein adaptor NET3C (Wang et al., 2014, 2017). In addition, SYT1 distribution depends on the cortical cytoskeleton because SYT1 is 24 primarily found in MT-depleted regions, and cannot diffuse laterally at EPCS when microtubules and F-actin are depolymerized (Pérez-Sancho et al., 2015; Lee et al., 2019). This relationship between SYT1 and the cytoskeleton is especially important when considering that salt stress induces many changes to the cytoskeletal elements, such as the transient depolymerization of the microtubules and the reduction of mobility and bundling of the actin filaments (Nunes et al., 2013, 2015; Lee et al., 2019). 3.1.2.3 Negatively Charged Phospholipids The activity of SYTs is influenced by the lipid composition at EPCSs. As indicated previously, the physical interactions between SYTs and the PM are mediated by electrostatic interactions between negatively charged phospholipids and the positively charged C2 domains. Using a protein-lipid overlay assay, it was shown that the C2 domains of SYT1 were strongly associated with phosphatidylserine (PS), phosphatidic acid (PA), lysophosphatidic acid (LPA), and negatively charged phosphatidylinositols (Pérez-Sancho et al., 2015). These interactions between SYTs and phospholipids contribute to the tethering between the cER and the PM, and are is also vital to mediate lipid exchanges between the cER and the PM via the SYTs SMP domain (Argyraki et al., 2018; Ruiz-Lopez et al., 2021). In mammals, the lipid exchange between the ER and the PM is well-characterized (Sohn et al., 2018). One important lipid exchange pathway involves the phosphorylation and dephosphorylation of phosphatidylinositol (PI) at the ER and plasma membrane (Figure 3.1). PI is transferred to the PM from the cER and becomes PI4P through phosphatidylinositol 4-kinase alpha (PI4KA)-mediated phosphorylation (Sohn et al., 2018). PI4P is then phosphorylated once again into phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) using phosphatidylinositol 4- 25 phosphate 5 kinase (PIP5K) (Nakatsu et al., 2012; Sohn et al., 2018). Nevertheless, PI and PI4P must transfer between the cER and the PM, which is a process facilitated by lipid-transfer proteins such as oxysterol-binding protein (OSBP)-related protein (ORP) 5 and ORP8 (Sohn et al., 2018). PI4P can be dephosphorylated back into PI due to Sac1 phosphatase in the ER. These processes fulfill a dynamic pathway of lipid-phosphorylation and lipid exchange between the cER and PM. In plants, an important lipid-modification step is the formation of diacylglycerol and inositol triphosphate (IP3) using the PI(4,5)P2 precursor, facilitated by phospholipase C (PLC) within the PM. PI(4,5)P2 accumulation in response to stress or PLC inhibitors (Lee et al., 2019), and the subsequent transfer of diacylglycerol at EPCSs is an essential process to maintain lipid homeostasis in plants (Ruiz-Lopez et al., 2021). Figure 3.1 Schematic summary of lipid transfer and associated proteins at the plasma membrane and endoplasmic reticulum in eukaryotes. Arrows represent the transfer of molecules. Orange represents phospholipids, green represents proteins. PI, phosphatidylinositol; PI4P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol triphosphate; DAG, diacylglycerol; PI4KA, phosphatidylinositol 4-kinase alpha; PIP5K, phosphatidylinositol 4-phosphate 5 kinase; PLC, phospholipase C; ORP, oxysterol-binding protein-related protein. Adapted from Sohn et al., 2018. 26 Research Questions for this Chapter: This chapter answers the following questions related to SYT1 promoter activity and SYT1 protein amounts: 1. When and where is the SYT1 expressed throughout seedling development? 2. Does the SYT1 gene expression change in response to abiotic stresses? 3. Does the SYT1 protein expression change in response to abiotic stresses? 4. Is there a direct correlation between SYT1 gene expression and SYT1 protein levels during abiotic stress? 3.2 Results 3.2.1 SYT1 expression throughout development 3.2.1.1. Changes to pSYT1:LUC throughout development To determine the levels of SYT1 gene expression throughout seedling development, I measured the activity of the SYT1 promoter in vivo using the expression of the luciferase gene as a reporter (pSYT1:LUC). I generated three independent pSYT1:LUC transgenic lines in Col-0 whose segregation in selection media was compatible with the presence of a single copy of the transgene (Table A2). I observed that the pSYT1:LUC activity was ubiquitous in Arabidopsis seedling tissues. The LUC intensity remained highest in true leaves and the shoot apex. The overall intensity observed in all seedlings increased at the same rate as the total mass (Figure A1). The intensity became uniform in cotyledons around 7 DAG (Figure 3.2a) and this time-point was used for quantitative measurements (Chapter 3.2.2). Expression within roots was also ubiquitous. LUC luminescence increased in secondary root tips after 8 to 9 days, the highest intensity found near the elongation zone and area of cell division (Figure 3.2b). 27 I also performed a preliminary characterization of SYT1 promoter activity in 6-week old pSYT1:LUC inflorescences and shoots to optimize the protocols in adult plants. When imaging detached rosette leaves, the highest expression was observed in the midrib, followed by the leaf veins and lamina, decreasing luminescence nearer the leaf apex (Figure A3). No difference was noticed when comparing the pSYT1:LUC signal in the adaxial and abaxial sides of both rosette leaves and cauline leaves. Of note are the increases in intensities found at the nodes along the primary stem, along the vasculature of cauline leaves, the petiole of cauline leaves, and young siliques. The pSYT1:LUC activity was ubiquitous and stable throughout the inflorescence, and slightly less intense in secondary stems compared to the primary inflorescence stem. pSYT1:LUC activity was high in axillary meristems, flowers, and abscission zones (Figure 3.5). Figure 3.2. pSYT1:LUC expression in the shoots and roots of Arabidopsis seedlings. A) pSYT1:LUC seedlings as a measure of SYT1 promoter activity in brightfield (top panels) and darkfield (bottom panels). B) Vertically grown pSYT1:LUC seedlings in brightfield (top panels) and darkfield (bottom panels). Seedlings were imaged directly above at 3 to 14 days after germination. Seedlings were grown in ½ LS agar, 16 hr light, 22°C. Scale: 2 cm. 28 3.2.1.2. Changes to SYT1 protein levels throughout development To determine the SYT1 accumulation throughout seedling development, I measured the in vivo SYT1 levels using a translational fusion of SYT1 with the luciferase reporter (proSYT1:SYT1-LUC). I generated four proSYT1:SYT1-LUC transgenic lines in Col-0, and three lines in syt1-2 that fully complemented the syt1-2 root length phenotype in salt stress (Figure 3.3). The segregation analysis suggested the presence of a single insertion for each line (Table A2). I observed that SYT1-LUC (established using the proSYT1:SYT1-LUC marker, hereafter SYT1-LUC) was present in all Arabidopsis seedling tissues. In general, the intensity of SYT1-LUC was high in shoot organs in regions of active growth, such as the hypocotyl, cotyledons at 8 days, and true leaf growth at 9 days (Figure 3.4). SYT1-LUC luminescence in secondary root tips was high after 8 -9 days, with the strongest luminescence at the root tip. The change in intensity between 3-14 DAG increased at a similar rate as the plant biomass (Figure A2). Similar to pSYT1:LUC, the intensity of SYT1-LUC was greatest in rosette leaf vasculature (Figure A4), at the nodes along the primary stem, along the vasculature of cauline leaves, the petiole of cauline leaves, and young siliques. The accumulated SYT1-LUC was also similar to pSYT1:LUC in the inflorescence, with a slight increase in the pedicel. There was low luminescence within the petals and sepals. In most cases, the expression of pSYT1:LUC was positively correlated with the accumulation of SYT1-LUC protein, indicating that SYT1-LUC accumulates in tissues where pSYT1:LUC is expressed. 29 Figure 3.3. Root elongation phenotypic complementation of the syt1 mutant with SYT1-LUC. Root elongation using Col-0, syt1, and SYT1-LUC/syt1. Seedlings were grown for 4 days on ½ LS growth agar, transferred to 100 mM NaCl, and measured five days later. The box represents the 25th to 75th percentile, “+” represents the mean, the line within the box represents the median, the error bars are the 10th to 90th percentile, and the dotted lines are outliers. A student’s T-test and Tukey test were used for statistical significance, p<0.05. 30 Figure 3.4. SYT1 protein levels in the shoots and roots of Arabidopsis seedlings. A) pSYT1:SYT1-LUC seedlings as a measure of SYT1 protein in brightfield (top panels) and darkfield (bottom panels). B) Vertically grown pSYT1:SYT1-LUC seedlings in brightfield (top panels) and darkfield (bottom panels). Seedlings were imaged from above at 3 to 14 days after germination. Seedlings were grown in ½ LS agar, 16 hr light, 22°C. Scale: 2 cm. Figure 3.5. pSYT1:LUC expression and SYT1-LUC protein levels in the shoot of adult Arabidopsis plants. A) pSYT1:LUC (left), Col-0 (center), and proSYT1:SYT1-LUC (right) at 4 weeks brightfield. B) pSYT1:LUC (left), Col-0 (center), and proSYT1:SYT1-LUC (right) at 4 weeks darkfield. Arabidopsis was grown in ½ LS agar, 16 hr light, 22°C and transferred to soil and identical conditions. Samples are held down using multiple segments of porous tape. D-luciferin incubated exogenously. Scale (left): 5 mm; scale (right): 2.5 mm. 31 3.2.2 Changes to the SYT1 promoter expression in response to abiotic stress Salt stress SYT1 has a functional role related to abiotic stress tolerance, as demonstrated by multiple mutant phenotypes (Schapire et al., 2008; Yamazaki et al., 2008; Pérez-Sancho et al., 2015). To examine the changes in the promoter activity of SYT1 after exposure to abiotic stress, pSYT1:LUC was used to measure promoter activity. After incubation in five salt treatments, 150 mM NaCl, 100 mM KCl, 50 mM Na2SO4, 50 mM MgCl, and 50 mM K2SO4, the luminescence of the pSYT1:LUC marker increased in almost all instances (Figure 3.6). The 16-hr liquid treatment in NaCl, KCl, Na2SO4 and MgCl2 significantly increased promoter activity (p<0.0001 for all treatments). pSYT1:LUC luminescence increased 3.9-fold in response to NaCl, 1.9-fold in response to KCl and MgCl2, and 1.3-fold in response to Na2SO4 (Figure 3.6). Surprisingly, K2SO4 reduced pSYT1:LUC luminescence 0.28-fold (p<0.0001). These results suggest that, in most cases, ionic stress activates pSYT1:LUC expression, but its effect is dependent on the ions that generate the stress. In particular, salts containing Cl- enhance the promoter activities to a higher level than salts containing SO42- which in some cases, have an inhibitory effect on pSYT1:LUC expression. 32 Figure 3.6. The change in pSYT1:LUC expression in response to salt stress. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in salt stress-inducing chemical treatments. A) Images of pSYT1:LUC plants used as a proxy to measure endogenous SYT1 promoter activity. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:LUC. Scale 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. 33 Osmotic stress The salts used in this study cause both ionic and osmotic stresses; to differentiate those stress components, I analyzed the pSYT1:LUC response to sorbitol and mannitol treatments that generate osmotic but not ionic stress. Figure 3.7 shows that the pSYT1:LUC luminescence showed a statistically significant 1.5-fold (p<0.0001) increase in response to mannitol, and a 1.7-fold (p<0.0001) increase in response to sorbitol compared to the control. These results suggest that osmotic stress partially contributes to the overall effect observed in the salt experiments. Figure 3.7. The change in pSYT1:LUC expression in response to osmotic stress. