Arts and Sciences, Irving K. Barber School of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Adal, Ayelign Mengesha
Doctor of Philosophy - PhD
University of British Columbia
Several species of the genus Lavandula (lavenders) are important plants grown for their essential oils (EO), which find extensive applications in cosmetics, personal care products and alternative medicines. The EO of these plants is mainly constituted of monoterpenes, the C₁₀ class of terpenoids. In this study, we investigated three aspects of lavenders using advanced genomic resources derived from EO-producing tissues of these plants. Firstly, we developed diagnostic simple sequence repeat (SSR) markers from expressed sequence tag (EST) databases to delineate closely related species. Over 250 SSRs (≥ 18 bp) were developed from L. angustifolia and L. x intermedia ESTs. Thirty-one EST-SSRs were validated, 24 of which had a strong discrimination power in donor plant species, and a considerable transferability into related species. Secondly, we identified and functionally characterized cDNAs encoding geranyl pyrophosphate synthase (GPPS), which produces GPP, the linear precursor to all regular monoterpenes. Lavender GPPS is a heterodimeric protein composed of a small subunit (LiGPPS.SSU1), and a large subunit (LiGPPS.LSU). Recombinant LiGPPS.SSU1 interacts with LiGPPS.LSU, catalyzing the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to produce GPP. Recombinant LiGPPS.SSU1 also interacts with lavender and N. benthamiana geranylgeranyl pyrophosphate synthase (GGPPS; the enzyme responsible for synthesis of GGPP, the linear precursor to diterpenes), modifying its activity to form GPP. Transcript levels for LiGPPS.SSU1 and LiGPPS.LSU parallel total monoterpene concentration, with more transcripts present in flowers where most of lavender EO accumulates. Finally, we cloned and functionally characterized lavender cDNAs encoding two monoterpene synthase genes, 3-carene synthase (Li3CARS) and S-linalool synthase (LiS-LINS), in vitro. Recombinant Li3CARS and LiS-LINS proteins converted GPP into 3-carene and S-linalool, respectively. Li3CARS transcripts and the levels of 3-carene were up-regulated by methyl jasmonate, suggesting a potential defensive role for 3-carene. The transcripts for LiS-LINS were also highly expressed in flowers, where S-linalool accumulates.
DEVELOPMENT OF MOLECULAR MARKERS AND CLONING OF GENES INVOLVED IN THE BIOSYNTHESIS OF MONOTERPENES IN LAVANDULA by Ayelign Mengesha Adal B.Sc., Jimma University, 2006 M.Sc., Leibniz University of Hannover, 2011 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) August 2019 © Ayelign Mengesha Adal, 2019 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: DEVELOPMENT OF MOLECULAR MARKERS AND CLONING OF GENES INVOLVED IN THE BIOSYNTHESIS OF MONOTERPENES IN LAVANDULA submitted by Ayelign Mengesha Adal in partial fulfillment of the requirements of the degree of Doctor of Philosophy Dr. Soheil S. Mahmoud, Biology, Irving K. Barber School of Arts and Sciences, UBC Supervisor Dr. Micheal Deyholos, Biology, Irving K. Barber School of Arts and Sciences, UBC Supervisory Committee Member Dr. Louise Nelson, Biology, Irving K. Barber School of Arts and Sciences, UBC Supervisory Committee Member Dr. Wesley Zandberg, Irving K. Barber School of Arts and Sciences, UBC University Examiner Dr. Jim Mattsson, Department of Biological Sciences, Simon Fraser University External Examiner Additional Committee Members include: Dr. Nasser Yalpani, Adjunct Professor, Biology, Irving K. Barber School of Arts and Sciences, UBC Supervisory Committee Member . iii Abstract Several species of the genus Lavandula (lavenders) are important plants grown for their essential oils (EO), which find extensive applications in cosmetics, personal care products and alternative medicines. The EO of these plants is mainly constituted of monoterpenes, the C10 class of terpenoids. In this study, we investigated three aspects of lavenders using advanced genomic resources derived from EO-producing tissues of these plants. Firstly, we developed diagnostic simple sequence repeat (SSR) markers from expressed sequence tag (EST) databases to delineate closely related species. Over 250 SSRs (≥ 18 bp) were developed from L. angustifolia and L. x intermedia ESTs. Thirty-one EST-SSRs were validated, 24 of which had a strong discrimination power in donor plant species, and a considerable transferability into related species. Secondly, we identified and functionally characterized cDNAs encoding geranyl pyrophosphate synthase (GPPS), which produces GPP, the linear precursor to all regular monoterpenes. Lavender GPPS is a heterodimeric protein composed of a small subunit (LiGPPS.SSU1), and a large subunit (LiGPPS.LSU). Recombinant LiGPPS.SSU1 interacts with LiGPPS.LSU, catalyzing the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to produce GPP. Recombinant LiGPPS.SSU1 also interacts with lavender and N. benthamiana geranylgeranyl pyrophosphate synthase (GGPPS; the enzyme responsible for synthesis of GGPP, the linear precursor to diterpenes), modifying its activity to form GPP. Transcript levels for LiGPPS.SSU1 and LiGPPS.LSU parallel total monoterpene concentration, with more transcripts present in flowers where most of lavender EO accumulates. Finally, we cloned and functionally characterized lavender cDNAs encoding two monoterpene synthase genes, 3-carene synthase (Li3CARS) and S-linalool synthase (LiS-LINS), in vitro. Recombinant Li3CARS and LiS-LINS proteins converted GPP into 3-carene and S-linalool, respectively. Li3CARS transcripts and the levels of 3-carene were up-regulated by iv methyl jasmonate, suggesting a potential defensive role for 3-carene. The transcripts for LiS-LINS were also highly expressed in flowers, where S-linalool accumulates. v Lay Summary This thesis describes the development of genetic markers for lavenders, and reports the cloning and functional characterization of genes that direct essential oil formation in these plants. First, we developed over 250 genetic markers, some of which were tested and had a strong discrimination power between two commercially grown lavender species, L. angustifolia and L. x intermedia. Next, we identified and studied genes encoding the small and large subunits for geranyl diphosphate synthase. This enzyme mediates the generation of geranyl diphosphate, the immediate precursor to lavender monoterpene essential oil constituents. Finally, we isolated and functionally characterized genes responsible for the production of 3-carene and S-linalool, two important monoterpene constituents of lavender essential oil. vi Preface Chapter 2 has been published in Planta and Bio-protocol journals. The contents published in Planta were reprinted/ adapted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs, Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014, License # 4466820330355. The content from Bio-protocol was published as Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud (2016) EST-SSR analysis and cross-species transferability study in Lavandula, Bio-protocol 6(15): e1891, © Authors 2016, Creative Commons Attribution License (CC BY 4.0.). I conducted the identification and validation of EST-SSRs and prepared manuscripts with guidance and inputs of Dr. Soheil Mahmoud in Dr. Soheil Mahmoud's lab. Dr. Zerihun A. Demissie who was a Post-Doctoral fellow in Dr. Soheil Mahmoud's lab at UBCO helped me on annotation and manuscript preparation. Chapter 3 was prepared for publication as Ayelign M. Adal and Soheil S. Mahmoud "Short-chain trans-isoprenyl diphosphate synthases in lavender (Lavandula)" and is under revision in Plant Molecular Biology. I designed the experiments, functionally characterized isoprenyl diphosphate synthases and prepared the manuscript. I conducted this study in Dr. Soheil Mahmoud's lab under the supervision of Dr. Soheil Mahmoud. In Chapter 4, two manuscripts were produced. A portion of this Chapter has been published in Plant Molecular Biology journal. The contents were reprinted/ adapted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia, Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017, License # 4466820619201. I identified 3-carene synthase candidate, designed and conducted the experiments, wrote the manuscript with the guidance of Dr. Soheil Mahmoud. Mr. Lukman S. Sarker helped on qPCR analysis. Ms. Ashley Lemke was an undergraduate student working vii with me on essential oil analysis from methyl jasmonate-treated plants. Another portion of this Chapter has been published in Planta and the contents were reprinted/ adapted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula), Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018, License # 4466811214167. I identified the S-linalool synthase candidate, designed and conducted the experiments, wrote the manuscript with the guidance of Dr. Soheil Mahmoud. Mr. Lukman S. Sarker assembled and annotated RNA-seq and helped on cloning of the candidate gene. Mr. Radesh PN Malli and Dr. Ping Liang analyzed genomic seq and copy numbers of S-linalool synthase in the genome. I studied both monoterpene synthases in Dr. Soheil Mahmoud's lab. While doing my thesis project, I also co-authored the following manuscripts: a) Malli RPN, [Adal AM], Sarker LS, Liang P and Mahmoud SS (2019) De novo sequencing of the Lavandula angustifolia genome reveals highly duplicated and optimized features for essential oil production, Planta, 249(1):251–256. (I collected leaf tissues, prepared genomic DNA for sequencing and provided ESTs and transcriptomes for annotation). b) Hind KR, [Adal AM], Upson TM and Mahmoud SS (2018) An assessment of plant DNA barcodes for the identification of cultivated Lavandula (Lamiaceae) taxa, Biocatalysis and Agricultural Biotechnology, 16:459-466. (I re-analyzed sequence-based barcoding comparison and modified the manuscript for publication). c) Wells R, Truong F, [Adal AM], Sarker LS and Mahmoud SS (2018) Lavandula essential oils: a current review of applications in medicinal, food, and cosmetic industries of lavender. Natural Product Communications, 13(10): 1403-1417. (I wrote a section of the review and helped with reference management and manuscript preparation). viii d) Demissie ZA, Tarnowycz M, [Adal AM], Sarker LS and Mahmoud SS (2019) A lavender ABC transporter confers resistance to monoterpene toxicity in yeast. Planta, 249(1):139–144. (I contributed on cloning of ABC transporter to yeast expression vector, yeast transformation and running yeast assay). ix Table of Contents Abstract..........................................................................................................................................iii Lay Summary................................................................................................................................. v Preface .......................................................................................................................................... vi Table of Contents..........................................................................................................................ix List of Tables ............................................................................................................................... xv List of Figures ............................................................................................................................ xvi List of Abbreviations ..................................................................................................................xix Acknowledgments ......................................................................................................................xxi Dedication ..................................................................................................................................xxiii Chapter 1: Introduction ................................................................................................................ 1 1.1 The genus Lavandula ................................................................................................. 1 1.2 Molecular Markers ...................................................................................................... 2 1.3 Lavandula essential oil constituents ........................................................................... 5 1.4 Monoterpene biosynthesis .......................................................................................... 7 1.4.1 Isoprenyl diphosphate synthases ........................................................................... 8 1.4.1.1 IDSs for regular monoterpene precursors biosynthesis................................. 9 1.4.1.2 IDSs for irregular monoterpene precursors biosynthesis............................. 10 1.4.1.3 IDSs for sesqueiterpene precursors biosynthesis........................................ 11 1.4.2 Terpene synthases (TPSs) ................................................................................... 11 1.4.3 Regulation of terpenoid biosynthesis ................................................................... 13 1.5 Ecological and economic importance of terpenoids................................................. 17 1.6 Dissertation objectives and hypotheses ................................................................... 18 1.6.1 Outline of Chapter 2 .............................................................................................. 19 1.6.2 Outline of Chapter 3 .............................................................................................. 20 1.6.3 Outline of Chapter 4 .............................................................................................. 21 x Chapter 2: Identification, validation and cross-species transferability of novel Lavandula EST-SSRs.................................................................................................................. 23 2.1 Overview ................................................................................................................... 23 2.2 Materials and methods.............................................................................................. 24 2.2.1 EST-SSR motif identification, characterization and primer designing ................. 24 2.2.2 Functional annotation of SSR containing unigenes ............................................. 25 2.2.3 Plant materials and genomic DNA extraction....................................................... 