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in osmotic stress-inducing chemical treatments. A) Images of pSYT1:LUC plants used as a proxy to measure endogenous SYT1 promoter activity. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:LUC. Scale: 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. 34 Ca2+ channel disruption To explore how changes in [Ca2+] influence SYT1 expression, I analyzed the effect of non-selective Ca2+ channel blockers (LaCl3 and GdCl3, Achary et al., 2013; Jacob et al., 2021) and known extracellular Ca2+ chelators (EGTA and BAPTA, Gilroy et al., 1986; Pierson et al., 1994) by measuring the luminescence of the pSYT1:LUC. Figure 3.8 shows that the pSYT1:LUC luminescence decreased by 0.48-fold (p<0.0001) in LaCl3 treated seedlings compared with the control, and decreased 0.63-fold (p=0.0002) in GdCl3 treated seedlings compared with the control. Similar reductions in pSYT1:LUC luminescence were observed when the seedlings were treated with the Ca2+ chelators EGTA (0.5-fold) (p<0.0001) and BAPTA (0.77-fold) (p<0.0001). I concluded that limiting the entry of Ca2+ in the cell causes a reduction in the activity of the SYT1 promoter. 35 Figure 3.8. The change in pSYT1:LUC expression in response to Ca2+ channel disruption and chelation. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in rare earth elements and Ca2+ chelators. A) Images of pSYT1:LUC plants used as a proxy to measure endogenous SYT1 promoter activity. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:LUC. Scale: 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. Cytoskeleton depolymerization To examine the change in SYT1 expression in response to cytoskeleton disruption, I exposed pSYT1:LUC seedlings to LatrunculinB and oryzalin, an F-actin depolymerizing drug and a microtubule polymerization inhibitor respectively. When treated with LatrunculinB, the pSYT1:LUC luminescence decreased by 0.67-fold (p<0.0001) compared to the control. However, disruption of the microtubules increased pSYT1:LUC by 1.38-fold (p<0.0001) (Figure 3.9). 36 These results suggest that the activity of the SYT1 promoter decreases in response to F-actin disruption, but increases in response to microtubule disruption. Figure 3.9. The change in pSYT1:LUC expression in response to actin and microtubule disruption. Luciferase intensity of 7-d Arabidopsis seedlings after a 2-hr incubation in the actin-depolymerizing LatB and 16-hr incubation in the microtubule polymerization inhibitor oryzalin. A) Images of pSYT1:LUC plants used as a proxy to measure endogenous SYT1 promoter activity. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:LUC. Scale: 2 cm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. PI(4,5)P2 accumulation PLC activity can be inhibited using the chemical U-73122, which inhibits the hydrolysis of PI(4,5)P2 into IP3 and diacylglycerol. Treatment of Arabidopsis seedlings with U-73122 causes an accumulation of PI(4,5)P2 in the PM (Lee et al., 2019). SYT1 has an affinity for PI(4,5)P2 in vitro, and therefore, to examine how changes in the phospholipid composition affect the expression of SYT1, the PLC inhibitor U-73122 was applied to pSYT1:LUC transgenic seedlings. After treatment, there was a statistically significant decrease in pSYT1:LUC luminescence by 0.67-fold (p<0.0001) compared to the control (Figure 3.10). I concluded that the accumulation of PI(4,5)P2 decreases the expression of the SYT1 promoter. 37 Figure 3.10. The change in pSYT1:LUC expression in response to PI(4,5)P2 accumulation. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in the PLC-inhibiting U-73122, inducing PI(4,5)P2 accumulation in the PM (Lee et al., 2019). A) Images of pSYT1:LUC plants used as a proxy to measure endogenous SYT1 promoter activity. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:LUC. Scale: 1 cm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. 3.2.3 Changes in SYT1 protein levels in response to abiotic stress Salt stress When examining changes to SYT1-LUC protein levels in response to salt stress, I found that NaCl, KCl, and Na2SO4 treatment did not cause significant changes in the SYT1-LUC luminescence, and the intensity of SYT1-LUC signal decreased by 0.77-fold and 0.81-fold after exposure to MgCl2 (p=0.0003) and K2SO4 (p<0.0001) respectively (Figure 3.11). These results suggest that, unlike the SYT1 promoter, ionic stresses do not induce the accumulation of SYT1 protein. 38 Figure 3.11. The change in SYT1-LUC protein levels in response to salt stress. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in salt stress-inducing chemical treatments. A) Images of pSYT1:SYT1-LUC plants used as a proxy to measure endogenous SYT1 protein accumulation. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:SYT1-LUC. Scale: 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. 39 Osmotic stress When measuring specific responses to osmotic stress, treatments with either mannitol or sorbitol did not show statistically significant differences in the luminescence of the SYT1-LUC marker between the treatments and controls (Figure 3.12). These results suggest that osmotic stress does not induce the accumulation of SYT1 protein. Figure 3.12. The change in SYT1-LUC protein levels in response to osmotic stress. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in osmotic stress-inducing chemical treatments. A) Images of pSYT1:SYT1-LUC plants used as a proxy to measure endogenous SYT1 protein accumulation. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:SYT1-LUC. Scale: 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. Ca2+ channel disruption When measuring the levels of SYT1-LUC protein in response to Ca2+ channel disruption, the pattern resembled that observed for pSYT1:LUC; LaCl3 decreased the SYT1-LUC protein amount by 0.59-fold (p<0.0001), and GdCl3 decreased the SYT1-LUC protein amount by 0.63-fold (p<0.0001) compared to the control (Figure 3.13). Remarkably, the treatment with either EGTA or BAPTA Ca2+ chelators did not change the amount of SYT1-LUC protein compared to 40 the control. These results suggest that SYT1 protein decreases during Ca2+ channel inhibition but remains unchanged in the presence of extracellular Ca2+ chelators. Figure 3.13. The change in SYT1-LUC protein levels in response to Ca2+ channel disruption and chelation. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in rare earth elements and Ca2+ chelators. A) Images of pSYT1:SYT1-LUC plants used as a proxy to measure endogenous SYT1 protein accumulation. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:SYT1-LUC. Scale: 5 mm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. Cytoskeleton depolymerization When the SYT1-LUC line was treated with LatrunculinB, an actin polymerization inhibitor, the SYT1-LUC luminescence decreased by 0.71-fold (p<0.0001) compared to the control. When the SYT1-LUC line was treated with oryzalin, a microtubule depolymerizing drug, there was no change in SYT1-LUC signal compared to the control (Figure 3.14). These 41 results suggest that SYT1 protein levels are reduced in response to actin depolymerization but are not affected by disruptions in the microtubules. Figure 3.14. The change in SYT1-LUC protein levels in response to actin and microtubule disruption. Luciferase intensity of 7-d Arabidopsis seedlings after a 2-hr incubation in the actin depolymerizing LatB and 16-hr incubation in the microtubule polymerization inhibitor oryzalin. A) Images of pSYT1:SYT1-LUC plants used as a proxy to measure endogenous SYT1 protein accumulation. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:SYT1-LUC. Scale: 2 cm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. PI(4,5)P2 accumulation PI(4,5)P2 accumulation by PLC inhibition also decreased SYT1-LUC luminescence by 0.45-fold compared to the control (p<0.0001) (Figure 3.15). These results suggest that PLC inhibition has the same down-regulating effect on the SYT1 promoter and SYT1 protein. 42 Figure 3.15. The change in SYT1-LUC protein levels in response to PI(4,5)P2 accumulation. Luciferase intensity of 7-d Arabidopsis seedlings after a 16-hr incubation in the PLC-inhibiting U-73122, inducing PI(4,5)P2 accumulation in the PM (Lee et al., 2019). A) Images of pSYT1:SYT1-LUC plants used as a proxy to measure endogenous SYT1 protein accumulation. B) Quantification of luminescence intensity in the hypocotyl and cotyledons of pSYT1:SYT1-LUC. Scale: 1 cm. Imaged using PIXIS 1300B CCD. A student’s T-test and Tukey test were used for statistical significance, denoted with an asterisks p<0.05. Each experiment was replicated three times using 60-80 seedlings. General conclusions of the luciferase experiments In most ionic and osmotic stress treatments used in this study an increase in the pSYT1:LUC promoter activity is not associated with a concomitant increase in SYT1-LUC protein accumulation suggesting that the transcriptional and translational regulation of SYT1 are uncoupled. A similar behavior was observed when Ca2+ channel blockers were used but not when Ca2+ chelators were added to the media. Actin depolymerization reduces the pSYT1:LUC activity and reduces the amount of SYT1-LUC protein suggesting that SYT1 gene expression is coupled to SYT1 protein accumulation. However, microtubules depolymerization increases the pSYT1:LUC activity, but has no effect in SYT1-LUC levels. Last, the accumulation of PI(4,5)P2 in the PM decreases the pSYT1:LUC activity and reduces the amount of SYT1-LUC protein 43 suggesting that the endogenous SYT1 transcription and translation are coupled in these conditions. 44 Table 3.1. Changes to pSYT1:LUC promoter activity and SYT1-LUC protein levels in response to abiotic stress-inducing chemicals. Green boxes represent increase, gray represents no change, red represents decrease. Interaction Treatment effect Chemical pSYT1 fold change SYT1 fold change Salt stress Ionic stress NaCl 3.97 ± 0.03 KCl 1.89 ± 0.05 NaSO4 1.31 ± 0.05 MgCl2 1.94 ± 0.03 0.80 ± 0.07 K2SO4 0.28 ± 0.07 0.83 ± 0.08 Osmotic stress Mannitol 1.48 ± 0.03 Sorbitol 1.72 ± 0.05 Ca2+ signalling Ca2+ channel blocker LaCl3 0.49 ± 0.25 0.59 ± 0.11 GdCl3 0.63 ± 0.17 0.62 ± 0.10 Ca2+ chelator EGTA 0.50 ± 0.11 BAPTA 0.76 ± 0.06 Cytoskeleton F-Actin depolymerizing LatB 0.68 ± 0.08 0.72 ± 0.07 Microtubule polymerization inhibitor Oryzalin 1.38 ± 0.03 Phosphoinositide concentration PLC inhibitor U-73122 0.66 ± 0.08 0.45 ± 0.08 3.3 Discussion When comparing both transgenic plant lines, the persistent pSYT1:LUC and SYT1-LUC expression throughout early development indicates a positive correlation between the promoter activity of SYT1 to the protein accumulation of SYT1; i.e. SYT1 accumulates in tissues where 45 the gene is expressed. In all cases, the expression of pSYT1 and SYT1 occurred in all measured tissues, with the highest luminescence intensity in young tissues. The high intensity in young tissues may reflect the high density of rapidly dividing and growing cells and/or an increased demand for intracellular communication in metabolically active tissues. Additionally, strong pSYT1:LUC expression was found in the root tips, hypocotyls, and cotyledons which is in agreement with published data using transgenic lines carrying a pSYT1:GUS construct (Schapire et al., 2008). This is expected considering that the density of SYT1-GFP labelled EPCS is significantly higher in young cells than mature cells, especially when comparing developmental regions such as the hypocotyl and young root (McFarlane et al., 2017). These results suggest that the pSYT1:LUC line accurately represents the transcriptional activity of the endogenous SYT1 promoter. The coordination and signalling implicated with salt tolerance must be tightly regulated and controlled. In