25 2.2.4 EST-SSR analysis and cross-species transferability study ................................. 26 2.2.5 Analysis of molecular marker efficiency ............................................................... 27 2.3 Results ...................................................................................................................... 31 2.3.1 Identification and in silico characterization of EST-SSRs .................................... 31 2.3.2 Functional annotation of unigenes containing SSRs ........................................... 35 2.3.3 Lavandula EST-SSR validation and polymorphism ............................................. 36 2.3.4 Cross-species transferability of EST-SSR markers in Lavandula ....................... 43 2.3.5 EST-SSR-based genetic relationship analysis..................................................... 44 2.4 Discussion ................................................................................................................. 47 2.4.1 Lavandula EST-SSRs identification and characterization ................................... 47 2.4.2 EST-SSR polymorphism ....................................................................................... 49 2.4.3 Cross-species transferability and application in genetic relationship analysis .... 51 Chapter 3: Cloning and functional characterization of short-chain trans-isoprenyl diphosphate synthases involved in the biosynthesis of precursors for monoterpenes in lavender (Lavandula) ................................................................ 54 3.1 Overview ................................................................................................................... 54 3.2 Materials and Methods.............................................................................................. 55 3.2.1 Plant growth conditions and chemicals ................................................................ 55 3.2.2 Analysis of trans-IDS sequences.......................................................................... 55 xi 3.2.3 Subcellular localization studies in N. benthamiana protoplasts ........................... 56 3.2.4 Cloning, expression and recombinant protein purification ................................... 58 3.2.5 Enzymatic assay and product identification ......................................................... 59 3.2.6 Bacterial genetic complementation assay ............................................................ 60 3.2.7 Generation of binary vectors and Agrobacterium preparation ............................. 61 3.2.8 N. benthamiana leaf transformation and regeneration......................................... 61 3.2.9 RNA isolation and qPCR analysis ........................................................................ 63 3.2.10 Analysis of pigments, GPPS activity and phenotypes in transgenic plants ..... 66 3.3 Results ...................................................................................................................... 67 3.3.1 Identification of cDNAs encoding short-chain trans-IDSs .................................... 67 3.3.2 Phylogenetic relationship of short-chain trans-IDSs ............................................ 70 3.3.3 Subcellular localization studies............................................................................. 72 3.3.4 The trans-IDSs expression and terpenoid composition in L. x intermedia .......... 74 3.3.5 Heterologous expression of LiGPPS and LiFPPS cDNAs in E. coli .................... 76 3.3.6 In vivo genetic complementation assay................................................................ 79 3.3.7 Expression of LiGPPS.SSU1 in N. benthamina ................................................... 80 3.3.8 Impacts of LiGPPS.SSU1 on GGPP-derived terpenoids and plant phenotypes . 82 3.3.9 Expression of LiGPPS.SSU2 in N. benthamiana ................................................. 85 3.4 Discussion ................................................................................................................. 86 3.4.1 Analysis of Lavandula short-chain trans-IDSs sequences................................... 86 3.4.2 GPP is synthesized by heteromeric LiGPPS ....................................................... 88 3.4.3 Transcripts of LiGPPS.SSU1 and LiGPPS.LSU correspond to the accumulation of monoterpenes .......................................................................................................... 89 3.4.4 LiGPPS.SSU1 can recruit GGPPS to enhance GPP formation........................... 89 3.4.5 LiFPPS catalyzed substrates to produce FPP ..................................................... 92 xii Chapter 4: Cloning and functional characterization of two monoterpene synthases from Lavandula x intermedia ........................................................................................... 93 4.1 Overview ................................................................................................................... 93 4.2 Materials and Methods.............................................................................................. 94 4.2.1 Plant growth condition and chemicals .................................................................. 94 4.2.2 Identification and cloning of monoterpene synthases .......................................... 94 4.2.3 Subcellular localization of LiS-LINS...................................................................... 96 4.2.4 Phylogenetic analysis ........................................................................................... 97 4.2.5 Recombinant protein expression and purification ................................................ 97 4.2.6 Enzyme assays and product identification ........................................................... 98 4.2.7 Induction of Li3CARS and 3-carene by methyl jasmonate .................................. 99 4.2.8 Relative expression............................................................................................... 99 4.2.9 EO extraction and GC-MS analysis.................................................................... 101 4.3 Results .................................................................................................................... 101 4.3.1 Characterization of 3-carene synthase from L. x intermedia ............................ 101 4.3.1.1 Sequence analysis ...................................................................................... 101 4.3.1.2 Genomic DNA organization and phylogenetic analysis ............................. 103 4.3.1.3 Functional characterization of Li3CARS..................................................... 107 4.3.1.4 Relative expression and level of the corresponding monoterpene ............ 111 4.3.2 Characterization of S-linalool synthase from L. x intermedia............................. 114 4.3.2.1 Analysis of sequences and phylogenetic relationship................................ 114 4.3.2.2 Subcellular localization studies................................................................... 118 4.3.2.3 Expression and assay of the recombinant LiS-LINS.................................. 119 4.3.2.4 Relative expression of the two linalool synthase genes............................. 122 4.4 Discussion ............................................................................................................... 124 4.4.1 L. x intermedia 3-Carene synthase..................................................................... 124 xiii 4.4.1.1 Li3CARS sequence has typical mTPS conserved motifs .......................... 124 4.4.1.2 The evolutionary inference from Lavandula TPS genes ............................ 124 4.4.1.3 Li3CARS has high affinity to GPP ............................................................. 125 4.4.1.4 Li3CARS is differentially expressed ........................................................... 127 4.4.2 L. x intermedia S-linalool synthase .................................................................... 129 4.4.2.1 LiS-LINS belongs to TPS-g subfamily ........................................................ 129 4.4.2.2 Recombinant LiS-LINS catalyzes S-linalool formation from GPP & NPP . 130 4.4.2.3 LiS-LINS is differentially regulated ............................................................. 130 4.4.2.4 The implication of S-linalool biosynthesis in Lavandula oil quality ............ 131 Chapter 5: General conclusion ............................................................................................... 132 5.1 Development of EST-SSR markers........................................................................ 133 5.2 Biosynthesis of precursor (GPP) for regular monoterpenes .................................. 134 5.3 Biosynthesis of 3-carene and S-linalool ................................................................. 136 5.4 Closing summary .................................................................................................... 138 Bibliography ...............................................................................................................................140 Appendices .................................................................................................................................169 Appendix A: Reagents and buffers used in EST-SSR validation ...................................... 169 Appendix B: List of Lavandula EST-SSR motifs classified under Category I (≥18 bp in length). ............................................................................................................ 172 Appendix C: Estimated effective multiplex ratio, mean heterozygosity and marker index for validated makers............................................................................................. 182 Appendix D: List of GenBank accessions for protein sequences used in phylogenetics .. 184 Appendix E: Maps of plasmids used in this thesis. ............................................................ 188 Appendix F: Bacteria strains used in this thesis................................................................. 193 Appendix G: Media recipes used for N. benthamiana transformation and regeneration .. 194 Appendix H: Multiple sequence alignment of short-chain trans-IDSs................................ 196 xiv Appendix I: Responses of N. benthamiana when over-expressing LiGPPS.SSU1 or LiGPPS.SSU2................................................................................................. 201 Appendix J: Kinetic analysis of Li3CARS using NPP with respect to different products... 203 Appendix K: Monoterpene profile of young leaf tissues of L. x intermedia (cv. Grosso)... 204 xv List of Tables Table 2.1 List of Lavandula species and cultivars used in this study. ......................................... 26 Table 2.2. The occurrence of different SSR motif types in unigenes of Lavandula EST databases derived from flower, leaf and glandular trichome cDNA libraries . ............. 33 Table 2.3 The occurrence and localization of category I SSRs in unigenes of Lavandula EST databases derived from flower, leaf and glandular trichome tissue cDNA libraries.... 35 Table 2.4 List of selected Lavandula EST-SSRs along with their primer pairs, motif type, location and putative function....................................................................................... 39 Table 2.5 Validation of 31 EST-SSR markers in L. angustifolia and L. x intermedia. ................. 42 Table 2.6 Cross-species transferability of 18 EST-SSR markers in six Lavandula species. ...... 44 Table 3.1 List of primers used in this study. ................................................................................. 64 Table 3.2 The Lavandula short-chain trans-isoprenyl diphosphate synthases (IDSs) with their conserved amino acid motifs........................................................................................ 69 Table 4.1 List of all primer sets used in this study. .................................................................... 100 Table 4.2 Distribution of products from in vitro enzymatic assay of recombinant Li3CARS using GPP and NPP as substrates. ..................................................................................... 109 Table 4.3 Comparison of the kinetic properties of recombinant Li3CARS using GPP and NPP as substrates. .................................................................................................................. 111 Table D.1 Short-chain trans-IDSs............................................................................................... 184 Table D.2 Selected plant TPSs and Li3CARS. .......................................................................... 185 Table D.3 S-linalool synthases from selected plants, LiS-LINS and TPSs from Lavandula. .... 186 Table I.1 GGPP-derived plant pigments from N. benthamiana expressing LiGPPS.SSU2. ..... 202 Table I.2 LiGPPS.SSU2 transgenic plant phenotypes. .............................................................. 202 xvi List of Figures Figure 1.1 Terpenoid biosynthesis pathways in plants. ............................................................... 15 Figure 1.2 Biosynthesis of Lavandula terpenoids. ....................................................................... 16 Figure 2.1 Overview of SSRs analysis from EST libraries of lavender species and validation of identified SSR on the source as well as related Lavandula species. ....................... 30 Figure 2.2 A flowchart showing a stepwise in silico analysis of ESTs to identify SSRs.............. 32 Figure 2.3 The overall frequency of SSR motifs in unigenes of Lavandula EST databases derived from flower, leaf and floral glandular trichome cDNA libraries. .................... 34 Figure 2.4 Molecular level functional annotation of Lavandula unigenes containing SSR markers using Blast2GO software. .......................................................................................... 36 Figure 2.5 The phylogenic relationship among Lavandula species and cultivars deduced from similarity index results of the EST-SSRs. .................................................................. 46 Figure 2.6 PCR amplification of SSR markers in selected Lavandula species and cultivars. .... 47 Figure 3.1 An overview of shoot regeneration from leaf discs N. benthamiana. ......................... 63 Figure 3.2 Conserved motifs in L. x intermedia GPPS subunits, GGPPS and FPPS. ................ 