many cases, the response to salt stress induces a cascade of genes and transcription factors that form complex pathways (Zhu, 2016; Kawa & Testerink, 2017). The expression of SYT1 in response to the salts (NaCl, KCl, Na2SO4, and MgCl2), and osmotic stress generators (mannitol and sorbitol), activates the pSYT1 promoter without changing the SYT1 protein levels. It is important to indicate that these analyses provide information about the total amount of SYT1 that is present during these stresses but cannot make inferences related to its activity. The differences between pSYT1 expression and SYT1 levels in response to salt and osmotic stresses could be explained by two different models. In the first model, the stability and half-life of SYT1 decreases in response to salt and osmotic stresses and pSYT1 activation is required to maintain constant SYT1 protein levels. In agreement with this model, the stability of 46 the salt-tolerance-related protein SERINE HYDROXYMETHYLTRANSFERASE1 (SHM1) is regulated by ubiquitin-specific-protease 16 (USB16) induced by salt stress. USB16 mediates the degradation of SHM1 effectively reducing its half-life (Zhou et al., 2013). There are many other examples of post-transcriptional regulation in response to abiotic stressors, addressed in further detail in Chapter 4 (Floris et al., 2009; Vélez-Bermúdez & Schmidt, 2014; Gibbs et al., 2016; Van Ruyskensvelde et al., 2018; Bahrman et al., 2019). In the second model, post-transcriptional regulatory mechanism(s) could reduce the SYT1 mRNA levels prior to translation. This model is illustrated by the increased stability of the Na+/H+ antiporter SOS1 mRNA during ionic stress, dehydration, and H2O2 treatment (Chung et al., 2008). The correlation between mRNA expression and protein expression has long been an uncertain relationship. Studies in yeast have demonstrated the insufficiency in predicting protein using the detected mRNA amounts, in some cases where mRNA increases by 30-fold with no change in protein (Gygi et al., 1999). This difference is not unique to yeast as there are reported cases of discordance between mRNA expression levels to protein abundance in Arabidopsis (Fukao, 2015). There are various explanations for this discordance. Vélez-Bermúdez & Schmidt propose a model whereby differences in transcript and protein abundance may be due to additional regulatory mechanisms unique to plants. Because of the diversity of ribosomal proteins (r-protein) specific to plants, environmental signals prioritize the translation of specific genes (Vélez-Bermúdez & Schmidt, 2014). These results presented here demonstrate that SYT1 is a protein where the activity of the promoter is not sufficient to predict change in the SYT1 pool. 47 The activity of the pSYT1:LUC marker decreased in response to rare earth elements (REE) and Ca2+ chelators, suggesting that the activity of the SYT1 promoter is influenced by decreasing Ca2+. Changes to plant gene expression in response to signals and hormones is common. Ca2+ signatures are known to influence gene expression in response to environmental changes (Lu & Huang, 2008; Krogan & Long, 2009). Similar to the decrease in SYT1 expression, the protein levels of SYT1 decreased in response to REEs. However, the protein amounts did not alter when exposed to Ca2+ chelators. The difference in protein levels between REE and chelator treatments may be attributed to the cellular accumulation of REEs directly influencing SYT1 protein function. In this model, the decrease in SYT1 is caused by interactions between the SYT1 protein and REEs. The chemical similarities of REE ions to Ca2+ is known to influence protein function through competitive binding. In phospholipase D (PLD), the binding of REEs to the C2 domain of PLCD increases affinity towards micelles 100-fold (Dressler et al., 2014). In this manner, rare-earth metals may compete with Ca2+ for SYT1 C2 domains, adjusting SYT1’s affinity towards the PM. Rare-earth ions are believed to bind to the C2 domain of SYT1, causing a 2- to 4-fold increase in measured SYT1-GFP-labelled “beads” in epidermal cells (Lee et al., 2020). Interestingly, this EPCS configuration is not observed when treated with Ca2+ chelators, perhaps because Ca2+ chelators are not interacting intracellularly with SYT1. Therefore, a lack of interaction between extracellular Ca2+ chelators does not impact the SYT1 protein. Overall, these experiments suggest that Ca2+ channel disruption and rare earth element accumulation decreases both pSYT1 activity and SYT1 protein, whereas extracellular Ca2+ chelation exclusively decreases SYT1. 48 In a similar fashion, when PI(4,5)P2 accumulation increases in the PM, which influences the frequency of binding events between SYT1 and the PM, the pSYT1:LUC activity and SYT1-LUC decreases, the same pattern that was observed for REEs. The relationship between PLC activity may be coupled to SYT1 activity, similar to the regulation of PLC-dependent EPCS by E-Syts in Drosophila (Nath et al., 2020). Additionally, SYT1 is known to transfer lipids at EPCS (Ruiz-Lopez et al., 2021). It is possible that changes to SYT1 function through increased affinity to the PM, caused by PI(4,5)P2 accumulation in the PM, are a cause of repression of the SYT1 gene transcription and SYT1 pool. When observing changes to SYT1 during cytoskeleton disruption, the gene expression and protein levels decrease in response to actin depolymerization (LatB). This pattern suggests a post-transcriptional regulatory mechanism that downregulates the SYT1 protein levels. Complex interactions between the cytoskeleton and SYT1 expression are highlighted in this experiment, ultimately affecting the protein pool. A protein complex that facilitates ER-cytoskeleton linkage was recently discovered and demonstrates the complex relationship between the cytoskeleton and Arabidopsis EPCS (Zhang et al., 2021). The family of cytoskeleton-interacting proteins have unique interactions with actin filaments and microtubules separately. In this manner, SYT1 expression likely responds differently to actin depolymerization compared to microtubule depolymerization. When disrupting F-actin, the ER morphology changes, which may be the cause for changes in SYT1 expression (Wang et al., 2017). Interestingly, considering that salt also disrupts microtubule assemblies, disruption using oryzalin led to the same pattern observed in most salt treatments: increased gene expression without change to protein levels. In these cases, SYT1 follows the same two explanations provided earlier: either (1) the stability of SYT1-LUC 49 is decreased and pSYT1 is induced to compensate for the decreased SYT1 protein pool, or (2) a post-transcriptional regulatory mechanism(s) decreases RNA levels before translation. Based on these results, I conclude that ionic stress reduces the stability or increases the turnover of the SYT1 protein and that increasing pSYT1 transcriptional activity is required to maintain a steady level of SYT1 protein. SYT1 has been reported to respond to many abiotic stresses, and these experiments suggest that the expression of SYT1 does not accurately predict the change in protein levels. 50 Chapter 4: SYT1 Stability and Degradation 4.1 Introduction The steady-state level of a protein is determined by its turnover, whereby the rate of protein synthesis is equal to the rate of protein degradation. This dynamic equilibrium allows for easy manipulation of the protein levels, such that the protein pool can be rapidly increased (increased synthesis, decreased degradation) or decreased (decreased synthesis, increased degradation). In addition to protein turnover, many post-transcriptional and post-translational regulatory mechanisms are involved in protein level adjustments (Floris et al., 2009). 4.1.1 mRNA processing and gene silencing in response to abiotic stress Following active transcription of protein-encoded genes, the pre-mRNA is capped, polyadenylated, and spliced in a highly regulated process that can change the steady-state levels of a protein (Zhang et al., 2008). During splicing there is a rearrangement and removal of different introns that are excised from the pre-mRNA generating different transcripts. In Arabidopsis, approximately 30% of transcripts are alternatively spliced, effectively increasing the diversity of mRNAs and proteins (Matlin et al., 2005). Alternative splicing is an important stress tolerance mechanism and is influenced by abiotic stress factors. For example, the disruption of the pre-mRNA splicing for COLD-REGULATED 15A causes hypersensitivity to chilling, salt stress, and ABA (Lee et al., 2006). Some RNA-Binding Proteins have been implicated in the expression and splicing of mRNAs highly expressed during stress (Kim et al., 2007). 51 Alternatively, mRNA can be inhibited from translation by the mechanism of RNA silencing, a conserved mechanism among Eukaryotes (Brodersen & Voinnet, 2006). RNA silencing occurs through the base pair hybridization between target mRNA and small interfering RNA (siRNA) loaded into the RNA Induced Silencing Complex (RISC), triggering the cleavage of the mRNA product. Similar to siRNA, microRNAs (miRNA) can influence gene expression through RNA silencing. The expression of some miRNAs is induced under stress, such as miR399 during phosphate starvation. The miR399 targets the 5’UTR of UBIQUITIN CONJUGATING ENZYME 24, involved in the protein degradation pathway (Jones-Rhoades & Bartel, 2004). On the other hand, some siRNAs decrease in expression during stress responses. Stress generates harmful ROS, which can be recovered using scavengers that detoxify the O2- radical. The scavengers CSD1 and CSD2 accumulate in response to intense light stress and heavy metal stress (Sunkar et al., 2006). The transcription of CSD, however, remains constant. Due to the RNA silencing from the action of miR398, CSD transcription remains unchanged under normal conditions. By reducing miR398 activity, CSD protein product increases during stress, allowing for the detoxification of ROS (Sunkar et al., 2006). 4.1.2 Post-translational regulatory mechanisms Once the fully processed mRNA reaches the ribosome, the amount of protein that is generated depends on the rate of translation and subsequent post-translational regulatory mechanisms. Initiation factors bind to ribosomes to form the synthesis complex that recognizes the genetic code at the entry site in the ribosome, the aminoacyl site (Clancy & Brown, 2008). The corresponding transfer RNA (tRNA) binds the corresponding amino acid and attaches to the polypeptide chain through peptide bonds at the peptidyl site (Clancy & Brown, 2008). Post- 52 translational protein modifications can occur non-enzymatically due to the cellular environment and reactivity of amino acids, or enzymatically on the residues and exposed protein surface (see Friso & Wijk, 2015 for a list of post-translational modifications for each residue). For example, the frequency of spontaneous deamidation of Asn to Asp and Gln to Glu has been observed to increase in response to biotic and abiotic stress (Di Pietro et al., 2013). The protein pool can also be regulated post-translationally through protein degradation. The action of protein degradation commonly occurs through the ubiquitin proteasomal system, a series of ligase enzymes that “tag” proteins for degradation. The E1 ligase transfers the activated form of the ubiquitin molecule to the E2 ligase. The E2 enzyme then transfers the ubiquitin molecule to the protein of interest, tagged by the E3 ligase. The high diversity of E3 ligase genes, especially when compared to E1 and E2 genes, suggests that E3 ubiquitin ligases provide the specificity of protein degradation in response to different stresses and development (Nelson & Millar, 2015). Repetition of the ubiquitination cycle forms a polyubiquitinated chain, ultimately recognized by the 26S proteasome complex composed of one 20S core and two 19S lids (Nelson & Millar, 2015). The 26S proteasome is located in the cytosol and nucleus of eukaryotic cells (Marshall & Vierstra, 2019). The central chamber in the barrel of the protein complex contains six active sites that cleave peptide bonds (Marshall & Vierstra, 2019). The degradation of proteins is the final step in post-translational modifications. The compilation of these processes dictates the activity and cellular reserve of proteins. 