69 Figure 3.3 A phylogenetic tree of the short-chain IDSs from Lavandula and other selected plants based on their amino acid sequences. ...................................................................... 71 Figure 3.4 Transiently expressed full-length cDNAs of GPPS subunits – fused separately to GFP – in N. benthamiana protoplasts. ...................................................................... 73 Figure 3.5 Relative expression of short-chain trans-IDSs and the composition of the corresponding total mono- and sesquiterpenes in L. x intermedia. .......................... 75 Figure 3.6 GC-MS analysis depicting the catalytic activity of recombinant Lavandula GPPS enzymes expressed in E. coli. ................................................................................... 77 Figure 3.7 The catalytic activity of recombinant Lavandula FPPS enzyme expressed in E. coli as detected by GC-MS. .................................................................................................. 78 Figure 3.8 Detection of GGPPS activity using in vivo genetic complementation assays............ 80 xvii Figure 3.9 Impact of LiGPPS.SSU1 expression on GPPS activity and phenotypes of N. benthamiana plants.................................................................................................... 82 Figure 3.10 Effects of LiGPPS.SSU1 expression on GGPP-derived terpenoids and plant growth in N. benthamiana transgenic lines. .......................................................................... 84 Figure 3.11 Dwarf phenotype complementation assay using exogenous GA3 in transgenic N. benthamiana expressing LiGPPS.SSU1. .................................................................. 85 Figure 3.12 Transgenic N. benthamiana plants expressing LiGPPS.SSU2. ............................... 86 Figure 4.1 Sequence alignment of Li3CARS (KX024762) with SoBPPS (AAC26017.1), LaLIMS (ABB73044.1), Ss3CARS (AF527416.1) and Pa3CARS (AAO73863.1). .............. 103 Figure 4.2 Schematic presentation of Li3CARS genomic DNA organization and intron length comparison............................................................................................................... 104 Figure 4.3 Phylogenetic analysis of Lavandula and other selected plant monoterpenes and sesquiterpene synthases. ........................................................................................ 106 Figure 4.4 GC-MS analysis of monoterpenes generated by recombinant Li3CARS from NPP and GPP substrates. ................................................................................................ 108 Figure 4.5 Kinetic assay of affinity-purified recombinant Li3CARS with GPP and NPP. .......... 110 Figure 4.6 qPCR analysis for temporal expression of Li3CARS from L. x intermedia foliage. . 112 Figure 4.7 Accumulation of monoterpenes in young L. x intermedia plant foliage in response to MeJA. ....................................................................................................................... 113 Figure 4.8 Multiple sequence alignment of LiS-LINS (MG870571) with its closest plant homologs AmNES/LINS1 (EF433761) and AmNES/LINS2 (EF433762). ............................... 115 Figure 4.9 Phylogenetic analysis of LiS-LINS and selected plant TPSs based on deduced amino acid sequences. ....................................................................................................... 117 Figure 4.10 Transiently expressed LiS-LINS-TP(50 aa)::GFP fusion in N. benthamiana protoplasts analyzed by fluorescent microscopy. ...................................................................... 119 xviii Figure 4.11 GC-MS analysis of reaction product generated by the recombinant L. x intermedia S-linalool synthase from GPP, and monoterpene standards. ................................. 121 Figure 4.12 Linalool enantiomer biosynthesis by recombinant linalool synthases from Lavandula, and chiral analysis using GC-FID. ........................................................ 122 Figure 4.13 qPCR analysis for the spatial expression of L. x intermedia linalool synthases (LiS-LINS and LiR-LINS). ................................................................................................ 123 Figure E.1 A map of p326::sGFP vector. ................................................................................... 188 Figure E.2 A map of pET32a(+) vector (Novagen). ................................................................... 189 Figure E.3 A map of pET41b(+) vector (Novagen). ................................................................... 190 Figure E.4 A schematic representation of pGA (pGA482) vector carrying GUS. ...................... 191 Figure E.5 A map of pGEM-T vector (Promega). ...................................................................... 192 Figure H.1 Selected GPPS.SSU1 from angiosperms. ............................................................... 196 Figure H.2 Sequence alignment of LiGPPS.SSU2 with other plant GPPS.SSU2 proteins. ..... 197 Figure H.3 Sequence alignment of selected plant GPPS.LSU and GGPPS proteins............... 199 Figure H.4 Multiple sequence alignment of selected plant FPPSs. ........................................... 200 Figure I.1 Boxplots depicting the phenotypic responses of LiGPPS.SSU1 expression in N. benthamiana. .............................................................................................................. 201 xix List of Abbreviations CDS Coding region DMAPP Dimethylallyl pyrophosphate D Discriminating power EO Essential oil EST-SSRs Expressed sequence tag-derived SSRs FPP Farnesyl pyrophosphate FPPS FPP synthase GC-MS Gas chromatography mass spectrometry GC-FID GC flame ionized detector GFP Green fluorescent protein GPP Geranyl pyrophosphate GPPS GPP synthase GPPS.LSU GPPS large subunit GPPS.SSU GPPS small subunit GGPPS Geranylgeranyl pyrophosphate synthase He Expected heterozygosity IDS Isoprenyl diphosphate synthase IPP Isopentenyl pyrophosphate Li3CARS L. x intermedia 3-carene synthase LiS-LINS L. x intermedia S-linalool synthase LiR-LINS L. x intermedia R-linalool synthase LPP Lavandulyl pyrophosphate LPPS LPP synthase MeJA Methyl jasmonate xx mTPS mono-TPS Ni-NTA Nickel–nitrilotriacetic acid OD Optical density ORF Open reading frame PCR Polymerase chain reaction PFR Primer flanked region PIC Polymorphic information content R Resolving power SSRs Simple sequence repeats TPS Terpene synthase UTR Untranslated region xxi Acknowledgments First and foremost, I would like to thank the faculty, staff and graduate students of UBC Okanagan who have been by my side throughout my studies. My special appreciation goes to my supervisor Dr. Soheil Mahmoud for giving me the opportunity to work in his lab under his supervision. His continuous generosity, guidance and unreserved support throughout my study were my special motivators to become successful. I am also highly grateful to my past and present committee members, Drs. Michael Deyholos, Louise Nelson, Nasser Yalpani, Mary Forrest and Michael Russello for their generosity and continuous technical advice and research input throughout my study. I would like to extend my sincere appreciation to the past and current members of Dr. Soheil Mahmoud's lab (Dr. Zerihun Demissie, Mr. Lukman Sarker, Dr. Lauren Erland, Ms. Rebecca Wells, Mrs. Elaheh Najafian, Ms. Ashley Lemke, Ms. Katie Del Buono, Ms. Felisha Truong, Mr. Christopher Bitcon, Mr. Dakshita Ranatunga, Mr. Mike Tarnowycz, Ms. Michelle Hawkins, Ms. Sarah Mahmoud, Mr. Gabriel Zavala and Ms. Lina Bauer) for their unreserved support throughout my study and kind friendship. This thesis would not have been possible without their generous assistance. In particular, I am grateful to Dr. Zerihun A. Demissie and Mr. Lukman Sarker for their openness and being there whenever I needed them and making my new working environment smooth. Additionally, I would like to express my sincere appreciation to Dr. Aaron Johnstone (Dr. Philip Barker's lab) for technical support with an inverted fluorescence microscope and Mrs. Rosemary Garner (UBCO Biology lab technician) for her generous support with teaching equipment whenever I needed it for my research. I also thank my best fellow friends who supported me directly or indirectly during my study, including Mr. Mansak Tantikachoniket (Ben), Dr. Dinesh Adikary, Mr. Antreas Pogiatzis, Dr. Vasilis Kokkoris, Mr. Bruno Carturan, Mrs. Isadora Quintans, Dr. Eric Vukicevich, Mr. Andre Alcantara, Dr. Krin Mann and Mr. Michael Morgon. I thank Mrs. Barb Lucente (Biology Unit 2 Assistant) for her tireless assistance with regard to administrative issues throughout my academic life at UBCO. xxii Lastly, I am highly grateful to Dr. Günther Scherer (Emeritus Professor and my M.Sc. thesis supervisor at The Leibeniz University of Hannover, Hanover, Germany) for his generosity and unreserved support in my higher education career. I would like to thank the UBC Okanagan for the financial assistance through subsequent awards of University Graduate Fellowship during my study. My thesis work, in particular, was supported through grants or in-kind contributions by the Investment Agriculture Foundation of British Columbia, Genome British Columbia, The University of British Columbia, the Natural Sciences and Engineering Research Council of Canada and the Canadian Foundation for Innovation to Dr. Soheil Mahmoud. I would also like to thank my family who are the source of my encouragement and the architect of who I am. In particular, my sincere appreciation goes to my younger brother, Engineer Addisu Mengesha Adal, for being with family when nobody is around. The last but certainly not least, special thanks to my beautiful wife, Tirhas M. Gebretsadikan, for being by my side and sharing all ups and downs during my academic and personal life. xxiii Dedication …My Wife and Family... 1 Chapter 1: Introduction 1.1 The genus Lavandula The genus Lavandula belongs to the mint (Lamiaceae) family along with mint (Mentha spp.), sage (Salvia spp.), and Oregano (Origanum spp.), among others. Lavandula species are widely distributed across the world, with a range from India in the east, across northern Africa, the Mediterranean, south-west Asia, Arabia and western Iran to Macaronesia in the west (Upson and Andrew 2004). The genus encompasses over 39 species along with numerous natural hybrids and registered cultivars, and these species are characterized by complex polyploidy with chromosome numbers ranging from 18 to 75 (Upson and Andrew 2004). The genus is further classified into three subgenera (Lavandula, Fabricia and Sabaudia) and eight sections based on their growth habit, leaf shape, inflorescence structure and other traits. For example, the subgenus Lavandula is comprised of three sections; Lavandula, Dentatae and Stoechas that are characterized by their multi-flowered cymes and are woody shrubs with narrow leaves. On the other hand, the subgenus Fabricia – comprising the sections Pterostoechas, Subnudae, Chaetostachy and Hasikenses – and the subgenus Sabaudia (with Sabaudia as the only section) have single-flowered cymes with the former section lacking bracteoles (Upson and Andrew, 2004; Paton et al. 2004). Intra- and intersection hybrids and hundreds of cultivars, containing unique morphologies and oil profiles, are also amongst the major members of the subgenus Lavandula. Humans have used lavenders since the ancient times. For example, the Greeks and Romans used parts of these plants or their EOs for personal hygiene, treatment of various ailments and cooking (Upson and Andrew 2004). Currently, several species are grown across the globe for the production of EOs and other horticultural applications. In particular, lavender EOs are of substantial economical value as they are extensively used in cosmetic, personal care, culinary, and alternative medicine products, among others (Cavanagh and Wilkinson 2 2002; Moon et al. 2006; Denner 2009; Aprotosoaie et al. 2014; Erland and Mahmoud 2015; Wells et al 2018). Further, the by-product of lavender oil distillation (distilled straw) is used for soil amendment (as organic fertilizer) and biogas production (Lesage-Meessen et al. 2015). The type of species and/or cultivars used in cultivation largely determine the market values and composition (bioactivity) of produced essential oils (EOs) (Cavanagh and Wilkinson 2002). For example, widely known lavender species – Lavandula angustifolia, Lavandula latifolia and their natural hybrids called Lavandula x intermedia – have been used for commercial EO production. The quality of the oil can vary among the species due to the relative content of specific EO constituents, particularly linalool/linalyl acetate and camphor ratios. For example, L. angustifolia produces higher quality and relatively lower amount of EOs compared to L. latifolia and L. x intermedia (Lis-Balchin, 2002). The oil profiles and morphologies of lavenders have been used as a means of species/cultivars identification (Upson and Andrew 2004; Do et al. 2015; Lafhal et al. 2016; Beale et al. 2017). However, these approaches are influenced by environmental cues and lack the power to discriminate cultivars/hybrids derived from genetically related species. Thus, there is a need for developing appropriate markers that could differentiate species and their cultivars irrespective of their geographic origin and environmental conditions. 