4.1.3 Techniques to study protein stability Many mechanisms regulate the transcription and translation of proteins. This long pathway allows researchers to manipulate specific points in the gene-to-protein pathway in vivo. 53 Disrupting protein synthesis by inhibiting protein translation halts the production of proteins, and the existing protein pool thus declines at the rate of degradation (Clancy & Brown, 2008). This can be studied with the most common laboratory reagent used to inhibit protein synthesis, cycloheximide (CHX) (Schneider-Poetsch et al., 2010). CHX inhibits protein synthesis through binding to the E site position of the ribosome, blocking translation elongation after peptidyl transfer of the second amino acid (Schneider-Poetsch et al., 2010). The CHX treatment on plant tissues inhibits all protein synthesis, allowing for the in vivo study of protein degradation. Similarly, the degradation of proteins via the 26S proteasome can be measured using the proteasome inhibitor MG132 (Vannini et al., 2014). The MG132 substrate binds to the catalytic subunits of the 26S proteasome, reversibly inhibiting proteolytic activity. This process causes the accumulation of damaged, misfolded, and short-lived proteins. The individual and combinatorial applications of CHX and MG132 can pinpoint protein stability in the presence and absence of proteasome activity. This chapter answers the following questions related to Arabidopsis SYT1 protein stability: 1. What is the half-life of SYT1 in normal conditions? 2. Does the half-life of SYT1 change in response to salt stress? 3. Does the 26S proteasome pathway facilitate SYT1 degradation? 4.2 Results 4.2.1 Leaf disc samples accurately measure in vivo protein degradation Changes to luciferase activity can be measured in tissues over extended periods of time. To validate the inhibition of protein synthesis in rosette leaf discs using CHX, I used the IAA1- 54 LUC marker as a convenient control for protein degradation because IAA1 encodes a well-characterized auxin response gene that produces short-lived proteins (Zenser et al., 2001; Gilkerson et al., 2009). The reported half-life of IAA1-LUC in wild type is approximately 14 minutes (Gilkerson et al., 2016). In our experimental conditions, using excised rosette discs, I determined the half-life of IAA1-LUC to be in the 12.9 to 13.5 minutes range (R2=0.96) (Figure 4.1). These results confirm that excised rosette leaf discs derived from transgenic LUC lines are suitable to measure protein degradation. Figure 4.1. The half-life of IAA1-LUC. Leaf discs of transgenic IAA1-LUC after the addition of the translational inhibitor cycloheximide. The half-life is 13 minutes (R2=0.96). The first half-life is indicated by a dashed 55 line. Leaf discs were measured in a Synergy 2 Multi-Mode Microplate Reader for 16 hours. The figure was generated in GraphPad Prism 8. 4.2.2 The stability of SYT1 increases in response to NaCl To examine changes to SYT1 protein degradation, I measured the half-life of SYT1 using CHX in the presence and absence of NaCl. Almost all transgenic plant lines had a good fit (R2>0.90) to the one-phase decay linear regression analysis. The LUC enzyme half-life was measured using pSYT1:LUC to ensure that LUC is not the driving force for protein degradation in SYT1-LUC. The 95% confidence interval for the half-life of LUC alone was 58-61 minutes, a ten-minute variation from the reported ~70 minutes (Gilkerson et al., 2009). The profile likelihood for the half-life of SYT1-LUC was measured as 45-47 minutes (R2=0.98) (Figure 4.2.2.). Compared to the luciferase enzyme alone (T1/2=59.9 minutes, R2=0.95), the degradation of SYT1-LUC does not seem to be driven by the chimerically attached LUC. There was no statistical difference between the mock half-life (58-62 minutes) and NaCl treated (65-69 minutes) of the LUC control (p=0.72). However, the stability of SYT1-LUC increased from 45-47 minutes to 51-56 minutes (T1/2=53.7 minutes, R2=0.92, p=0.0068). These results demonstrate that NaCl increases the SYT1-LUC stability in vivo. 56 Figure 4.2. Changes to SYT1 protein stability in response to NaCl. CHX was used in both conditions to determine the half-life as 46 minutes for SYT1-LUC (R2=0.98) and 54 minutes for SYT1-LUC in NaCl (R2=0.92). The first half-life is indicated by a dashed line. Leaf discs were measured in a Synergy 2 Multi-Mode Microplate Reader for 16 hours. GraphPad Prism 8 was used for a one-phase decay analysis. 4.2.3 SYT1 stability in response to proteasome inhibition To determine if SYT1-LUC degradation involves the 26S proteasome, I measured the luminescence of SYT1-LUC leaf discs in CHX in the presence and absence of the proteasomal inhibitor MG132. When measuring the half-life of the LUC control, there was no statistical difference between the mock (58-62 minutes) and the MG132 treatment (63-68 minutes) 57 (p=0.34), suggesting that the 26S proteasome is not the driving force behind LUC degradation. In contrast, the half-life of SYT1-LUC, increased from 45-47 minutes in the mock to 67-72 minutes (R2=0.94, p=0.0002) in the presence of MG132 (Figure 4.3). These results demonstrate that inhibition of the 26S proteasome decreases the rate of degradation for SYT1-LUC, suggesting that SYT1 is degraded through the 26S proteasome pathway. Figure 4.3. Changes to SYT1 protein stability in response to the proteasome inhibitor MG132. CHX was used in both conditions to determine the half-life of 46 minutes for SYT1-LUC (R2=0.98) and 70 minutes for SYT1-LUC after adding MG132 (R2=0.94). The first half-life is 58 indicated by a dashed line. Leaf discs were measured in a Synergy 2 Multi-Mode Microplate Reader for 7.5 hours. GraphPad Prism 8 was used for a one-phase decay analysis. Table 4.1. Summary of protein half-life and associated statistics in the presence of NaCl and MG132. Protein Treatment Half-life (min) Half-life 95% confidence (min) R2 p (compared to mock) IAA1-LUC 13.1 12.9 to 13.5 0.96 LUC Mock 59.9 58.1 to 61.7 0.95 NaCl 67.1 65.4 to 68.9 0.96 0.7161 MG132 65.4 63.2 to 67.7 0.94 0.3357 SYT1-LUC Mock 46.3 45.4 to 47.3 0.98 NaCl 53.7 51.5 to 56.0 0.92 0.0068 MG132 69.7 67.3 to 72.2 0.94 0.0002 4.3 Discussion The many steps involved in protein regulation allow for a tight regulation of the protein amounts in response to environmental stressors. To distinguish the stability of SYT1 in the fused SYT1-LUC protein, the unfused LUC was measured. In cellular conditions, the stability of LUC decreases due to the catalytic reaction of the substrate D-luciferin into the product oxyluciferin (Thompson et al., 1991; Feeney et al., 2016). The half-life reported in these experiments was shorter than the half-life reported by Gilkerson et al. (2009), using seedlings less than 7 DAG (~70 minutes). This reported difference in the half-life of LUC is likely due to the difference in luciferin sorption of seedlings vs leaf discs, whereby different sorption rates influence the binding of luciferin to the catalytic domain of LUC, influencing the rate of LUC degradation (Finkenstädt et al., 2008; Feeney et al., 2016; Gilkerson et al., 2016). I established the half-life of 59 LUC to be 59 minutes in these experimental conditions. Because the half-life of SYT1-LUC (46 minutes) is shorter than LUC, the degradation determinants must be present in the residues of the SYT1 protein. Due to the difference in SYT1 expression and SYT1 protein (Chapter 3), I predicted that SYT1 stability might decrease in response to salt stress and increase SYT1 expression to maintain SYT1 protein levels. However, the stability of SYT1 increased slightly after NaCl stress. There are many examples of protein stability increasing and decreasing in response to NaCl stress (Jiang et al., 2007). These results classify SYT1 as a protein that increases stability under salt stress. When treated with MG132, the SYT1 half-life increased 1.5-fold, suggesting that SYT1 degradation is impacted by the 26S proteasome. Interestingly, the half-life of SYT1 may be longer than the observed 69 minutes (in the presence of MG132) because the -LUC fusion is susceptible to initiating degradation (Gilkerson et al., 2016). The increase in proSYT1 activity without changes to SYT1 protein after NaCl stress suggests that there must be a regulatory mechanism between transcription and degradation that accounts for this difference. Additionally, changes to protein degradation are not uncommon to plants; different environmental conditions can change the rate of proteolysis and the activity of the ubiquitination pathway (Zhang et al., 2015; Zhai et al., 2016). From these results, I conclude that the increase in proSYT1 activity is not compensatory for SYT1 protein and that NaCl stress increases SYT1 stability. Therefore, an additional and unknown regulatory mechanism exists between transcription and translation that compensates for the increased pSYT1 activity. 60 Chapter 5: Comparative Gene Expression Analysis of the Arabidopsis Synaptotagmins 5.1 Introduction 5.1.1 Regulation of promoter activity and gene expression The coordinated expression of genes in space and time is essential for cellular and organ differentiation (Ruuska et al., 2002; Schmid et al., 2005; You et al., 2017). The expression of genes commonly leads to the synthesis of proteins (and microRNAs) in a spatially and temporally regulated process controlled by developmental (e.g. hormones) and environmental (e.g. stress) signals. The integration of those signals, in turn, activates and coordinates the expression of specific genes that adjusts cellular development and metabolism to the prevailing conditions. The timely expression of genes is essential for salt stress tolerance, as illustrated by the regulation of the Na+ homeostasis processes in response to NaCl stress mediated by the SOS pathway. In this pathway, transient NaCl-induced Ca2+ spikes in the cytosol are sensed by the EF-hand Ca2+-binding protein SOS3 in roots and the Ca2+-binding protein SCaBP8 in shoots (Zhu, 2016). The SOS2-SOS3 and SOS2-SCaBP8 protein complexes then activate the Na+/H+ antiporter SOS1, which mediates Na+ cytoplasmic export outside the cell, and the vacuolar Na+/H+ Exchanger (NHX) antiporter for Na+ compartmentation in the vacuole. The SOS pathway is just one example of the complex gene regulatory networks activated in response to salt stress that have been recently described (Henriquez-Valencia et al., 2015; Yang et al., 2019; Rolly et al., 2020). Other examples include the activation of DNA-binding miRNAs, such as RD29A, that bind to the promoter region of stress regulated genes and regulate their activity in response to salt stress (Hsieh et al., 2010). RD29A contains a cis-acting 61 regulatory element in the 5’ region, inducing gene expression during cold stress (Jeong et al., 2010). Combined, these examples illustrate the importance of analyzing the DNA sequence motifs in the promoter regions of putative stress regulated genes to elucidate how their expression is regulated. Promoter regions usually contain conserved cis-regulatory DNA sequences around 100 bp upstream from the transcriptional start site known as the core promoter. These conserved sequences are recognition sites for RNA polymerase II and general transcription factors (GTFs) (Haberle & Stark, 2018). The promoters also contain variable DNA sequences upstream or downstream of the transcriptional start site known as the regulatory promoter. These variable DNA sequences serve as recognition sites where specific transcription factors bind to modulate (either enhance or repress) the RNA polymerase II activity. The RNA polymerase II activity is also regulated by co-activators (increased expression) and co-repressors (decreased expression), which directly bind and regulate the activity of GTFs and the RNA Poly II. The characterization of transcription factors and DNA binding sequences have significantly expanded in the past two decades, making the computational investigation of promoters more accessible to scientists (Hudson & Quail, 2003; Sandelin et al., 2004; Ramirez & Basu, 2009; Mistry et al., 2021). Now, transcription factor databases are routinely used to predict the regulatory mechanism that controls the expression of specific genes, and here I will use some of them to understand the regulation of the SYT family members. 5.1.2 Techniques to measure gene expression. Gene expression can be measured using various techniques that measure the mRNA amounts produced, such as RNA-sequencing and quantitative PCR. There are also publicly 62 accessible databases that summarize these experiments (Austin et al., 2016). Gene expression can also be monitored using hybridization-based methods and promoter-reporter assays (Avison, 2008). A standard promoter-reporter assay frequently used in plants fuses the promoter region of a gene of interest with a gene encoding the β-glucuronidase (GUS) enzyme (Jefferson et al., 1987). The GUS enzyme hydrolyzes β-glucuronides, and when the 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate is present, the enzymatic activity produces a blue-color product that can be visualized within plant tissues. In this assay, gene expression can be estimated semi-quantitatively by measuring the intensity of the blue colour compound generated which is a proxy for the promoter activity. Using the promoter GUS assay described above, it was shown that SYT1 is ubiquitously expressed in Arabidopsis (Schapire et al., 2008). SYT2 is primarily expressed in pollen (Wang et al., 2015), and SYT3 is expressed in cotyledons and roots (Ruiz-Lopez et al., 2021). In this chapter, I used in silico analyses to predict the expression pattern of different SYTs throughout development and in response to environmental stresses and chemical treatments. I used GUS histochemical assays to validate the most interesting results obtained. This chapter answers the following three questions related to Arabidopsis SYT gene expression: 1. When and where are the SYT genes expressed throughout development using gene expression databases? 2. How much does the gene expression change for the SYT genes in response to abiotic stresses or chemical treatments? 63 3. Are the reported expression patterns of the SYT genes derived from microarray and RNAseq databases consistent with the expression obtained using the GUS reporter? 4. Can we infer any sub-functionalization/neo-functionalization for the SYT genes based on the observed expression patterns? 5.2 Results 5.2.1 Arabidopsis SYTs change expression throughout development The Arabidopsis SYTs can be defined by the presence of (1) a single transmembrane domain that anchors the protein to the PM, (2) an SMP domain that is a conserved lipid-transfer domain in the tubular lipid-binding (TULIP) superfamily (Alva & Lupas, 2016; Reinisch & De Camilli, 2016) involved in the transfer of diacylglycerol between the cER and the PM (Ruiz-Lopez et al., 2021), and (3) a variable number of Ca2+-dependent phospholipid-binding domains C2 that anchor the SYTs to the PM (Pérez-Sancho et al., 2016). Using this definition, I identified seven putative SYTs in the Arabidopsis genome (Table 5.1) that were used for in silico analyses to determine their expression profiles and response to different environmental and developmental conditions. For these analyses, I used microarray and RNA Sequence datasets available at eFP Browser [http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi] and TraVA [http://travadb.org/] (Winter et al., 2007; Klepikova et al., 2016; Kasianov et al., 2017). The in silico analyses show that SYT1, SYT5, and CLB1 are ubiquitously expressed throughout development and, in most tissues, SYT1 shows the highest expression followed by CLB1, SYT5, SYT6, SYT3, SYT4 (Figure 5.2a). Remarkably, the SYT2 transcripts are undetectable in most tissues but show high expression in reproductive tissues (Figure 5.2a), including mature pollen and stamens (Figure 5.2b and Figure 5.2c). 64 Table 5.1. The Arabidopsis family of synaptotagmins investigated in this thesis. Gene name Alias AT2G20990 SYT1 AT1G20080 SYT2 AT5G04220 SYT3 AT5G11100 SYT4 AT1G05500 SYT5 AT3G18370 SYT6 AT3G61050 CLB1 65 Figure 5.1. Gene expression of Arabidopsis SYT1-6 and CLB1 throughout development. A) Relative expression of Arabidopsis SYTs in different tissues. The x-axis represents a variety of tissues samples of different microarray/RNA seq databases. The details of the x-axis and replicates are presented in Table A3. B) Arabidopsis SYTs average expression throughout development organized by proportional representation. C) Arabidopsis SYTs average expression in flowers, organized by proportional representation. Data were normalized within each database for comparison between databases. 5.2.2 Most Arabidopsis SYTs do not change expression in response to abiotic stress Many abiotic stress-related genes in Arabidopsis are often differentially regulated and respond to abiotic factors through the change in gene expression (Cheong et al., 2003). To compare changes in gene expression during abiotic stress, I organized the expression of the Arabidopsis SYTs from the eFP Browser (Winter et al., 2007). I compiled data of samples that were measured up to 24 hours during cold, drought, genotoxic treatment with drugs that induce 66 DNA damage (bleomycin and mitomycin), heat, a drug that induces ROS-mediated oxidative stress (methyl viologen), salt stress, UV-B stress, and wounding (Winter et al., 2007). When I used a 1.5-fold expression change as a significance threshold, a total of 91 abiotic stress treatments changed the expression above the significance level for at least one SYT (Table 5.2). In this selection, 61 out of the 91 treatments increased gene expression and 30 treatments decreased gene expression below the 0.67-fold threshold. When I used a 2-fold-expression change as a threshold, 26 treatments increased gene expression and only two treatments decreased gene expression (Table 5.). Noteworthy changes in SYT3 expression include a 2.6-fold increase in response to cold, a 2-fold increase in response to genotoxic stress, and a 3.5-fold increase in response to osmotic stresses. Noticeable changes in SYT4 expression include an increase up to 3.0-fold in expression during genotoxic stress, up to 6.4-fold in heat, and 2.4 -fold and 2.0-fold in osmotic stress and salt stress. SYT6 increased in expression by 4.1-fold during osmotic stress and 3.3-fold during UV-B stress in shoot samples. Remarkably, neither SYT1 nor SYT5 change expression above the 1.5-fold threshold activity in response to any environmental abiotic stresses analyzed. Interestingly, SYT3 decreased by 0.38-fold during drought, SYT6 decreased by 0.32-fold in heat, and CLB1 decreased by 0.48-fold in oxidative stress. 67 Figure 5.2. The change in gene expression of Arabidopsis SYTs after exposure to abiotic stressors. Data was collected and summarized from tissue samples available at eFP Browser (Winter et al., 2007). The details of the x-axis are summarized in Table A4. Samples were measured in duplicates (Winter et al., 2007). Table 5.2. Frequency of the Arabidopsis SYT expression up-regulation and SYT expression downregulation in response to abiotic stress. * Many changes in SYT2 were filtered out as the expression levels for that gene were often indistinguishable from noise. Change in expression SYT1 SYT2 SYT3 SYT4 SYT5 SYT6 CLB1 Total Total* 1.5-fold 0 47 19 27 2 13 0 108 61 0.67-fold 0 18 7 3 4 5 11 48 30 2-fold 0 20 8 14 0 4 0 46 26 0.5-fold 0 10 0 0 0 1 1 12 2 5.2.3 GUS Expression of the Arabidopsis SYTs are controlled by the respective upstream promoter regions To validate and expand upon the in silico expression data for each Arabidopsis SYT, I conducted a reporter-based gene expression analysis of the different SYTs using the histochemical GUS reporter system. I cloned an approximate 1.5 Kb region upstream from the 68 translational start site into a vector containing the GFP-GUS marker (Figure 5.4). The resulting vector was used for Agrobacterium transformation. I isolated and confirmed stable transgenic lines using segregation analysis, summarized in Table A5, and the selected transgenic lines were subject to a β-Glucuronidase histochemical assay using the 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) substrate (see materials and methods) to determine the patterns of expression. Figure 5.3. Schematic diagram of gene structure for Arabidopsis SYT1-6/CLB1. Promoter regions highlighted in green were amplified from Col-0 gDNA and cloned into pENTR/D-TOPO. Arabidopsis accession numbers for neighbouring genes are listed left of each gene structure. The gene loci were adapted from JBrowse 1.16.6. SYT Expression in seedling shoots One-week-old seedlings stained with X-Gluc were variable in SYT gene expression in the cotyledons and the hypocotyl (Figure 5.4). Expression for SYT1 was strong in the vasculature, true leaf development, and apex. The expression pattern, however, was spotted rather than uniform throughout the cotyledon. The forming true leaves contained deeply stained trichomes 69 of SYT1 and SYT4. The genes SYT2, SYT5, and SYT6 were not expressed in cotyledons nor the apical meristem. Specifically, SYT6 did not have any expression in the shoots at 1-week seedlings. These experimental results show that the difference in expression between SYTs is greater than predicted by the databases (Figure 5.1). Figure 5.4. GUS expression of SYT1-6 and CLB1 one week after germination. Scale 5 mm. SYT Expression in seedling roots The expression of SYT1 was ubiquitous in most regions of the root. Expression was high in the maturation zone and along and throughout the primary root. Blue staining was also observed in root hairs and emerging lateral roots. Expression of SYT2 was low throughout the roots, with faint signals in the vasculature of the roots. The strongest SYT2 signal was observed in the root tip of the primary root, specifically the columella cells. SYT3 expression was common in the vasculature and epidermis of the primary root. SYT3 expression in root hairs was exclusive to the maturation zone. Remarkably, different from the other SYTs, no SYT3 and SYT5 expression was observed in the root tip. SYT4 was found in the vasculature along the entire root, excluding the root tip. SYT4 was expressed in the columella cells of the root cap. SYT5 was expressed in the meristem region, the vasculature of the primary root, and the tips of secondary roots. SYT6 was expressed faintly along the epidermal tissue of the primary root and root hairs, 70 as well as a strong expression found in the meristematic zone and elongation zone. CLB1 was strongly expressed throughout all regions of the root, primary and secondary roots and all regions of the root apical meristem, with the strongest expression found in the meristem. Figure 5.5. GUS expression of SYTs 1-6 and CLB1 in seedling roots. Scale: 0.5 mm SYT expression in reproductive tissues I examined the expression for each SYT gene in developing reproductive organs. The expression for SYT1 was found throughout many organs, including