1.2 Molecular Markers Genetic variations – variation in alleles of genes – are retained through generations and can be detected with the aid of molecular markers, reflecting diversities within and among species. A molecular marker refers to a site of heterozygosity for genetic variations, particularly silent DNA variations that are not associated with measurable phenotypic variations. Abundance and stability of the markers contribute to their polymorphism ability in various applications, including genetic diversity, fingerprinting, genetic mapping, and marker-assisted selection (Duran et al. 2009; Mondini et al. 2009; Pourmohammad 2013). Molecular markers have 3 evolved from non-PCR-based (e.g., Restriction fragment length polymorphism (RFLP) and allozymes) to the current PCR-based markers (Schloterrer 2004; Lübberstedt and Varshney 2013). PCR-based arbitrarily amplified dominant markers (e.g., rapid amplified polymorphic DNA (RAPD)) have been widely used in plant species with no sequence information. However, its applications have been limited due to the rapid development of genomic resources that allow developing high-throughput molecular markers, including simple sequence repeats (SSRs) (or microsatellites) and single nucleotide polymorphisms (SNPs) (Schloterrer 2004; Lübberstedt and Varshney 2013). The availability of genomic resources also facilitates the quick discovery and analysis of molecular markers, depending on the type of markers. The choice of molecular markers varies depending on the objectives, the level of polymorphism to be detected, their genomic coverage, and the tools for assays and detections (Schloterrer 2004; Lübberstedt and Varshney 2013). Although different types of DNA markers have been developed and employed to characterize diverse crops, SSRs are the preferred markers next to SNPs in current plant genetics and breeding studies (Li et al. 2004; Duran et al. 2009). SSRs, composed of 1– 6 bp tandemly repeated motifs, are useful for species identification, paternity analysis, intellectual property right protection, construction of genetic mapping, marker-assisted selection, and for establishing genetic and evolutionary relationships, among others (Li et al. 2004; Duran et al. 2009). Depending on the source databases, SSRs can be Expressed Sequence Tag-derived SSRs (EST-SSRs) and/or genomic-SSRs. EST-SSRs are repeats located in expressed regions of a genome and are mainly derived from ESTs/ transcriptome databases. Although repeat numbers and total lengths of EST-SSRs are relatively small compared to genomic-SSRs (Li et al. 2004), they are: (1) uniquely informative due to alterations in structural and regulatory genes of an organism (Li et al. 2004; Chabane et al. 2005), (2) easily transferable among closely related species, and (3) identified from EST databases that are cost-efficient to develop and also not limited to model organisms. Genomic-4 SSRs, on the other hand, are the most abundant in nature, often located in non-coding regions of the genome. They are generally mined from SSR-enriched and non-enriched (random) genomic libraries, or from genome-wide sequencing. SSR-enriched genomic DNA libraries are constructed either through selective hybridization of genomic portions containing SSRs or by selective amplification of microsatellites containing genomic DNA fragments using SSR specific primers. Non-enriched libraries are derived from randomly cloned partial or whole genomic DNA fragments (Senan et al. 2014). In the genus Lavandula, very few genetic studies have been reported using molecular markers, such as RAPD and allozyme in Lavandula multifida (a single-flowered cyme species) (Hnia and Mohamed 2010; Hnia et al. 2012), nuclear internal transcribed spacer (ITS) in major multi-flowered cymes species (Upson and Andrew 2004), plastid DNA barcoding (trnH-matK) in different lavender species (Moja et al. 2015), and plastid DNA barcoding and ITS in cultivated lavender species (Hind et al. 2018). However, there are high-throughput molecular markers that emerge occasionally and are extensively being applied in diverse plant species, such as genic- and genomic SSRs (Duran et al. 2009; Senan et al. 2014) and SNPs (Hayward et al. 2012). SSRs have been successfully developed and applied in genetic diversity, fingerprinting and evolutionary relationship analyses of several species in Lamiaceae, including Salvia spp (Radosavljević et al. 2011; Radosavljević et al. 2012; Karaca et al. 2013), Rosmarinus spp (Segarra-Moragues and Gleiser 2008) and Origanum vulgare (Novak et al. 2008). Particularly, Karaca et al. (2013) have reported the cross-genera amplification of Salvia officinalis EST-SSRs to L. x intermedia, but only 7 of the 75 loci were positively transferred. These studies highlighted the presence of ample potentials to develop species-specific SSRs and use these markers across related species. By contrast, since SSRs are widely developed for diploids, it may not be fully exploited in Lavandula due to the complex polyploidy characteristics of lavender species. However, there has been a proposed strategy to use SSRs in species with 5 different ploidy levels using as a dominant marker (multi-locus marker) (López-Vinyallonga et al. 2015). 1.3 Lavandula essential oil constituents Essential oils, in general, are a mixture of volatile organic compounds produced from plant tissues. Lavandula EOs comprise mainly of monoterpenes and a lesser amount of sesquiterpenes. Monoterpenes, particularly regular types, such as linalool, linalyl acetate, 1,8-cineole, borneol, and camphor, are the major components in Lavandula EOs, whereas irregular monoterpenes (e.g., lavandulol and lavandulyl acetate) and sesquiterpenes (e.g., caryophyllene, alpha-bergamotene and others) exist in trace amount in the oil (Lis-Balchin 2002; Upson and Andrews 2004; Aprotosoaie et al. 2017). Among the major monoterpenes found in Lavandula EOs, linalool and linalyl acetate are the key desired monoterpenes, while camphor and to a lesser extend borneol and 1,8-cineole contribute undesired odor. In this context, EOs with a high linalool/linalyl acetate to camphor ratio, (e.g., EO from L. angustifolia) are considered to be of high quality (Aprotosoaie et al. 2014; Aprotosoaie et al. 2017). In particular, linalool exists in two stereochemistry isomers (chiral), R and S types, in which R-linalool has a woody lavender-like scent and is the dominant monoterpene in Lavandula oils. In contrast, S-linalool (sweet and pleasant odor) occurs in a lesser amount in these plants (Renaud et al. 2001; Aprotosoaie et al. 2014). The EO quality can be associated with the proportion of EO constituents, depending on plant species and environmental cues, including temperature and light intensity (Angioni et al. 2006; Woronuk et al. 2011; Dudareva et al. 2013; Carvalho et al. 2016). Lavender species that produce marketable EO vary by their oil yield and quality, relying primarily on the relative content of linalool, linalyl acetate, camphor, and 1,8-cineole (Lis-Balchin 2002; Upson and Andrews 2004; Aprotosoaie et al. 2014; Aprotosoaie et al. 2017), which also differ across cultivars within a species (Renaud et al. 2001; Boeckelmann 2008; Ontario Lavender 6 Association 2012). For example, a cultivar trial in Ontario, Canada (Ontario Lavender Association 2012) revealed that all cultivars of L. x intermedia contain high camphor contents with the top level in cultivar Hidcote Giant. However, some cultivars, like Super, produce relatively similar high-quality oil compared to most L. angustifolia cultivars. Despite the presence of some undesirable terpenoids, its high EO yield makes L. x intermedia an important commercial species for essential oil production. The proportion of EO constituents influences the ultimate uses of lavender EOs relying on the types of plant species and their cultivars (Renaud et al. 2001; Lis-Balchin 2002; Upson and Andrews 2004; Ontario Lavender Association 2012). For instance, L. angustifolia produces high-quality oil and is known as a source of major raw materials for the perfume and aromatherapy industries. Such preference is mainly associated with the highest contents of linalool and linalyl acetate that produce a mild aroma (floral and fruity), allowing generation of sweeter scents from EO. In contrast, camphor-rich L. x intermedia is less suitable for such purposes, instead, it is more frequently used in medicinal, personal care products and so forth. Camphor combined with borneol and 1,8-cineole provide a strong aroma (spicy), in which the variations in these component concentrations strongly determine the pleasantness of an EO. There are also some outlier cultivars containing mild aromas (e.g., Super) and high borneol (e.g., Provence) from L. x intermedia, and strong aromas (e.g., Rosea) and high lavandulol (e.g., Maillette) from L. angustifolia (Ontario Lavender Association 2012). However, EO yield is not always associated with oil quality as some high EO yielding varieties produce low-quality oil and vice versa. Despite such variability, there have been several attempts to improve the lavender EO yield and quality through the development of new improved lines. Urwin et al. (2007) and Urwin (2014) used chromosome doubling through tissue culture systems to produce novel fertile L. x intermedia lines. Erland and Mahmoud (2015) also used Ethyl 7 Methanesulfonate-induced mutagenesis through tissue culture regeneration to develop improved L. x intermedia (cv. Grosso) lines. 1.4 Monoterpene biosynthesis Monoterpenes are the C10 class of terpenoids, the largest family of plant specialized metabolites. When produced at high levels (e.g., in mints and lavenders), monoterpenes are secreted and stored in glandular trichomes, modified epidermal hairs covering the surface of tissues. The glandular trichomes can be capitate (small in size with a basal cell, a short stalk, and 1-2 cell heads) or peltate (6-8 secretory cells, a stalk, and a storage cavity; exist in Lavandula), and both types of glandular trichomes are localized on the surface of different plant tissues (e.g., leaves, flower, and stem tissues) (Turner et al. 2000; Iriti et al. 2006; Tissier 2012). The outer surface of the glands is covered by a cuticle, and the oil accumulates in the subcuticular space. Monoterpenes are secreted from the secretory cells, particularly in leucoplasts (i.e., non-photosynthetic plastids of oil glands) (Turner et al. 2000; Lange and Turner 2013). The number of trichomes distributed on the surface of EO-producing tissues can impact the EO yield and thereby its terpenoid contents, as trichome number and size are directly associated with EO yield in peppermint leaves (Turner et al. 2000; Lange and Turner 2013). Monoterpene biosynthesis involves several stages that start with the formation of isoprene units and ends with a modification of terpenoid skeletons into other products. Initially, C5 isoprene units, isopentenyl diphosphate (IPP) and its allylic isomer, dimethylallyl diphosphate (DMAPP) are synthesized through two pathways: the plastidial 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, and the cytosolic mevalonate (MVA) pathway (Figure 1.1) (Tholl and Lee 2011; Dudareva et al. 2013). The MEP starts with the condensation of glyceraldehyde-3-phosphate (GAP) and pyruvate to produce several intermediates and then eventually IPP and DMAPP. The MVA pathway, on the other hand, begins with the condensation of acetyl Co-A 8 (three molecules) and then produces other intermediates prior to ending with the formation of isoprene units. The isoprene units produced from each pathway are often responsible for the branching of specific terpenoids. For example, the MEP pathway gives rise to C10 monoterpenes, C20 diterpenes, and other polyterpenes, while the MVA pathway leads to the synthesis of C15 sesquiterpenes, C30 triterpenes, and other polyterpenes. IPP is isomerized to DMAPP by IPP isomerases (IPPI), which can be localized to different subcellular compartments. For example, in Arabidopsis, two isomerases (IPPI1 and IPPI2) are found in the cytosol, but only IPPI1 is targeted to plastids, and IPPI2 is transported to mitochondria (Phillips et al. 2008). Both isomerases are partially functionally redundant and seemed to be essential for the plants, in which double-mutation of these genes can cause nonviable plants (Phillips et al. 2008) or dwarf and male sterile plants (Okada et al. 2008). IPP and DMAPP are condensed into isoprenyl diphosphates (precursors) by chain length-specific isoprenyl diphosphate synthases (IDS) (Figure 1.2) (Tholl and Lee 2011). The coupling of the isoprene units can be head-to-tail or non-head-to-tail, depending on the type of precursor formation or plant species. The head-to-tail condensation of IPP and DMAPP produces isoprenyl diphosphates – the linear precursors to various terpene classes – including geranyl pyrophosphate (GPP), neryl pyrophosphate (NPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) (Tholl and Lee 2011), and geranylfarnesyl pyrophosphate (GFPP) (Nagel et al. 2015; Liu et al. 2016b). The non-head-to-tail condensation of two DMAPP forms unique prenyl diphosphates, such as lavandulyl pyrophosphate (LPP) (Demissie et al. 2013) and chrysanthemyl pyrophosphate (CPP) (Rivera et al. 2001). 1.4.1 Isoprenyl diphosphate synthases Isoprenyl diphosphate synthases (IDSs) catalyze the condensation of IPP with its allylic isomer, DMAPP to produce different chain lengths of isoprenyl diphosphates (Dewick 2002; Dudareva et al. 2013; Jia and Chen 2015). The enzymes can be either trans (E)-IDSs or cis (Z)-9 IDSs, depending on the stereochemistry of the isoprenyl diphosphate produced and the encoded protein sequences. The trans-IDSs are the common IDSs and are believed to synthesize various chain lengths of isoprenyl diphosphate ranging from C10 (e.g., GPP) to C50 (e.g., polyprenyl diphosphate) in plants. By contrast, there are few cis-IDSs involved in the synthesis of short-chain (C10) (e.g., NPP in tomato) and long-chain (>C50) isoprenyl diphosphates reported in plants (Schilmiller et al. 2009; Demissie et al. 2013; Jia and Chen 2015). The trans (E)-IDSs consist of a family of structurally related proteins with conserved motifs found in many organisms (Wang and Ohnuma 2000), while the cis-IDSs are structurally related to each other but they are distinct to the trans-IDSs and appear to use different mechanisms (Kharel et al. 2006). Several cis-IDSs have been identified and characterized in bacteria and yeast (Apfel et al. 1999; Sato et al. 1999). The cis-IDSs are often involved in the formation of the Z-prenyl diphosphates precursor directly from IPP and DMAPP units and/ or E,E-FPP and IPP units, depending on the length of the chains (Asawatreratanakul et al. 2003; Sallaud et al. 2009). The cis-prenyl diphosphates serve as precursors for the synthesis of short and long-chain terpenoids (Asawatreratanakul et al. 2003; Sallaud et al. 2009). 