the stamen, pistil, sepals, and peduncle. The expression was low in the sepals, primarily stained in a spotted pattern throughout epidermal tissue. The expression for SYT1 was low in the stamen filament and stigma. Some developed pollen grains inside a closed anther were stained blue, though most expression occurred in the early stages of the anther development. SYT2 was only found in developing anthers and the pollen grains of developed anthers. When observing individual developed pollen grains from anthers, SYT2 had the strongest expression uniformly across the cytoplasm of the vegetative cell. SYT3 was expressed at low levels in the anther but did not have any noticeable 71 expression patterns in the other floral tissues. SYT3 had slight staining in the center of the pollen grain, possibly the generative cell. SYT4 expression was intense in early anther development, though this expression decreased as stamens matured. There was, however, the expression of SYT4 along the filament in matured stamens. Expression was also observed in the vasculature of the peduncle and some spotted regions of the epidermal tissue. The expression of SYT5 was highest in the petals, vasculature of the sepals, stamen filament vasculature, and the style. No expression was observed below the receptacle. Additionally, unlike all the remaining SYTs, SYT5 was expressed in developing seeds and vasculature of the silique. In contrast, the expression of SYT6 was not observed in flowers. The expression of CLB1 was only observed in the vasculature of petals. Figure 5.6. GUS stains in floral tissues. Flower scale: 1mm; pollen scale: 25 µm. 5.2.4 The Arabidopsis SYTs are grouped into four major clades The differences in expression among SYTs and their tissue specificity suggest that different SYTs might have sub- or neo-functionalized. To gain insight into how different SYTs could acquire different functions, I analyzed the phylogenetic relationships of the different SYT 72 family members. I used their conserved SMP domains for these analyses as this is the protein domain with a known function in lipid transfer (Ruiz-Lopez et al., 2021) (Figure 5.7a). Using a pairwise alignment, I calculated the percentage of amino acid identity between each SMP (Figure 5.7b) and identified the amino acid residues that are conserved across all SYTs. The conserved SMP residues were the W (7), N (9), W (16), Y (19), L (55), G (56), P (60), G (64), W (85), I (92), G (154), P (161), G (167), P (187), and I hypothesize that those residues could be essential to maintain the SMP structure and function. The sequence alignment derived from MUSCLE produced four clades, whereby SYT1, SYT2, and SYT3 were grouped in the same clade (clade I) separated from SYT4 and SYT5 (clade II) CLB1 (clade III) and SYT6 (clade IV) (Figure 5.7). The amino acid identity was highly conserved, greater than 50%, within each clade, suggesting their evolutionary conservation (Dalal & Atri, 2014). (Figure 5.7a). Of note is a low identity, less than 25%, between SYT6 and the remaining SYTs. 73 Figure 5.7. The amino acid similarity between the Arabidopsis SYTs SMP domain. A) Amino acid alignment of the SMP domain region using MUSCLE. The DNA sequence logo is shown above the consensus sequence, with the size determined on the proportion of amino acids. The frequency of similar amino acids at each position is highlighted in darkening blue. B) Percent identity between the Arabidopsis SYTs SMP domains using pairwise alignment. Alignment and logos were generated in Jalview. I also compared the promoter region of the seven putative SYTs using pairwise alignment of the nucleotide sequence (Figure A5). When analyzing the promoter region, the pairwise alignment did not produce the four SYT clades described earlier; i.e. the amino acid identity between SYTs does not translate to the regulatory promoter regions To elucidate regulatory elements that control SYT expression, I analyzed the promoter region 2Kb upstream from the translational start site for previously identified DNA sequence motifs for TFs and recurring motif binding sequences. Using a co-expression network of the 50 topmost co-expressed genes, each SYT gene was analyzed for known TF-binding sequences. I used a variety of co-expression networks for various developmental stages (seed germination, 74 roots; a full list is summarized in Chapter 2) and exogenous treatments (chemical stress and abiotic stress). Sequence motifs repeatedly found in co-expression networks were recorded (Table A5). Known sequences were primarily found in SYT1, SYT2, SYT3, and SYT4, while motifs were not identified in SYT5, SYT6, and CLB1. The dehydration-related ACGTAERD1 and the light-responsive IBOXCORE were identified in SYT3 (Table A5). The auxin-responsive factor SUPERCOREATSULTR11, root-hair specific cis-element RHERPATEXPA7, and etiolation-related ABRELATERD1 were identified in SYT1. Potential binding sites can be predicted by detecting recurrent DNA motifs common across different promoters in a co-expression gene network (Usadel et al., 2009). A familiar sequence in the promoter region of co-expressed genes suggests evolutionary conservation; a promoter DNA sequence that is conserved among alludes to possible regulatory element-binding sequences. I searched for repeated sequences in each network for each SYT gene to predict possible regulatory binding sites. Nine unique sequence motifs were found between all SYTs: three sequence motifs were found in the promoter of CLB1, two motifs were found in SYT1 and SYT4 each, and one motif was found in SYT3 and SYT5 each (Table A6). No over-represented sequence motifs were identified in co-expression networks for SYT2 and SYT6. 5.3 Discussion 5.3.1 The Arabidopsis SYTs are variably expressed throughout development and in response to abiotic stress It is well-established that the variation of gene expression patterns plays a critical role in the phenotypes and traits observed in organisms, and it is crucial to study expression profiles to elucidate tissue-specific functions (Romero et al., 2012). Determining the expression of 75 Arabidopsis SYTs is important to determine which genes are active, where the proteins are expressed, and the spatial relationship between them. Using analyses of datasets available at eFP Browser [http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi] and TraVA [http://travadb.org], SYT1 was found to be the most highly expressed SYT, followed by CLB1 and SYT5 respectively. The expression of SYT1, SYT5, and CLB1 in most tissues is consistent with previously reported expression profiles (Schapire et al., 2008; Ishikawa et al., 2020; Ruiz-Lopez et al., 2021). The ubiquitous and relatively high expression for these three genes suggests protein functionality (Butte et al., 2001; Eisenberg & Levanon, 2013). This is also supported by the recent discovery of a protein complex between SYT1, SYT5, and CLB1 (Lee et al., 2020; Ishikawa et al., 2020). The formation of a tethering complex, such as the SMP domain of E-Syt2, provides the spatial distance between membranes for the transfer of lipids, two models that have been described as the “tunnel model” and “shuttle model” (Giordano et al., 2013; Argyraki et al., 2018). Interestingly, SYT1 and SYT5 have low expression levels in pollen, a pattern opposite to SYT2 (Ishikawa et al., 2020). In this context, SYT2 expression throughout development is different from all other SYTs, as it is absent in most tissues and highly expressed in pollen (Figure 5.2). SYT2 also contains a promoter-binding sequence that is recognized by the AP2 DNA-binding domain, AP2R2, likely activating the expression of SYT2 in floral tissues (Dinh et al., 2012). A promoter deletion analysis can be used to confirm the AP2 binding sequence in the promoter, an avenue of future research. Differences in gene expression between previously reported GUS-based analyses, RNASeq databases, and the GUS stains presented in this report can be attributed to differences in the regulatory region of the gene. Differences between databases and marker-based analyses arise in the promoter size, which can predict the inclusion or exclusion of regulatory regions. For 76 example, SYT1 expression is spotted and dispersed in the cotyledons, which is different compared to the expression databases that were used for the bioinformatics analysis. These differences suggest that either an enhancing sequence exists upstream or downstream from the promoter or that the promoter includes a repressor for SYT1 that is usually deactivated or repressed by upstream or downstream elements of the promoter. 5.3.2 The Family of Arabidopsis SYTs classifies into four clades Bioinformatic analyses often utilize the protein sequence to determine the homology between the coded protein sequence, with greater accuracy when compared to the nucleotide sequence. The degeneracy of the genetic code and the expanse of the amino acid alphabet contribute to a preference for amino acid alignments in bioinformatics (Abascal et al., 2010). The promoter region, nucleotide sequence, and amino acid sequence of the SYTs were aligned and analyzed. The SMP domain alignment corresponds to the phylogeny of SYTs most recently published by Ishikawa et al., 2020. SYT6 is more related to the SYT homolog from the green algae Micromonas commoda (XP002502920) than to any other Arabidopsis SYT, implying that the genes from classes I-III (SYT1-5 and CLB1) resulted from multiple gene duplications that occurred later in the phylogeny of green plants (Ishikawa et al., 2020). Further in-depth phylogenetic analysis of the SYT family would estimate when these duplications likely happened and how they corresponded to the major events in the plant phylogeny (such as the emergence of flowering plants and, thus, new plant organs). The greater SYT2 expression in pollen suggests a functional role related to pollen, which has been demonstrated with syt2-1, syt2-2, syt2-3 loss-of-function mutants and RNAi mutant lines that exhibit a reduction in pollen germination and pollen tube growth (Wang et al., 2015). 77 Given the high similarity in amino acid identity and the predicted protein structure, similar cellular functions related to EPCS between SYT1 and SYT2 are plausible. However, the effect of SYT1 on normal pollen development/germination is not known. So far, the potential differential function of these two genes can only be based on their different expression levels in pollen and anthers. On the other hand, SYT3 has been determined to be functionally redundant to SYT1 under cold stress conditions, since the syt1 cold hypersensitive phenotype has been rescued in a 35S:SYT3-GFP transgenic line (Ruiz-Lopez et al., 2021). It remains to be tested whether overexpression of SYT2 would have the same rescuing effect as the overexpression of SYT3-GFP does on syt1-2. It is interesting that SYT1, SYT5, and CLB1 have the highest expression in most of the tissues, and belong to different clades, as well as forming a functional complex. This suggests that further sub-functionalization arose within the separate clades, where the more recent duplicates, such as SYT2 and SYT4, obtained functions that are specific to certain tissues and developmental stages. Further functional analyses are required to determine the unique roles of all Arabidopsis SYT proteins. It is important to note that overlapping and complementing protein functions between paralogs does not exclude neo- and sub-functionalization; rather, predictions of genetic redundancy involve many criteria (e.g., duplication patterns, epigenetic markers, posttranslational modifications) (Cusack et al., 2021). Despite the redundancy of protein function between SYT1 and SYT3, the difference in spatial-temporal expression between SYTs throughout development and abiotic stress highlights that there is not complete redundancy, but a generalized overlap in function. The redundancy of SYTs is continuous, as evidenced by the severity of phenotype of the syt1 single mutant compared to the syt3 single mutant and compared to the syt1/syt3 double mutant, or the syt1/syt5/clb1 (Lee et al., 2020; Ruiz-Lopez et al., 2021). 78 Using co-expression networks, the spatial-temporal expression can also elucidate gene function. Genes expressed in the same cell type, at the same time during development and stress, suggests a shared regulatory network. Similar proteins can be found using this method, which can assist in function prediction. When measuring additional RNASeq data, the SYT1 orthologue in Pisum sativum is positively induced in response to cold (Bahrman et al., 2019). Although SYT1 may not be responsive in the RNASeq data presented here, expression may change more than was reported in different tissues and different conditions. When comparing promoter activity measured through the firefly luciferase (Chapter 3) to the publicly accessible microarray and RNAseq databases, it was determined that SYT1 is induced under salt stress (Figure 4.3). For example, all abiotic stressors do not induce an increase in SYT1 greater than two-fold, yet, the results from these experiments demonstrate an increase by approximately 3.9-fold after treatment with NaCl. The discrepancy of SYT1 expression may be due to multiple factors. The difference in mRNA abundance compared to luciferase activity may be due to post-transcriptional regulatory processes. The luciferase experiments exclusively examine the change in promoter activity, whereas Winter et al., (2007) measured the absolute mRNA abundance, which will depend on transcription rates and stability of mRNA. Additionally, the observed expression depends on the age of the seedlings and the tissues sampled. This project exclusively measured activity in cotyledons and the hypocotyl of 8-day old seedlings, whereas Winter et al., 2007 measured whole tissue of 18-day Arabidopsis. These differences in measurement suggest that SYT1 is not induced at 18 days after germination but increases approximately 3.9-fold at 8 days after germination. Nonetheless, these results presented in this thesis suggest the promoter region for SYT1 is induced in 8-day Arabidopsis seedlings. 79 Figure 5.8. Simplified schematic of SYT expression in Arabidopsis seedlings. 80 Chapter 6: Conclusions and Future Directions Using luciferase markers, I determined that SYT1 expression is ubiquitous during seedling growth, with some higher expression levels around the nodes during adult growth. The expression of pSYT1:LUC is uncoupled to SYT1-LUC accumulation, especially in the presence of salt stress, whereby the increased gene expression does not reflect the change in protein amounts. To further understand the relationship between SYT1 gene expression and protein levels, stress tests using non-chemical abiotic stressors (e.g. temperature) could elucidate unknown patterns. It was demonstrated that SYT1 gene expression changes under cold, and this could be confirmed and further compared to SYT1 protein accumulation (Ruiz-Lopez et al., 2021). Additionally, using an EMS mutant screen of the luciferase lines can identify changes to SYT1 expression, ultimately identifying candidates for suppressors and/or enhancers of SYT1. SYT1 protein degrades through the 26S proteasome pathway, and decreases degradation in the presence of NaCl. Due to the half-life limitations of luciferase, it cannot be determined if SYT1-half life is equal or greater than the observed 69 minutes. Future experiments using other methods, such as Western blotting of SYT1, can identify a more accurate SYT1 half-life in the presence of MG132. Additionally, the change in protein stability should be tested in the presence and absence of additional stressors, such as other salts (KCl, MgCl2) and in response to temperature. The Arabidopsis SYT family is diverse in gene expression throughout development and in response to stress. Future research can further validate the bioinformatic analyses that predict changes in gene expression, such as the use of the GUS lines produced in this thesis in response to stressors. Identification of noticeable changes to gene expression will suggest a function or response in stress. However, as was discovered in Chapter 3, the activity of the promoter does 81 not reflect the changes in SYT1 protein. Therefore, I recommend that the protein amounts of the other Arabidopsis SYTs be compared to the change in gene expression. 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Plant line (T2 generation in Col-0) Positive samples Negative samples p 1-pSYT2:GUS 70 30 0.28 2-pSYT2:GUS 71 20 0.51 1-pSYT3:GUS 69 21 0.72 2-pSYT3:GUS 97 29 0.61 3-pSYT3:GUS 85 27 0.83 1-pSYT4:GUS 94 36 0.48 2-pSYT4:GUS 116 36 0.71 1-pSYT5:GUS 69 31 0.17 2-pSYT5:GUS 114 37 0.89 3-pSYT5:GUS 100 39 0.41 4-pSYT5:GUS 119 45 0.47 1-pSYT6:GUS 86 30 0.83 2-pSYT6:GUS 91 26 0.49 3-pSYT6:GUS 111 36 0.89 4-pSYT6:GUS 83 30 0.70 1-pCLB1:GUS 68 25 0.68 2-pCLB1:GUS 68 24 0.81 Table A2. Segregation analysis of pSYT1:LUC in Col-0 in the T2 generation. 95 All samples were confirmed in the T3 generation. Segregation analysis for pSYT1:LUC (Col-0) had already been completed for one line by EunKyoung Lee, I segregated and found two additional stable transgenic lines. The Chi-square test was conducted for each line expecting a 3:1 ratio of positive to negative samples, the p-value for the test is listed in the rightmost column. Plant line (T2 generation) Positive samples Negative samples p 1-pSYT1:LUC (Col-0) 67 24 0.76 2-pSYT1:LUC (Col-0) 70 22 0.81 1-pSYT1:SYT1-LUC (Col-0) 66 25 0.59 2-pSYT1:SYT1-LUC (Col-0) 51 16 0.83 3-pSYT1:SYT1-LUC (Col-0) 51 18 0.83 4-pSYT1:SYT1-LUC (Col-0) 61 22 0.75 5-pSYT1:SYT1-LUC (Col-0) 69 27 0.72 1-pSYT1:SYT1-LUC (syt1-2) 76 21 0.45 2-pSYT1:SYT1-LUC (syt1-2) 65 25 0.54 3-pSYT1:SYT1-LUC (syt1-2) 67 23 0.90 96 Table A3. Tissue samples, in order from left to right of the x-axis in Figure 5.1 and the respective source for each datum. Samples measured as triplicates for AtGenExpress eFP, duplicates for Klepikova eFP, and duplicates for Abiotic Stress eFP. Sample Source Dry seed Schmid et al. 2005; Winter et al. 2007 Dry seeds Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Dry Seeds Klepikova et al. 2016 Imbibed seed, 24 h Schmid et al. 2005; Winter et al. 2007 Germinating seeds 1 Klepikova et al. 2016 Germinating seeds 1 (first day after soaking) Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Germinating seeds 2 Klepikova et al. 2016 Germinating seeds 2 (second day after soaking) Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Germinating seeds 3 Klepikova et al. 2016 Germinating seeds 3 (third day after soaking) Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Meristem at 10 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Meristem at 11 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Meristem at 12 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Hypocotyl Winter et al. 2007 Cotyledon Winter et al. 2007 1st Node Winter et al. 2007 Stem, 2nd Internode Winter et al. 2007 Seedling cotyledons Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Seedling Cotyledons Klepikova et al. 2016 Seedling hypocotyl Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Seedling Hypocotyl Klepikova et al. 2016 Seedling meristem Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Seedling Meristem Klepikova et al. 2016 SAM at 7 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 SAM at 8 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 SAM at 9 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Shoot Apex, Vegetative Winter et al. 2007 Vegetative Rosette Winter et al. 2007 Vein of the mature leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Whole mature leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Vein of the senescent leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Internode Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Internode Klepikova et al. 2016 Cauline Leaf Winter et al. 2007 Leaf 1 + 2 Winter et al. 2008 Leaf 7, Distal Half Winter et al. 2009 Leaf 7, Petiole Winter et al. 2010 Leaf 7, Proximal Half Winter et al. 2011 Leaf blade of the young leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Leaf blade, intermediate 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Leaf blade, intermediate 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Leaf blade of the mature leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Leaf Lamina of the young leaf Klepikova et al. 2016 Leaf Lamina, intermediate 1 Klepikova et al. 2016 Leaf Lamina, intermediate 2 Klepikova et al. 2016 Leaf Lamina of the mature leaf Klepikova et al. 2016 Leaf Petiole of the young leaf Klepikova et al. 2016 Leaf Petiole, intermediate 1 Klepikova et al. 2016 Leaf Petiole, intermediate 2 Klepikova et al. 2016 Leaf Petiole of the senescent leaf Klepikova et al. 2016 Leaf Petiole of the mature leaf Klepikova et al. 2016 Leaf vein, intermediate 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Leaf Vein, intermediate 2 Klepikova et al. 2016 Leaf Vein of the mature leaf Klepikova et al. 2016 97 Leaf, mature Klepikova et al. 2016 Leaf Vein of the senescent leaf Klepikova et al. 2016 Rosette Leaf 10 Winter et al. 2007 Rosette Leaf 12 Winter et al. 2007 Rosette Leaf 2 Winter et al. 2007 Rosette Leaf 4 Winter et al. 2007 Rosette Leaf 6 Winter et al. 2007 Rosette Leaf 8 Winter et al. 2007 Petiole of the young leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2017 Petiole, intermediate 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2018 Petiole, intermediate 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2019 Petiole of the mature leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Petiole of the senescent leaf Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2021 Senescent internode Klepikova et al. 2016 Senescent internode. Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Senescing Leaf Winter et al. 2007 Root Winter et al. 2007 Root Winter et al. 2007 Root apex Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Root Apex Klepikova et al. 2016 Root without apex Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Root without apex Klepikova et al. 2016 Seedling root Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seedling Root Klepikova et al. 2016 Shoot Apex, Transition Winter et al. 2007 Shoot Apex, Inflorescence Winter et al. 2007 Inflorescence meristem at 13 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Inflorescence meristem at 14 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Inflorescence meristem at 15 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Inflorescence meristem at 16 days after germination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Entire Rosette After Transition to Flowering Winter et al. 2007 Axis of inflorescence Klepikova et al. 2016 Axis of the inflorescence Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pedicel Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pedicel Klepikova et al. 2016 Flower 1 Klepikova et al. 2016 Flower 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flower 2 Klepikova et al. 2016 Flower 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flower 3 Klepikova et al. 2016 Flower 3 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flower 4 Klepikova et al. 2016 Flower 4 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flower 5 Klepikova et al. 2016 Flower 5 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Carpel of 6th and 7th flowers Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Carpel of the 6th and 7th flowers Klepikova et al. 2016 Ovules from 6th and 7th flowers Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Ovules from