1.4.1.1 IDSs for regular monoterpene precursors biosynthesis Among IDSs, GPP synthase (GPPS) is a multi-subunit enzyme that catalyzes the head-to-tail fusion of IPP and DMAPP in the trans (E)- configuration to form GPP, the precursor to most common monoterpenes. To date, different subunits of homodimeric and heterodimeric GPPS have been identified in plants. Homodimeric GPPS enzymes found in both gymnosperms and angiosperms species have specific and multi-product activities in the presence of excess supply of IPP to DMAPP, GPP and FPP (Schmidt et al. 2010; Hsieh et al. 2011). The heteromeric GPPS enzymes are found only in angiosperms and typically have a large and a small subunit (Burke et al. 1999; Wang and Dixon 2009; Chang et al. 2010). The two subunits 10 interact physically to catalyze the condensation of isoprene units into GPP. The large subunit of heteromeric GPPS contains two aspartate-rich motifs (DD(X)2-4D) that are crucial for prenyl-substrate binding (Kellogg and Poulter 1997). The large subunit shares about 50 % sequence identity to geranylgeranyl diphosphate synthase (GGPPS), the enzyme responsible for the production of GGPP, the linear precursor to diterpenes. However, the (DD(X)2-4D) motifs are absent in small subunits of heteromeric GPPS, which alone are functionally inactive (Burke et al. 1999). The head-to-tail condensation of IPP and DMAPP in the cis (Z) configuration also produces NPP. NPP (a cis-isomer of GPP) serves mostly as a substrate for cyclic monoterpenes. Although there are many mono-TPSs that accept NPP as a substrate in vitro, only a single gene encoding NPP synthase has been isolated from plants (i.e., tomato) and functionally characterized (Schilmiller et al. 2009). Although several short-chain IDSs have been identified in diverse plants and functionally studied, short-chain IDS involved in the formation of regular monoterpene precursors (GPP/NPP) have not been investigated in Lavandula. 1.4.1.2 IDSs for irregular monoterpene precursors biosynthesis A few IDSs catalyze a non-head-to-tail coupling of two DMAPP molecules to produce C10 prenyl diphosphates, including LPP (Figure 1.2) (Demissie et al. 2013) and CPP (Rivera et al. 2001). The only gene encoding LPP synthase (LPPS) was identified in Lavandula and characterized in vitro to have a function of LPP production from two DMAPP units (Demissie et al. 2013; Liu et al. 2016a). However, monoterpenes derived from LPP are minor products in chrysanthemum (Rivera et al. 2001; Yang et al. 2014) and Lavandula. The mechanisms behind the low concentration of irregular monoterpenes and their precursor (LPP) produced in Lavandula have yet to be investigated. 11 1.4.1.3 IDSs for sesqueiterpene precursors biosynthesis The trans-IDSs (e.g., E-FPP synthase) catalyze the coupling of either one DMAPP and two IPP units or one GPP and one IPP unit to form FPP, the common precursor for sesquiterpenes (Figure 1.2). The C15 FPP serves as a precursor for a vast majority of sesquiterpene synthases to produce sesquiterpenes (Bohlmann et al. 1998). Unlike the universal cytosolic-based E-FPP synthase, a cis-IDS (z-FPP synthase) that has transit peptide targeting the plastids was identified and found to be involved in the formation of FPP in tomato (Sallaud et al. 2009). Short-chain IDSs involved in the formation of FPP have not yet been investigated in Lavandula. 1.4.2 Terpene synthases (TPSs) Once the prenyl diphosphates (substrates) are formed, they can be converted into diverse terpenoids through the actions of specific terpene synthases (TPS). TPSs are important enzymes that have high specificity to various precursors as their substrates and generate diverse terpenoids, including major and minor products. For example, mono-TPSs, such as 1,8-cineole and ß-phellandrene synthases identified from Lavandula could use both C10 GPP and NPP substrates in vitro to produce multiple products with the same major compounds (1,8-cineole and ß-phellandrene, respectively) (Demissie et al. 2011; Demissie et al. 2012). Structurally, TPSs contain DDxxD and (N,D)D(L,I,V)x(S,T)x3E) conserved motifs that are essential in the coordination of divalent metal ions for substrate binding. Most TPSs also contain a key motif (RRx8W), involved in isoprenyl diphosphate ionization (Williams et al. 1998), at the N-terminus of the proteins. Plants contain a TPS gene family, which ranges in size from a minimum of single TPS in Physcomitrella patens (moss) to 113 TPSs in Eucalyptus grandis (Martin et al. 2010; Chen et al. 2011; Butler et al. 2018). Based on their phylogenetic relationship, the TPS genes can be classified into eight different subfamilies, designated from TPS-a to h. Most TPSs are also 12 grouped into a specific subfamily based on the structurally distinct terpenoids produced from specific precursors, for example, TPS-a for angiosperm sesquiterpenes, and TPS-b and TPS-g for angiosperm monoterpenes (b- cyclic & g- acyclic). TPS-c and TPS-e are groups of diverse TPSs, such as copalyl diphosphate, ent-kaurene, diterpene, monoterpene, and sesquiterpene synthases, while TPS-f includes ent-kaurene, and diterpene, monoterpene and sesquiterpene synthases (Chen et al. 2011). There are also TPSs that are categorized separately into TPS-d (gymnosperm plants, e.g., Picea abies) and TPS-h (Selaginella spp) subfamilies in line with specific taxonomic sources (Chen et al. 2011). Specific TPSs transform prenyl diphosphates of various chain length into different classes of terpenes. For example, mono-TPSs catalyze GPP/ NPP to monoterpenes, sesqui-TPSs convert FPP to sesquiterpenes and di-TPSs produce diterpenes from GGPP (Figure 1.2). Some mono-TPSs also use LPP as a substrate for synthesis of irregular monoterpenes, for instance, lavandulol in lavenders (Demissie et al. 2013) and in chrysanthemum (Rivera et al. 2001). To date, several mono- and sesqui-TPSs genes involved in the synthesis of terpenes have been identified from Lavandula and functionally characterized in vitro (Lane et al. 2010; Demissie et al. 2011; Demissie et al. 2012; Sarker et al. 2013). The terpenes produced by TPSs can further be diversified into different metabolites (i.e., terpenoids) through the action of enzymes, such as acetyltransferases, cytochrome P450 hydroxylases and so forth (Lange 2015; Sarker and Mahmoud 2015). Terpenoids can also be modified into distinct non-volatile terpenoid derivatives through glucosylation (terpenoid glycosides) and prenylation. Prenylated phenolic and flavonoid compounds can be formed by the coupling of isoprene units on aromatic compounds (Yazaki et al. 2017). For example, linalool is transformed into glycosylated linalool derivatives with low volatility in Arabidopsis (Boachon et al. 2015). Overexpression of a gene encoding a strawberry nerolidol/S-linalool 13 synthase in Chrysanthemum morifolium also resulted in higher volatile S-linalool and non-volatile S-linalool glycoside derivatives (Yang et al. 2013; Yazaki et al. 2017). 1.4.3 Regulation of terpenoid biosynthesis Terpenoids have specialized functions in plants, in which their biosynthesis can be triggered by different factors such as plant developmental stages, biotic factors, phytohormones and elicitors (Cheng et al. 2007; Boeckelmann 2008; Dudareva et al. 2013). Although most TPSs are spatio-temporally regulated by primarily developmental growth stages, there are certain TPSs that are triggered by inducers. Direct herbivory attacks and pathogen infections activate the transcriptional expression of some mono- and sesqui-TPSs genes in Arabidopsis, Sitka spruce, Norway spruce and Lima bean (Fäldt et al. 2003; Miller et al. 2005; Navia-Giné et al. 2009; Huang et al. 2010). The activation of these genes in response to the attacks is potentially triggered by endogenous plant phytohormones and elicitors from the defense signal transduction cascades (Farmer et al. 2003; Halitschke and Baldwin 2004). Induced-TPS expression and terpenoids accumulation have been widely studied using simulating inducers, such as methyl jasmonate, jasmonic acid, wounding and salicylic acid in various angiosperms (e.g., tomato) (Van Schie et al. 2007b; Heiling et al. 2010; Menzel et al. 2014; Taniguchi et al. 2014) and gymnosperms (e.g., Sitka spruce) (Miller et al. 2005; Boone et al. 2011; Ott et al. 2011; Byun-McKay et al. 2012; Sarrou et al. 2015). The amount of supplied precursors (substrates) and the levels of TPS expression can regulate the abundance of specific terpenoids, as observed in snapdragon and Nicotiana tabaccum (Dudareva et al. 2000; Orlova et al. 2009). The levels of supplied isoprenyl diphosphates (precursors) are controlled by the expression of IDSs, and this can eventually control the amount of terpenoid production. A recent study showed that a glandular trichome-specific transcription factor (TF) (R2R3-MYB) suppressed IDS (e.g., GPPS large subunit) and then negatively regulated the monoterpene production in spearmint (Reddy et al. 2017). 14 Similarly, TPS expressions can be regulated by TFs specifically localized in tissues where a large amount of terpenoids accumulate, and these expressions control the levels of specific terpenoids in those particular plant tissues. For example, AP2/ERF and NAC TFs isolated from fruit tissues activated expression of TPS and subsequently increased the level of monoterpenes produced from sweet orange fruit (e.g., E-geraniol) and kiwifruit (e.g., 1,8-cineole), respectively (Nieuwenhuizen et al. 2015; Li et al. 2017). In Lavandula, the biosynthesis of terpenoids is regulated spatio-temporally. Flowers and leaves are the key sources of Lavandula monoterpenes, and flowers are the major sources of monoterpenes with a lesser amount of sesquiterpenes depending on plant developmental stages (Boeckelmann 2008). There are also monoterpenes that are abundantly produced in leaf tissues, for example, 1,8-cineole and camphor in L. x intermedia. Corresponding to the respective terpenoid levels in these tissues, the transcripts of TPSs identified in this genus have been regulated spatially. For instance, higher amounts of transcripts for R-linalool synthase and 1,-8 cineole synthases were found in flowers and leaves, respectively (Demissie et al. 2012). To date, little is known about regulation of terpenoid biosynthesis by inducers (e.g., pathogens, elicitors or hormones), the amount of supplied precursors, and regulatory elements (i.e., TFs) in this genus. Recently, candidate TFs that can potentially be involved in the regulation of specific monoterpene biosynthesis were identified via RNA-Seq from EO-producing tissues of Lavandula (Lukman Sarker and Soheil Mahmoud, personal communication). However, the roles of these identified TFs that may activate/ suppress particular TPS expressions and the corresponding terpenoid levels are yet to be investigated in Lavandula. 15 Figure 1.1 Terpenoid biosynthesis pathways in plants. Biosynthesis of IPP and DMAPP through the plastidial-localized MEP pathway (right) and the MVA pathway (left) in the cytosol. In the MEP pathway, seven enzymes are involved in forming IPP/DMAPP, starting from the synthesis of DXP from pyruvate and glyceraldehyde-3-P by DXS and ending with reduction of HMBPP to IPP/DMAPP by HDR in plastids. IPP/DMAPP pools are also synthesized via the cytosolic MVA pathway in six enzymatic steps. Initially, acetoacetyl-CoA (AAC) is formed from Acetyl CoA by AAC thiolase, and is subsequently converted to mevalonate-5-diphosphate (MVAPP), which is the last step to give IPP by MVA decarboxylase (MVD). IPP is then isomerized to DMAPP by IPPI. Overall, MEP and MVA are independent pathways, but interactive, exchanging IPP/DMAPP pool between the two compartments although the trafficking mechanisms are yet unknown. The intermediate products and enzymes in the MEP pathway: DXP - 1-deoxyxylulose-5-phosphate; DXS - DXP synthase; DXR - DXP reductoisomerase; MEP - 2-C-methylerythritol-4-phosphate; CDP-ME - 4-diphosphocytidyl-2-C-methylerythritol; MCT - CDP-ME synthase; CDP-ME2P - 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; CMK - 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MEcPP - 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; MDS - MEcPP synthase; HMBPP - 4-hydroxy-3-methylbut-2-enyl diphosphate; HDS - HMBPP synthase; HDR - HMBPP reductase; IPP - Isopentenyl diphosphate; IPPI - IPP isomerase; DMAPP - Dimethylallyl diphosphate. The MVA pathway products and enzymes are: HMG - 3-hydroxy-3-methylglutaryl-CoA; HMGS - HMG synthase; HMG - 3-hydroxy-3-methylglutaryl-CoA; HMGR- HMG reductase; MVA - mevalonate; MK - MVA kinase; MVAP - phosphomevalonate; PMK - MVAP kinase. (From Radesh PN Malli, Ayelign M. Adal, Lukman S. Sarker, Ping Liang, Soheil S. Mahmoud (2019) De novo sequencing of the Lavandula angustifolia genome reveals highly duplicated and optimized features for essential oil production, Planta, 249(1):251–256, © Authors 2018, the Creative Commons Attribution 4.0 International License (http://creativecommons.org/l icenses/by/4.0/)). 16 Figure 1.2 Biosynthesis of Lavandula terpenoids. IPP and its allylic isomer, DAMPP, are the common precursor of all terpenes. In plastids, DMAPP fuses either with one DMAPP unit to give LPP or with one IPP unit to form GPP. While LPP in the cis-configuration has been believed to be the natural precursor of irregular monoterpenes, the trans-prenyl diphosphate (GPP) serves as a primary substrate for different regular monoterpenes. Regular monoterpene synthases catalyze GPP not only to several regular monoterpenes but also to another prenyl diphosphate (e.g., BPP), which is then converted to other monoterpenes. Further, the condensation of DMAPP and three IPP units forms GGPP, which is subsequently converted to diterpenes by diterpene synthases in plastids. In the cytosol, one unit of DMAPP and two IPP units are condensed in a head-to-tail configuration to give FPP that serves as a substrate for sesquiterpenes. Sesquiterpene synthases convert FPP to specific sesquiterpenes. However, there are still unknown terpene synthases that are indicated by dashed arrows and "?". The prenyl diphosphates and enzymes include: LPP - lavandulyl diphosphate; LPPS - LPP synthase; GPP - geranyl diphosphate; GPPS - GPP synthase; GGPP - geranylgeranyl diphosphate; GGPPS - GGPP synthase; FPP - farnesyl diphosphate; FPPS - FPP synthase; BPP - bornyl diphosphate; AAT - monoterpene acetyltransferase; PhilS - β-phellandrene synthase; rLinS - (R)-linalool synthase; LimS - limonene synthase; CinS - 1,8-cineole synthase and BDH - borneol dehydrogenase; BerS - α-bergamotene synthase; GerS - germacrene D synthase; CadS - cadinol synthase; -CaryS - -caryophyllene synthase and 9-CaryS - 9-epi-caryophyllene synthase. (Adapted from Radesh PN Malli, Ayelign M. Adal, Lukman S. Sarker, Ping Liang and Soheil S. Mahmoud (2019) De novo sequencing of the Lavandula angustifolia genome reveals highly duplicated and optimized features for essential oil production, Planta, 249(1):251–256, © Authors 2018, the Creative Commons Attribution 4.0 International License (http://creativecommons.org/l icenses/by/4.0/)). 