the 6th and 7th flowers Klepikova et al. 2016 Flowers 6-8 Klepikova et al. 2016 Flower 6-8 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flower Stage 9 Winter et al. 2007 Flower 9-11 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flowers 9-11 Klepikova et al. 2016 Flower Stage 10/11 Winter et al. 2007 Flower Stage 12 Winter et al. 2007 Flower Stage 12, Carpels Winter et al. 2007 Flower Stage 12, Petals Winter et al. 2007 Flower Stage 12, Sepals Winter et al. 2007 Flower Stage 12, Stamens Winter et al. 2007 98 Flower 12-14 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flowers 12-14 Klepikova et al. 2016 Flower Stage 15 Winter et al. 2007 Flower Stage 15, Carpels Winter et al. 2007 Flower Stage 15, Petals Winter et al. 2007 Flower Stage 15, Sepals Winter et al. 2007 Flower Stage 15, Stamen Winter et al. 2007 Flowers Stage 15, Pedicels Winter et al. 2007 Flowers 15-18 Klepikova et al. 2016 Flower 15-18 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Flowers 19 and following Klepikova et al. 2016 Flower 19+ Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Petals of the mature flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Petals of the mature flower Klepikova et al. 2016 Sepals of the mature flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Sepals of the mature flower Klepikova et al. 2016 Sepals of the young flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Sepals of the young flower Klepikova et al. 2016 Stigmatic tissue Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Stigmatic tissue Klepikova et al. 2016 Carpels of the young flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Carpels of the young flower Klepikova et al. 2016 Carpels of the mature flower (before pollination Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Carpels of the mature flower (before pollination) Klepikova et al. 2016 Stamen filaments of the mature flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Stamen filaments of the mature flower Klepikova et al. 2016 Anthers of the young flower Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Anthers of the young flower Klepikova et al. 2016 Anthers of the mature flower (before opening) Klepikova et al. 2016 Anthers of the mature flower (before opening). Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Opened anthers Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Opened anthers Klepikova et al. 2016 Mature Pollen Winter et al. 2007 Young seeds 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Young seeds 1 Klepikova et al. 2016 Young seeds 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Young seeds 2 Klepikova et al. 2016 Young seeds 3 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Young seeds 3 Klepikova et al. 2016 Young seeds 4 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Young seeds 4 Klepikova et al. 2016 Young seeds 5 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Young seeds 5 Klepikova et al. 2016 Seeds 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seeds 1 Klepikova et al. 2016 Seeds 3 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seeds 3 Klepikova et al. 2016 Seeds 5 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seeds 5 Klepikova et al. 2016 Seeds 7 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seeds 7 Klepikova et al. 2016 Seeds Stage 3 w/ Siliques Winter et al. 2007 Seeds Stage 4 w/ Siliques Winter et al. 2007 Seeds Stage 5 w/ Siliques Winter et al. 2007 Seeds Stage 6 w/o Siliques Winter et al. 2007 Seeds Stage 7 w/o Siliques Winter et al. 2007 Seeds Stage 8 w/o Siliques Winter et al. 2007 Seeds Stage 9 w/o Siliques Winter et al. 2007 Seeds Stage 10 w/o Siliques Winter et al. 2007 Silique 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 99 Silique 2 Klepikova et al. 2016 Silique 4 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Silique 4 Klepikova et al. 2016 Silique 6 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Silique 6 Klepikova et al. 2016 Silique 8 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Silique 8 Klepikova et al. 2016 Pod of the silique 1 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pod of the silique 1 Klepikova et al. 2016 Pod of the silique 3 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pod of the silique 3 Klepikova et al. 2016 Pod of the silique 5 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pod of the silique 5 Klepikova et al. 2016 Pod of the silique 7 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Pod of the silique 7 Klepikova et al. 2016 Seeds of first yellowing silique. Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Seeds from the senescent silique 1 Klepikova et al. 2016 Senescent silique 2 Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Senescent silique 2 Klepikova et al. 2016 Pod of the senescent silique 1 Klepikova et al. 2016 Pod of the senescent silique 1. Klepikova et al. 2015; Klepikova et al. 2016; Klepikova et al. 2020 Table A4. Tissue samples and respective abiotic treatment in order from left to right of the x-axis in Figure 5.2. Samples measured as triplicates for AtGenExpress eFP, duplicates for Klepikova eFP, and duplicates for Abiotic Stress eFP. Cold Root After 30 Minutes Heat Root After 1 Hour Salt Root After 3 Hours Cold Root After 1 Hour Heat Root After 3 Hours Salt Root After 6 Hours Cold Root After 3 Hours Heat Root After 4 Hours Salt Root After 12 Hours Cold Root After 6 Hours Heat Root After 6 Hours Salt Root After 24 Hours Cold Root After 12 Hours Heat Root After 12 Hours Salt Shoot After 30 Minutes Cold Root After 24 Hours Heat Root After 24 Hours Salt Shoot After 1 Hour Cold Shoot After 30 Minutes Heat Shoot After 15 Minutes Salt Shoot After 3 Hours Cold Shoot After 1 Hour Heat Shoot After 30 Minutes Salt Shoot After 6 Hours Cold Shoot After 3 Hours Heat Shoot After 1 Hour Salt Shoot After 12 Hours Cold Shoot After 6 Hours Heat Shoot After 3 Hours Salt Shoot After 24 Hours Cold Shoot After 12 Hours Heat Shoot After 4 Hours UV-B Root After 15 Minutes Cold Shoot After 24 Hours Heat Shoot After 6 Hours UV-B Root After 30 Minutes Drought Root After 15 Minutes Heat Shoot After 12 Hours UV-B Root After 1 Hour Drought Root After 30 Minutes Heat Shoot After 24 Hours UV-B Root After 3 Hours Drought Root After 1 Hour Osmotic Root After 30 Minutes UV-B Root After 6 Hours Drought Root After 3 Hours Osmotic Root After 1 Hour UV-B Root After 12 Hours Drought Root After 6 Hours Osmotic Root After 3 Hours UV-B Root After 24 Hours Drought Root After 12 Hours Osmotic Root After 6 Hours UV-B Shoot After 15 Minutes Drought Root After 24 Hours Osmotic Root After 12 Hours UV-B Shoot After 30 Minutes Drought Shoot After 15 Minutes Osmotic Root After 24 Hours UV-B Shoot After 1 Hour Drought Shoot After 30 Minutes Osmotic Shoot After 30 Minutes UV-B Shoot After 3 Hours Drought Shoot After 1 Hour Osmotic Shoot After 1 Hour UV-B Shoot After 6 Hours Drought Shoot After 3 Hours Osmotic Shoot After 3 Hours UV-B Shoot After 12 Hours Drought Shoot After 6 Hours Osmotic Shoot After 6 Hours UV-B Shoot After 24 Hours Drought Shoot After 12 Hours Osmotic Shoot After 12 Hours Wounding Root After 15 Minutes Drought Shoot After 24 Hours Osmotic Shoot After 24 Hours Wounding Root After 30 Minutes Genotoxic Root After 30 Minutes Oxidative Root After 30 Minutes Wounding Root After 1 Hour Genotoxic Root After 1 Hour Oxidative Root After 1 Hour Wounding Root After 3 Hours Genotoxic Root After 3 Hours Oxidative Root After 3 Hours Wounding Root After 6 Hours Genotoxic Root After 6 Hours Oxidative Root After 6 Hours Wounding Root After 12 Hours Genotoxic Root After 12 Hours Oxidative Root After 12 Hours Wounding Root After 24 Hours Genotoxic Root After 24 Hours Oxidative Root After 24 Hours Wounding Shoot After 15 Minutes Genotoxic Shoot After 30 Minutes Oxidative Shoot After 30 Minutes Wounding Shoot After 30 Minutes Genotoxic Shoot After 1 Hour Oxidative Shoot After 1 Hour Wounding Shoot After 1 Hour Genotoxic Shoot After 3 Hours Oxidative Shoot After 3 Hours Wounding Shoot After 3 Hours Genotoxic Shoot After 6 Hours Oxidative Shoot After 6 Hours Wounding Shoot After 6 Hours Genotoxic Shoot After 12 Hours Oxidative Shoot After 12 Hours Wounding Shoot After 12 Hours 100 Genotoxic Shoot After 24 Hours Oxidative Shoot After 24 Hours Wounding Shoot After 24 Hours Heat Root After 15 Minutes Salt Root After 30 Minutes Heat Root After 30 Minutes Salt Root After 1 Hour Table A5. Promoter binding sequences found in plant promoter databases, with the respective location in SYT upstream from the translational start site. Perturbation Network Gene Cis-element name Sequence Site Z-score TFBS Function Reference Development SYT1 POLASIG2 AATTAAA -349, -354, -363 3.2 PolyA signal in alpha-amylase (rice) O’Neil et al., 1990 SYT3 WBOXATNPR1 TTGAC -508, -1250 3.31 NPR1-related WRKY Box Yu et al., 2001 TAAAGSTKST1 TAAAG -34, -771, -1024, -1508, -1643, 4 Guard cell regulation and K+ influx Plesch et al., 2001 SYT4 DOFCOREZM AAAG -223, -232, -318, -599 5.73 Maize Dof protein regulation Yanagisawa, 2001 Chemical Stress SYT1 ABRELATERD1 ACGTG -192, -705 3.09 Etiolation-induced erd1 expression Simpson et al., 2003 SYT2 INRNTPSADB YTCANTYY -666, -1320 3.03 “Inr”; light-responsiveness is impaired when the INR is replaced with a TATA box Nakamura et al., 2002 SYT3 ACGTATERD1 ACGT -269, -876, -976, -1115, -1703 3.02 Etiolation- and dehydration-induced erd1 expression Simpson et al., 2003 IBOXCORE GATAA -285, -633, -904, -930, -1226, -1537 3.05 “I-Box”; found in many light-responsive genes, but also non-light responsive genes (CaMV35S) Terzaghi & Cashmore, 1995 Abiotic Stress SYT1 RHERPATEXPA7 KCACGW -222, -236, -821, -961 4.19 Root-hair (type 3=positionally cued) specific cis-elements Kim et al., 2006 SURECOREATSULTR11 GAGAC -910, -1010 3.62 Auxin- response factor; core of SURE; adaptation to S deficiency Maruyama-Nakashita et al., 2005 Pollen Germination SYT1 POLASIG2 AATTAAA -349, -354, -363 3.66 PolyA signal in alpha-amylase (rice) O’Neil et al., 1990 Stigma and Ovaries SYT1 POLASIG2 AATTAAA -349, -354, -363 3.51 PolyA signal in alpha-amylase (rice) O’Neil et al., 1990 Table A6. Predicted Motifs in the upstream promoter region of Arabidopsis NTMCs. Perturbation Network Gene Cis-element name Motif Sequence SYT Sequence BP from ATG Development SYT1 Motif 1 T G G G C Y B T G G G C T T -237, -631 SYT4 Motif 2 C G C G C C G C G C G C C G CLB1 Motif 4 C Y R A A C C C Y R A A C C -1765 Chemical stress SYT4 Motif 5 C G C C G G A C G C C G G A CLB1 Motif 6 G T G G M G G G T G G A G G -1162 Abiotic Stress SYT1 Motif 8 C C R C C W S C C A C C T G -647 SYT3 Motif 9 M C A C G T S A C A C G T C -878 Pollen Germination SYT1 Motif 1 T G G G C Y B T G G G C T T -237, -631 SYT4 Motif 2 C G C G C C G C G C G C C G CLB1 Motif 4 C Y R A A C C C T G A A C C -1765 Stigma and Ovaries SYT1 Motif 1 T G G G C Y B T G G G C T T -237, -631 SYT4 Motif 2 C G C G C C G C G C G C C G CLB1 Motif 4 C Y R A A C C C T G A A C C -1765 Roots SYT5 Motif 3 T G G G C Y B T G G T G T G -62 CLB1 Motif 7 C C G G T Y Y C C G G T T T -115 101 Figure A1. Relationship between luciferase light intensity compared to the total mass of the seedlings for pSYT1:LUC. Figure A2. Relationship between luciferase light intensity compared to the total mass of the seedlings for SYT1-LUC. 102 Figure A3. pSYT1:LUC expression in rosette leaves of 3 week old Arabidopsis plants. Figure A4. SYT1-LUC expression in rosette leaves of 3-week-old Arabidopsis plants. 103 Figure A5. The DNA nucleotide similarity between the Arabidopsis SYTs promoter region.
Thesis/Dissertation
eng
Botany
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Rosado, Abel
Analysis of the Arabidopsis synaptotagmin gene expression and protein accumulation during stress using luciferase-based markers
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