17 1.5 Ecological and economic importance of terpenoids Plants produce a large assortment of terpenoids that perform ecological and physiological roles. Terpenoids grouped under primary metabolites provide several cellular and physiological functions, for example as growth regulators (e.g., gibberellins, abscisic acid and brassinolides) and components of photosynthetic pigments (e.g., phytol – chlorophylls and carotenoids), sterols and others (Tholl and Lee 2011; Tetali 2018). Several terpenoids also play ecological roles in response to biotic and abiotic factors and are grouped as secondary metabolites. They facilitate interactions of plants with their environment, for example, attracting pollinators, providing defenses and priming the neighbor plants (Cheng et al. 2007; Frost et al. 2007; Menzel et al. 2014; De Vega et al. 2014; Yazaki et al. 2017). In many plants, terpenoids have been found to have direct defensive roles as phytoalexins, antimicrobials, insecticides, deterrent of herbivores and allelopathic compounds, as well as indirect defensive functions as attractors of enemy predators and parasitoids (Fäldt et al. 2003; Hasegawa et al. 2010; Heiling et al. 2010; Menzel et al. 2014). Certain terpenoids may also be involved in plant protection against abiotic stresses, such as exposure to high temperature, and reactive oxygen species-derived oxidative damages (Vickers et al. 2009). Constitutive and induced terpenoids have been found to be involved in chemical defense against important pests in rice, tomato, maize, and Arabidopsis (Wittstock and Gershenzon 2002; Van Schie et al. 2007b; Huang et al. 2010; Fontana et al. 2011; Taniguchi et al. 2014) and in various coniferous plants (Miller et al. 2005; Boone et al. 2011; Ott et al. 2011; Byun-McKay et al. 2012). More specifically, 3-carene has been reported as a major induced defensive monoterpene in lodgepole pine and Norway spruce trees (Fäldt et al. 2003; Byun-McKay et al. 2012). Numerous terpenoids have been widely used in commercial products, including fragrances (cosmetics, flavored food products, personal care products), pharmaceuticals, alternative medicine, biofuel precursors, industrial materials (e.g., rubber) and others (Tetali 18 2018). Examples of terpenoids used in flavor/fragrance industries include limonene (e.g., in lime beverages) or linalool and santalol (e.g., in perfumes and other cosmetics). There are also terpenoids widely used in the pharmaceutical industry, including the diterpene taxol (as an anticancer drug) and the sesquiterpene lactone artemisinin (as an antimalaria drug) (Xie et al. 2016; Tetali 2018). In this context, several lavender terpenoids are believed to have various physiological and ecological roles, for example in allelopathy, plant defense (with antimicrobial and insecticidal activities) and pollinator attraction (Erland et al. 2015; Erland and Mahmoud 2015; Aprotosoaie et al. 2017). Lavenders provide excellent models for investigating the metabolism of monoterpenes since they produce large amounts of regular and irregular monoterpenes in glandular trichomes (or oil glands), which cover the surfaces of flowers and leaves. The oil glands contain a group of secretory cells that are specialized for monoterpene metaboplism, and strongly express the structural and regulatory genes involved in the process. Further, the oil gland seceretory cells can be mechanically removed and studied in isolation. In this context, Mahmoud group has recently developed extensive transcriptomic and genomic databases for L. angustifolia and L. x intermedia plants to facilitate the discovery of EO biosynthetic genes (Lane et al. 2010; Adal et al. 2019; Malli et al. 2019). Through minig of these databases, the main goals of the studies reported in this thesis were to develop molecular markers for lavenders, and to clone and characterize isoprenyl diphosphate synthase and terpene synthase genes involved in monoterpene metabolism. 1.6 Dissertation objectives and hypotheses The specific objectives of this thesis were to: 1. develop molecular markers for lavenders, 2. clone and characterize the isoprenyl diphosphate synthase (IDS) gene(s) responsible for monoterpene precursor (GPP) production, and 19 3. clone and characterize novel lavender monoterpene synthase genes. We hypothesized that: 1. EST-SSRs can be used as molecular markers in lavenders, 2. The gene responsible for GPP synthesis in lavenders is homologous to those reported from other plants, and 3. TPS-like sequences present in the lavender EST and other sequencing databases encode functional terpene synthase enzymes. To achieve these objectives, the following general strategies were employed: 1. Identify and characterize simple sequence repeat (SSR) markers from three expressed sequence tag (EST) databases previously developed in the Mahmoud lab, and evaluate their polymorphism and cross-species amplification. 2. Clone cDNAs encoding putative short-chain trans-isoprenyl diphosphate synthases (IDSs) in the above sequencing databases, and functionally characterize them in vitro and in vivo. 3. Clone and characterize cDNAs encoding putative monoterpene synthases from our sequencing databases, and functionally characterize them in vitro. Specific objectives and general research approaches are also discussed in sections 1.6.1 – 1.6.3, which provide overviews of forthcoming thesis chapters. 1.6.1 Outline of Chapter 2 Most lavender species are phenotypically closely related with limited variations in oil profiles and developmental growth stages. The subtle differences across species/ cultivars demand high-resolution molecular markers that are able to detect genetic variations, as no efficient markers are available for lavenders. So far, though lavenders display substantial 20 economic importance and social value, little attempt has been made to exploit advanced molecular tools in developing diagnostic markers for use in various applications, including fingerprinting, genetic variation studies, phylogenetic studies, genetic mapping and marker-assisted selection in the genus Lavandula. Recently, the Mahmoud group has reported EST databases corresponding to cDNA libraries of L. angustifolia leaf and flower (Lane et al. 2010), and L. x intermedia glandular trichome tissues (Demissie et al. 2012; Sarker et al. 2012). These libraries have already been used to isolate and functionally characterize key cDNAs involved in Lavandula terpene biosynthesis (Demissie et al. 2011; Demissie et al. 2012; Sarker et al. 2012; Demissie et al. 2013). Most of the identified cDNAs encoding TPSs had shown almost similar sequences and in vitro functions across related species and cultivars. In addition to their utility in cloning genes, EST databases have been widely used to screen SSR markers to be used in diverse genetic applications in many plants (Gao et al. 2004; Varshney et al. 2005; Peng and Lapitan 2005; Huang et al. 2011; Li et al. 2012; Xin et al. 2012; Zhang et al. 2014; Asadi and Rashidi Monfared 2014). Therefore, this portion of the thesis aimed to develop EST-derived SSR markers for lavenders. We demonstrated that most validated SSR markers delineated tested cultivars of L. angustifolia and L. x intermedia, and were cross-amplified in related species to be used as potential markers. 1.6.2 Outline of Chapter 3 Terpenoid metabolism can be regulated at the level of TPS expression and through precursor supply (Dudareva et al. 2000; Orlova et al. 2009). Several genes and enzymes responsible for the formation of terpenoids and their precursors have been reported in different plants (Tholl et al. 2004; Wang and Dixon 2009; Rai et al. 2013). However, over the last few years, most studies of Lavandula terpenoid metabolism have primarly focused on isolation and functional characterization of TPSs (Demissie et al. 2012; Sarker et al. 2012; Sarker et al. 2013; Demissie et al. 2013; Jullien et al. 2014; Benabdelkader et al. 2015; Despinasse et al. 2017). 21 Although monoterpenes are the dominant components of Lavandula oils, particularly in flowers (Boeckelmann 2008; Aprotosoaie et al. 2017), the biosynthesis of a precursor for regular monoterpenes (i.e., GPP) has not been studied. Hence, the main objective of the experiments reported in Chapter 3 was to identify and functionally characterize isoprenyl diphosphate synthase (IDS) responsible for the biosynthesis of GPP in lavenders. cDNAs encoding small and large subunits for lavender GPPS were isolated and functionally characterized in vitro and in vivo. 1.6.3 Outline of Chapter 4 Although numerous lavender TPS genes have been reported, those responsible for the production of 3-carene (an important defensive monoterpene) and S-linalool (a key scent-determining monoterpene) have not been reported. The objective of the studies reported in Chapter 4 was to clone and functionally characterize these genes from lavender. Constitutive and induced terpenoids play key roles in plant defense against important pests (Miller et al. 2005; Boone et al. 2011; Ott et al. 2011; Byun-McKay et al. 2012). Among these, 3-carene has been reported as a major induced defensive monoterpene in lodgepole pine and Norway spruce trees (Fäldt et al. 2003; Byun-McKay et al. 2012), in which its accumulation corresponds to plant resistance to white weevil (Robert et al. 2010). In Lavandula, the EOs and aqueous extracts from various lavender species have been reported to exhibit in vitro antimicrobial and insecticidal activities (Moon et al. 2006; De Rapper et al. 2013; Erland et al. 2015). However, the specific terpenoids involved in Lavandula defensive mechanisms in response to various pests have not been investigated. Thus, in efforts to identify and functionally characterize cDNAs encoding mono-TPSs with potential defensive roles, this portion of the thesis targeted 3-carene synthase. We demonstrated that the expression of 3-carene synthase is induced by methyl jasmonate and its transcripts correspond to the levels of 3-carene in the foliage of L. x intermedia plants. 22 Two linalool isomers (R and S types) exist in Lavandula oils. R-linalool has a woody lavender-like odor and is one of the two most abundant EO constituents in Lavandula oils. Despite its presence in much lower levels, S-linalool that has a sweet and pleasant odor contributes to the overall scent of lavender EOs (Renaud et al. 2001). There is, therefore, an interest in enhancing S-linalool composition in oils through metabolic engineering, as shown in L. latifolia overexpressing Clarkia breweri S-linalool synthase cDNA (Mendoza-Poudereux et al. 2014). This portion of the thesis aimed to clone and functionally characterize S-linalool synthase from L. x intermedia. 23 Chapter 2: Identification, validation and cross-species transferability of novel Lavandula EST-SSRs 2.1 Overview Lavenders (Lavandula) are economically important crops grown around the globe for essential oil production and other horticultural purposes. In an attempt to develop molecular markers for these plants, we analyzed sequences of over 13,000 unigenes (>22,000 ESTs) derived from flowers and leaves of L. angustifolia and L. x intermedia plants using web-based SSR mining tools, and identified 3,459 simple sequence repeats (SSRs) (1 - 6 bp) with lengths of above 12 bp. The identified SSRs were dominated by trinucleotides (41.2 %) and dinucleotides (31.45 %). Approximately 19 % of the unigenes contained at least one SSR marker, over 60 % of which were localized in the untranslated regions. However, only 252 EST-SSRs had primer flanked regions and were 18 bp or longer, from which 31 SSRs were randomly selected and validated for their polymorphism and transferability. Twenty-four of these SSRs amplified discrete multi-fragments with 88 % polymorphism in L. angustifolia and 85 % in L. x intermedia, with the average number of polymorphic alleles of 3.7 and 3.4 per marker, respectively. The utility of positive markers was further assessed by determining marker efficiency indices, such as expected heterozygosity (He), polymorphic information content (PIC), discriminating power (D) and resolving power (R). All indices showed high informativeness of the markers to distinguish the tested samples. The cross-species transferability rate of the markers was also examined in eight related species, including the donor species, and was in the range of 50 - 100 % across these species. The utility of these markers was assessed in 15 cultivars of L. angustifolia and L. x intermedia, and the dendrogram deduced from their similarity indices successfully delineated the cultivars into their respective species. Overall, we identified and characterized EST-SSRs with strong discrimination power against L. angustifolia and L. x 24 intermedia. The markers also showed a considerable cross-species transferability rate into related Lavandula species. 2.2 Materials and methods 2.2.1 EST-SSR motif identification, characterization and primer designing The 13,625 unigenes (6,316 and 3,193 from L. angustifolia flower and leaf libraries, respectively, and 4,116 from L. x intermedia gland library) with an average length of 714 bp were screened in silico to identify SSR motifs using the web-based SSR mining tool ‘SSR server’ available at the Genome Database for Rosaceae (GDR) (Jung et al. 2008). The following parameters were used during the screening: minimum motif length of 15 base pairs (bp) for mono- and pentanucleotides, 12 bp for di-, tri- and tetranucleotides, and 18 bp for hexanucleotides, modified from Iorizzo et al. (2011). In addition to searching motifs from our unigenes, the SSR server was also used to calculate the number, frequency and size of repeats and location of motifs (coding region (CDS) or UTR). The frequency of motifs residing in CDS and UTR were then manually calculated based on their location determined by the mining tool. Further, the average distance between two SSR motifs in ESTs was calculated as: EST-SSR density = (number of unigenes/number of SSRs)*average bp per unigene. SSRs with primer-flanked regions (PFRs) identified by the SSR server tool often do not meet primer designing criteria. Thus, all unigenes with all SSR motifs, except mononucleotides, were submitted to BatchPrimer3 software to screen PFRs that meet primer designing criteria and pick their corresponding primers using the default parameters of the “SSR screening and primers” module (You et al. 2008). Depending on repeat length determined by BatchPrimer3 for PFRs meeting primer designing criteria, motifs were classified into the category I and II. Category I included tri-nucleotide repeats with ≥ 18 bp and di-, tetra-, penta and hexanucleotides with ≥ 20 bp. Di, tetra, penta and hexanucleotide motifs with 12 - 20 bp and trinucleotides with 12 - 18 bp length were classified in category II. Since the high rate of 25 polymorphism is generally expected in longer SSRs (Temnykh et al. 2001), only category I members were advanced for further analysis. The frequency of each motif located in CDS or 5’ and 3’ UTR was calculated manually based on the location of the motif after determining the ORF location of unigenes using the National Center for Biotechnology Information (NCBI) ORF-finder software. From the 252 EST-SSRs classified in category I, 31 EST-SSRs were randomly selected and used for detailed validation. 2.2.2 Functional annotation of SSR containing unigenes The functional annotation of Lavandula unigenes containing SSR motifs was performed using the Blast2GO online platform. Briefly, the sequences were blasted against the non-redundant (nr) NCBI database using the BlastX module default parameters, gene ontology (GO) identifiers are mapped to the blast results, putative functions were assigned to the unigenes and analyzed, and finally the annotation of results was visualized using the direct acyclic graph (DAG) module (Conesa et al. 2005). 2.2.3 Plant materials and genomic DNA extraction The plant materials used in this study comprise of fifteen cultivars, eight from L. angustifolia and seven from L. x intermedia for validation, and of six cultivars from six other species (one cultivar per species) for transferability studies (Table 2.1). Leaf tissues of these cultivars were collected from The Okanagan Lavender and Herb Farm and The Greenery Garden Centre (Kelowna, BC, Canada), freeze-dried in liquid nitrogen and genomic DNA was extracted using Geneaid Genomic DNA extraction kit (plant) (Geneaid Biotech Ltd. Taiwan) as per the manufacturer's instructions. The quantity and quality of extracted DNA were determined using NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and agarose gel electrophoresis (1 %), and the DNA was stored at – 20 °C until used. 26 Table 2.1 List of Lavandula species and cultivars used in this study. Section Species Chromosome numbers* Cultivars Source Lavandula L. angustifolia 2n = 36, 42, 48, 50, 54 Tucker's early purple OLHF Betty's Blue OLHF Folgate OLHF Sachet OLHF Sharon Robert OLHF Royal Velvet OLHF Subsp. angustifolia OLHF Maillette OLHF L. x intermedia 2n= 51 Grosso OLHF unknown Provence OLHF Supper OLHF Hidcote Giant OLHF Fred Boutin OLHF Seal OLHF Abrialli OLHF L. latifolia 2n=36, 48, 50, 54, 75 Latifolia CUBG Inter-section hybrid L. x ginginsii unknown Goodwin Creek Grey GGC Dentatae L. dentata 2n= 42, 44, 45 Dentata GGC Stoechas L. lusitanica 2n = 30 Lusi pink GGC L. stoechas Anouk GGC Pterostoechas L. buchii 2n= 22 Jagged GGC *Chromosome numbers were adopted from Upson and Andrew (2004); OLHF = The Okanagan Lavender and Herb Farm, Kelowna, BC; GGC = The Greenery Garden Centre, Kelowna, BC; CUBG= Cambridge University Botanic Garden, UK. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.2.4 EST-SSR analysis and cross-species transferability study PCR conditions (mainly annealing temperatures) for each primer set were optimized by amplifying the corresponding DNA fragment from genomic DNAs extracted from leaf tissues of L. angustifolia cv. Maillette and L. x intermedia cv. Grosso individuals harvested from The University of British Columbia Okanagan lavender farm. The primers were then used for assessing genetic relationship by amplifying their respective loci from genomic DNAs isolated from L. angustifolia (eight cultivars) and L. x intermedia (seven cultivars). PCR amplifications were carried out at optimal conditions in a 25 μL reaction volume containing 70 ng of genomic 27 DNA, 0.3 μM of each forward and reverse primer, 2.5 μl of the 10x reaction buffer, 250 μM each of dNTP, 1.5 mM MgCl2, 0.5 μg of BSA and 1.25 unit of Taq DNA polymerase (New England Biolabs). The PCR program used was: initial denaturation at 95 °C for 15 min, followed by 11 cycles of denaturation at 95 °C for 30 s, annealing temperature stepping down every cycle by 1 °C from either 64 °C to 54 °C or 62 °C to 52 °C, depending on the primer type, and extension at 72 °C for 2 min. This was followed by a second round amplification of 24 cycles with the following program: denaturation at 95 °C for 30 s, annealing temperature of 54 °C or 52 °C (depending on the primer type), and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 10 min. Amplified fragments were visualized using 6 % polyacrylamide gels (PAGE), stained with SYBR safe (Life Technologies). Clear and indisputable bands that were consistent in three separate amplifications were scored as present (1) and absent (0). Fragments with identical molecular weight across all species or cultivars were considered as monomorphic. Cross-species amplification of EST-SSRs generated from L. angustifolia and L. x intermedia were tested in six Lavandula species (Table 2.1): in PCR conditions described above. The genetic relationship among eight species, including the two EST-SSRs donor species, was analyzed using 18 cross transferred EST-SSR markers. Similarly, 24 SSRs were used to analyze the genetic diversities among eight L. angustifolia and seven L. x intermedia cultivars. Detailed recipes and procedures for preparation of buffers and other reagents used in the validation of selected EST-SSRs are mentioned in Appendix A. 2.2.5 Analysis of molecular marker efficiency Lavandula species are characterized by complex polyploidy with significant variation in chromosome numbers, ranging from 18 – 75 chromosomes (Upson and Andrew 2004). Hence, to avoid errors associated with distinguishing alleles of homologous chromosomes, the co-dominant SSR markers were considered as dominant markers. Assuming every allele as a 28 single locus, all alleles detected in all species were recorded as present (1) and absent (0) in a binary data matrix. The binary data matrix was then used to determine the total number of alleles, the number of alleles per marker (Na), polymorphic alleles per marker (Pa) and other marker efficiency indices using PowerMarker v3.25 software (Liu and Muse 2005) and iMEC, an online marker efficiency calculator (Amiryousefi et al. 2018). The marker efficiency indices used in this study are defined as follows:  Expected heterozygosity (He) or gene diversity (the probability of a genotype that is heterozygous for the locus in the population); He = 1 - ∑pi2, where p is the allele frequency for the i-th allele (Belaj et al. 2003).  Polymorphic information content (PIC) (the probability of the genotype of a given offspring that will allow deduction of the two alleles of the affected parents it received in the absence of crossing over); PIC = 1 - ∑pi2 - ∑ ∑ pi2 pj2, where pi and pj are the population frequency of the i-th and j-th allele (Botstein et al. 1980).  Effective multiplex ratio (E) (the product of the fraction of polymorphic loci for an individual assay): E = n *β; where β = np / (np + nnp), n= total loci, p = the polymorphic and np = nonpolymorphic fraction of the markers, and np and nnp represent the corresponding counting numbers (Powell et al. 1996).  Mean heterozygosity (Havp) (the average heterozygosity calculated for polymorphic markers); Hav p = Σ Hn / np. Where Hn = the heterozygosity of the polymorphic fraction of markers, and np = over all of the polymorphic loci (Powell et al. 1996).  Marker index (MI) (the product of the effective multiplex ratio and the average heterozygosity for polymorphic markers); MI = E *Hav p, where Hav p = average heterozygosity for the polymorphic markers as Σ Hp / np, p= polymorphic loci (Powell et al. 1996). 29  Discriminating power (D) (the probability of two randomly chosen individuals exhibiting different banding patterns and are distinguishable from one another); D = 1 – C, where C = the confusion probability, C = Σ pi [(Npi−1)/ (N−1)] for all of the patterns generated by the primer, and N = individuals, the i-th pattern of the given j-th primer at frequency pi in a set of varieties (Tessier et al. 1999).  Resolving power (R) (ability of the marker to distinguish between genotypes. It is based on the distribution of alleles within the sampled genotype and strongly correlates with the ability to distinguish between analyzed samples based on the presence or absence of a band); R = Σ Ib, where Ib = band informativeness was scaled as 0 or 1 and Ib = 1 – (2 × |0.5 – p|), where p is the portion of the samples containing the observed band (Prevost and Wilkinson 1999). The genetic similarity level among L. angustifolia and L. x intermedia cultivars, and among related species representing four sections and one inter-section hybrid was estimated based on Jaccard’s similarity coefficient (Dalirsefat et al. 2009), which is expressed as Nxy / (Nxy + Nx + Ny ), where Nxy = the number of bands shared by both x and y samples, Nx= amplified bands in sample x and Ny = amplified bands in sample y. Using Jaccard’s similarity coefficient values, the cultivars and species were then clustered with the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis and SAHN procedure of the NTSYS-PC v2.10t (Rolf 2000). The UPGMA dendrogram confidence limits were determined from 2000 bootstraps using a WinBoot software program (Yap and Nelson 1996). Overall, the overview of SSR mining and validation steps is shown briefly in Figure 2.1. 30 Figure 2.1 Overview of SSRs analysis from EST libraries of lavender species and validation of identified SSR on the source as well as related Lavandula species. Major steps of SSR analysis from ESTs and subsequent validation include: (1) EST library development; (2) SSR motif identification using web-based mining tools; (3) SSR characterization, primer design and custom-synthesis of primers; (4) genomic DNA isolation; (5) PCR amplification of expected SSR motif-containing fragments; and (6) resolution of DNA fragments on polyacrylamide gel for scoring of amplicons as presence (1) or absence (0). (From Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud (2016) EST-SSR analysis and cross-species transferability study in Lavandula, Bio-protocol 6(15): e1891. © Authors 2016, Creative Commons Attribution License (CC BY 4.0.)). 31 2.3 Results 2.3.1 Identification and in silico characterization of EST-SSRs A total of 3,459 EST-SSR motifs were identified in our unigene libraries, of which 1,641 were from flower database, 953 from leaves and 865 from oil glands (Figure 2.2). The 3,459 SSR motifs were identified from 2,556 (18.8 %) unigenes because more than one SSR motif (up to five per unigene) were identified in some instances (Table 2.2). The distribution density of the EST-SSRs in Lavandula unigenes was one SSR for every 2.81 kb distance and the number of repeat units per SSR ranged from 3 for tetranucleotides - 40 for dinucleotides. The majority of the identified motifs (~60 %) were located in the UTR of the unigenes. Although the unigenes of flower tissues resulted in the highest number of SSR motifs, the number of SSR motifs identified in each tissue was proportional to the number of unigenes present in the database. For example, the proportion of SSR motifs in L. angustifolia flower database was 26 % and that of the leaf was 29 %. Trinucleotide SSRs were the most abundant motifs in Lavandula unigenes, with 41 % occurrence frequency, followed by di- (31.45 %) and tetra-nucleotides (11.51 %), while the frequencies of mono-, penta- and hexanucleotides were 6.07 %, 3.79 % and 5.98 %, respectively (Figure 2.3). However, when different repeat types were considered, the dinucleotide motif type AG/GA/CT/TC showed the highest occurrence (26.2 %), followed by the trinucleotide motif groups GGC/GCG/CGG/GCC/CCG/CGC and AAG/AGA/GAA/CTT/TTC/TCT with occurrence frequencies of 10 % and 8.09 %, respectively (Table 2.2). All other motif types had relatively low distribution, 0.35 - 5.72 %, while the GC/CG motif types were completely absent in Lavandula unigenes. The same motif types also dominated the three tissue-specific databases at comparable frequencies. For example, the dinucleotide motif group AG/GA/CT/TC is the abundant marker in flower, leaf and oil gland accounting for 26 %, 27.2 % and 25.6 % of the total SSR motifs, respectively (Table 2.2). 32 Figure 2.2 A flowchart showing a stepwise in silico analysis of ESTs to identify SSRs. cDNA libraries derived from flower and leaf tissues of L. angustifolia and floral-based glandular trichome tissues of L. x intermedia were initially used for sequencing of ESTs. PFR- primer flanking region, category I- long SSR repeats as ≥ 18 bp for trinucleotide and ≥ 20 bp for others ; category II - short SSR repeats 12 - 18 bp for trinucleotides and between 12 and 20 bp for other motifs . (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). SSRs are generally characterized by the presence of conserved flanking sequences. However, often not all EST-SSRs contain these sequences due to limitations associated with the nature of the EST database or because some conserved sequences are too short to satisfy primer-designing parameters. From the 3,459 SSR motifs we identified, only 1,812 (about 52.4 %) were flanked by conserved sequences, also called primer-flanking regions (PFRs), of which 33 1,108 were mined from the flower unigene library, 300 from leaf and 404 from oil gland (Figure 2.2). The 1,812 motifs were grouped into two categories based on their repeat length where 252 (13.9 %) EST-SSRs were clustered into the category I and the rest, 1,560 (86.1 %), were clustered into category II. Category II type EST-SSRs were excluded from subsequent analysis because previous research established that they are inefficient in detecting a polymorphism (Singh et al. 2009). Table 2.2. The occurrence of different SSR motif types in unigenes of Lavandula EST databases derived from flower, leaf and glandular trichome cDNA libraries . Flower tissues Leaf tissues Oil glands Overall Motif types (repeat unit) Occur. % Occur. % Occur. % Occur. % Mononucleotides A/T 105 3.03 69 1.99 24 0.69 198 5.72 G/C 7 0.2 3 0.09 2 0.06 12 0.35 Subtotal 112 3.23 72 2.08 26 0.75 210 6.07 Dinucleotides AG/GA/CT/TC 427 12.34 259 7.49 221 6.39 907 26.22 AC/CA/TG/GT 27 0.78 10 0.29 14 0.40 51 1.47 AT/TA 56 1.62 38 1.10 36 1.04 130 3.76 GC/CG - - - - - - - - Subtotal 510 14.74 307 8.88 271 7.83 1088 31.45 Trinucleotides AAT/ATA/TAA/ATT/TTA/TAT 34 0.98 14 0.40 27 0.78 75 2.17 AAC/ACA/CAA/GTT/TTG/TGT 24 0.69 14 0.40 13 0.38 51 1.47 AAG/AGA/GAA/CTT/TTC/TCT 138 3.99 76 2.20 66 1.91 280 8.09 ACC/CCA/CAC/GGT/GTG/TGG 80 2.31 41 1.19 31 0.90 152 4.39 AGG/GGA/GAG/CCT/CTC/TCC 87 2.52 49 1.42 44 1.27 180 5.2 ATG/TGA/GAT/CAT/ATC/TCA 49 1.42 33 0.95 34 0.98 116 3.35 ACG/CGA/GAC/CGT/GTC/TCG 16 0.46 9 0.26 13 0.38 38 1.1 AGC/GCA/CAG/GCT/CTG/TGC 77 2.23 54 1.56 37 1.07 168 4.86 AGT/GTA/TAG/ACT/CTA/TAC 7 0.20 7 0.20 5 0.14 19 0.55 GGC/GCG/CGG/GCC/CCG/CGC 167 4.83 107 3.09 72 2.08 346 10.00 Subtotal 679 19.63 404 11.68 342 9.88 1425 41.19 Tetranucleotides TAAA/TTTA 10 0.29 3 0.09 5 0.14 18 0.52 AAAT/ATTT 11 0.32 4 0.12 10 0.29 25 0.72 Others 148 4.28 78 2.25 129 3.73 355 10.26 Subtotal 169 4.89 85 2.46 144 4.16 398 11.52 Pentanucleotides (sum) 67 1.94 31 0.90 33 0.95 131 3.79 Hexanucleotides (sum) 104 3.01 54 1.56 49 1.42 207 5.98 Overall sum 1641 47.44 953 27.55 865 25.01 3459 Occur. = Occurrence. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 34 Figure 2.3 The overall frequency of SSR motifs in unigenes of Lavandula EST databases derived from flower, leaf and floral glandular trichome cDNA libraries. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). Category I member EST-SSRs were dominated by the dinucleotide repeat type AG/GA/CT/TC motif (99 of the 252 loci) followed by hexanucleotide repeats (30), the AT/TA repeat type (21), the trinucleotide repeats GGC/GCG/CGG/GCC/CCG/CGC and AAG/AGA/GAA/CTT/TTC/TCT (14 each), in their respective order (Table 2.3). Of the 252 category I loci, 140 (55.2 %) were located in the CDS of the unigenes while 80 loci (32.1 %) were found in the 5’ UTR and 32 loci (12.7 %) were in the 3’ UTR region. This was contrary to the localization of the overall SSRs where only 40 % of them were located in the CDS region. When the locations of different motif types were compared, dinucleotides were predominantly found in the 5’ UTR region while all the other motif types were predominantly located in the CDS region. Unlike the overall SSRs, the distribution of the dominant category I member motif types showed considerable variation among the three databases. For example, out of the 99 AG/GA/CT/TC motif types 48 (48.4 %) were identified from L. angustifolia flower database and 35 only 26 and 15 loci were identified from L. x intermedia gland and L. angustifolia leaf databases, respectively. Similarly, tri- and hexanucleotides were distributed unevenly in the three databases whereas, except for a few outliers, tetra- and pentanucleotides were generally less abundant in all databases (Table 2.3). Table 2.3 The occurrence and localization of category I SSRs in unigenes of Lavandula EST databases derived from flower, leaf and glandular trichome tissue cDNA libraries. L. angustifolia Flowers ESTs L. angustifolia Leaves ESTs L. x intermedia Oil glands ESTs SSR repeats CDS 5'UTR 3'UTR CDS 5'UTR 3'UTR CDS 5' UTR 3'UTR Total % Dinucleotides AT/TA 5 3 8 - 1 1 1 - 2 21 8.33 AG/GA/CT/TC 15 32 11 4 9 2 15 11 - 99 39.29 AC/CA/TG/GT 1 3 - - - 1 - - - 5 1.98 GC/CG - - - - - - - - - - 0.00 Trinucleotides AAT/ATA/TAA/ATT/TTA/TAT 1 1 1 - 1 - 1 - - 5 1.98 AAG/AGA/GAA/CTT/TTC/TCT 6 1 - 6 - - - - 1 14 5.56 AAC/ACA/CAA/GTT/TTG/TGT 1 - - 3 - - - - - 4 1.59 ATG/TGA/GAT/CAT/ATC/TCA - 1 - 1 - - 5 - - 7 2.78 AGT/GTA/TAG/ACT/CTA/TAC 4 - - - - - 1 - - 5 1.98 AGG/GGA/GAG/CCT/CTC/TCC 3 - - 1 - - 2 - - 6 2.38 AGC/GCA/CAG/GCT/CTG/TGC 5 1 - - 1 - 1 - 1 9 3.57 ACG/CGA/GAC/CGT/GTC/TCG 1 - - 1 - - - - 1 3 1.19 ACC/CCA/CAC/GGT/GTG/TGG 6 - - 2 - - 4 - - 12 4.76 GGC/GCG/CGG/GCC/CCG/CGC 3 1 - 1 - 1 5 3 - 14 5.56 Tetranucleotides 2 1 - - - - - - - 3 1.19 Pentanucleotides 2 4 - 1 2 - 4 2 - 15 5.95 Hexanucleotides 15 3 2 3 - - 7 - - 30 11.90 Total 70 51 22 23 14 5 46 16 5 252 % 27.78 20.24 8.73 9.13 5.56 1.98 18.25 6.35 1.98 CDS- coding regions and UTR- untranslated regions. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.3.2 Functional annotation of unigenes containing SSRs From the 2,556 unigenes containing SSRs, BLAST hits were obtained for 2,321 (90.8 %) of them while putative functions could be assigned to 1,933 (83.3 %) unigenes. The majority of unigenes containing SSRs have homology to nucleic acid binding and catalytic activity, 41.23 % 36 and 38.9 %, respectively, and other functions-like transporter (5.02 %), transcription factors (4.24 %), structural molecule activity (3.72 %) and so forth (Figure 2.4). Some of the polymorphic loci were associated with functional genes including monoterpene synthases, implying their potential for functional adaptation genetic studies in addition to classical genetics (Table 2.4). Figure 2.4 Molecular level functional annotation of Lavandula unigenes containing SSR markers using Blast2GO software. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.3.3 Lavandula EST-SSR validation and polymorphism Since the SSR markers were derived from L. angustifolia and L. x intermedia libraries, we used genomic DNAs extracted from the two species to study the polymorphism of 31 category I loci as a proof of concept. The remaining 223 markers along with their locations, sizes and other features are shown in Appendix B. Among the selected motifs, 13 were dinucleotides, 12 were trinucleotides and 6 belong to tetra-, penta- and hexanucleotides. In addition, 20 of the 31 randomly selected EST-SSRs were located in the CDS while seven of them were located in the 5’ UTR and the remaining four in 3’ UTR (Table 2.4). Of the total 37 primer sets tested, 24 of them successfully amplified genomic DNA fragments in three different attempts, while seven markers failed to do so in five different attempts. All positive SSRs were multi-allelic with expected allele sizes, except LINT5 in L. angustifolia and LAF8 and LAF16 in L. x intermedia that were mono-allelic (Table 2.5). Exceptionally, three markers, such as LAF8, LINT5 and LINT14, produced alleles greater than expected sizes. A total of 201 alleles (with 85 % polymorphism), 103 (with 86 % polymorphism) of which were from L. angustifolia and 98 (with 84 % polymorphism) from L. x intermedia, were detected with an average of 4.3 and 4.1 alleles per marker, respectively. In particular, the LAF6 locus was the only monomorphic marker in L. angustifolia, but was polymorphic in L. x intermedia genomic DNA. The efficiency of positive markers was examined by determining marker efficiency indices and summarized in Table 2.5. One of the indicators was expected heterozygosity (He), in which its values ranged from 0 (no variation) to 0.5 (high variation) among samples. In L. angustifolia, the SSR locus LAF6 produced the minimum (0) He level, while marker LAF5, LAF10, LAF15 and LAF20 produced the maximum He value (0.5). For L. x intermedia, the minimum He value (0.2) was obtained at the LAF6 locus, while the maximum value (0.5) was detected at the LAF10 and LAF20 loci. Of the total positive markers, 75 % of them had higher He than the average value (0.42) in L. angustifolia, and over 65 % had above the corresponding average mean value (0.44) in L. x intermedia. This implies that these novel markers were able to detect relatively high genetic variations in both species. The polymorphic information content (PIC) value ranges from zero (for monomorphic markers) to 0.5 for polymorphic markers that are present in 50 % of the samples and absent in the other 50 %. The PIC values of L. angustifolia plants were between 0.0 (LAF6) to 0.38 (LAF10) with an average value of 0.32. The PIC values for L. x intermedia plants were between 0.12 (LAF21) and 0.38 (LAF10) with an average value of 0.34. These values indicate that the selected markers have high levels of informativeness in samples. Over 60 % of tested EST-SSRs (15) had greater PIC values (above 38 average > 0.35) in both donor species samples, suggesting that they were the most informative markers in both L. angustifolia and L. x intermedia. Discriminating power (D) that shows the probability of two individual samples with different band patterns was determined with values ranging from 0 (no variation) to 1 (high variation). The values varied from 0 (LAF6) to 1 (LINT5) in L. angustifolia and 0.14 (LAF21) to 0.91 (LAF1) in L. x intermedia, with the same average value of 0.64 in both species (Table 2.5). The results suggest that most of the markers have greater discriminating power, in which about 60 % (14-15 markers) of them had D values above the average values in both species. We also assessed the resolving power (R) showing the ability of informative primers to distinguish sampled genotypes. The R values were between 0 (LAF6) and 4.22 (LAL4) in L. angustifolia and 0.29 (LAF21) and 4.29 (LAF13) in L. x intermedia, with average values of 1.95 and 1.90, respectively (Table 2.5). More than 50 % of the positive markers had R values above the average, suggesting that they are efficient markers to differentiate samples of L. angustifolia and L. x intermedia. Additionally, the overall marker utility was then estimated by Marker Index (MI) after measuring mean expected heterozygosity (H.av) and the multiplex ratio (E) (Appendix C). The values of MI ranged from 0 (LF6) to 0.033 (LF1, LINT4 and LINT10) in L. angustifolia, and 0.018 (LAF21) to 0.042 (LAF2, LAF15, LAF16, LAF18, LAL4, LINT5 and LINT10) in L. x intermedia, with overall average values of 0.026 and 0.036, respectively. Of the total positive markers, over 60 % of them had MI with values greater than the overall mean in both species, suggesting their comparable ranges of marker utilities. 39 Table 2.4 List of selected Lavandula EST-SSRs along with their primer pairs, motif type, location and putative function. Genbank Acces. No. Locus Primers (5'==>3') Motif SSR location Tm * (oC) Expected allele (bp) Functional annotation Protein name Acces. no. L. angustifolia flowers KM288520 LAF1 F: GGGATTCGACTGCCTTGATA R: AATGCCATTTCCCACCACT (AG)15 3' UTR 60.04 246 Uncharacterized protein EYU29981.1 KM288521 LAF2 F: GAAAATGATTCCGGACGAGA R:TGGCCCTGGTGATTGAAATA (TA)11 3' UTR 60.01 245 Cytochrome P450 AHL46848.1 KM288522 LAF3 F: TCTCGATGCAAACTGAATGC R: ATACTCGGTGCCCAGATCAC (AG)19 CDS 59.96 230 ABC transporter G family member 11-like XP_006351519.1 KM288523 LAF4 F: AGCACGACGAGCTTTCAAGT R: TTGTGCTGTTAAACCATAAGTCC (AT)26 CDS 60.2 168 Two-component response regulator-like APRR1-like XP_006354770.1 KM288524 LAF5 F: CAAATGACCCCATCAACAA R: GTATGATCCCATCCCGTGAG (CT)18 5' UTR 59.75 225 Root phototropism protein XP_002525040.1 KM288525 LAF6 F: GATGATGGGCTACTCGTGGT R: GGCGATGACAGAAGAGAGA (AGC)9 CDS 59.96 217 SQUAMOSA promoter binding protein-like 14 AIE89803.1 KM288526 LAF7 F: GCTGATTCATCTTGGCCTTG R: CAAATCGTTTGGAAGCACA (ATT)15 3' UTR 60.74 219 Sugar phosphate/ phosphate translocator XP_004245685.1 KM288527 LAF8 F: TGCAATCCTCCTATGTGTTGTC R: TTGGAGTTGCTGTGGATGAG (CT)15 CDS 60 228 No hit KM288528 LAF9 F: GAGCTGCGAGTGTGAGTCAG R: TTTACTTGGGGGACGTTGAG (AG)13 CDS 59.92 149 Uncharacterized protein EYU38816.1 KM288529 LAF10 F: TGTCAGGATCGAAACTCGTG R: ATGCACCTTTGGGATTTCAG (AT)23 CDS 59.83 227 Filament-like plant protein 4 XP_008221294.1 KM288530 LAF11 F: GCAATGTTGGAATGTGATGC R: AAGCGGCAATCTTGGTAGTG (CA)13 5' UTR 59.94 193 Monodehydroascorbate cytoplasmic isoform 2-like XP_007031494.1 KM288531 LAF12 F: AGAGCAGGCCTCTTTTGCTA R: ATGAAAGGCTCAAGGGCTTC (ATT)17 5' UTR 59.36 194 No hit KM288532 LAF13 F: TCCTTCTCCCTCTCCCTCTC R: AGCTATCCGAGCACGACAAT (TCTCCC)5 5' UTR 59.88 188 Cyclin-d1-1-like CAB61221.1 40 Genbank Acces. No. Locus Primers (5'==>3') Motif SSR location Tm * (oC) Expected allele (bp) Functional annotation Protein name Acces. no. KM288533 LAF14 F: GTCCCACTCCCACAACTCAT R: TCCTTCTTGGTCAACAATTTCA (CT)35 3' UTR 59.82 237 Uncharacterized protein XP_002299474.1 KM288534 LAF15 F: AAGCGGAAGTGGATTCATGT R: GCAAGATTGCATTAGCACGA (TGC)7 CDS 59.56 154 39S ribosomal protein L27, mitochondrial-like XP_006356646.1 KM288535 LAF16 F: CGCTCTATCCCTTTCCCTCT R: AGCCGCTTGTGACTTTCTCT (TCCCTC)5 CDS 59.81 139 E3 ubiquitin-protein ligase XP_008236747.1 KM288536 LAF18 F: TTCACCCGGAATCTTTACCA R: CAAATTCCCTGCAACCAATC (CGTT)6 CDS 60.3 256 Transcription factor ABA-Inducible bHLH-type XP_003596884.1 KM288537 LAF19 F: TCATGGAGCAGCAGAACATC R: CGAAGTCCTGGTCCAATAGC (GCA)10 CDS 59.95 153 Uncharacterized protein EYU24058.1 KM288538 LAF20 F: TATCGGACACCGCTCCAAT R: AAGAGGGGAATCAACCAAGC (CCG)9 CDS 61.43 187 RHOMBOID-like 1 XP_007027004.1 KM288539 LAF21 F: GAGGAGGAGGAGGAAGTGCT R: ATACTCGGTGCCCAGATCAC (GAC)10 CDS 59.95 222 Nucleolin-like XP_006366238.1 L. angustifolia leaves KM288540 LAL2 F: TTGGCAGCATCAGAGACAAC R: CTTGG AGGTTGGGAAGTTT (ACA)12 CDS 59.99 201 Nucleic acid binding XP_002531176.1 KM288541 LAL3 F: TCTGTTGTTGCCTCCTCTCC R: AAGTGGGAAAACGGAAAAGG (TCTGC)4 5'UTR 60.39 190 No hit KM288542 LAL4 F: AAGTTTCCCTCTGCCTCCTC R: AGAGGCCGTAGCTGTCTTCA (AAC)8 CDS 59.82 186 Uncharacterized protein EPS61380.1 L. x intermedia oil glands KM288543 LINT1 F: GGCACATTGGGGTACAAGAT R: GCTGATCCAGGCTTCTCATC (CAC)10 CDS 59.68 191 N-alpha-acetyltransferase 10-like XP_004514569.1 KM288544 LINT4 F: CATCTCCTCCCCTCCCTACT R: CGACGGAGAAGAGTGACGAC (CT)15 CDS 59.51 185 No hit KM288545 LINT5 F: TGCGAGAAGACCGTTTATCC R: CCAAGCATCAAAACAGCAAA (TC)18 CDS 60.21 214 Pollen-specific protein SF3-like XP_004138434.1 KM288546 LINT6 F: TTTTCTCTTTGGTTTCATGAGC R: AATCCTGGTTCCTCGGAGTT (GA)11 5'UTR 58.49 170 Uncharacterized protein EYU26600.1 41 Genbank Acces. No. Locus Primers (5'==>3') Motif SSR location Tm * (oC) Expected allele (bp) Functional annotation Protein name Acces. no. KM288547 LINT10 F: TTAGCCGAGGCACTAGAGGA R: CTTGGCCTCCTTCTTCTTCA (ATG)7 CDS 60.11 142 Uncharacterized protein CDSP13488.1 KM288548 LINT11 F: AAGGCGAGAAAAGCATTTGA R: CTGAGCCTTCACGGTTCTTC (GATTT)4 5' UTR 59.96 263 Ras-related protein raba2a-like XP_004230439.1 KM288549 LINT12 F: GAAACCCTCCAATCCATCCT R: GCCGAAAGCAATTCAATGTT (ATC)7 CDS 60.13 226 1,8-cineole synthase AFL03421.1 KM288550 LINT14 F: CCTCACGAACCACACCATC R: TTAGGAAGGAGGAGCAACCA (CCACAT)6 CDS 59.95 232 No hit Acces. No. = accession number; *Tm = the melting temperature for every primer pair. However, best PCR amplification done using touchdown PCR (TD-PCR) programs: (a) Tm= 64-54 oC (-1 in every cycle) for 11 cycles and 54 oC for 24 cycles worked well for majority, (b) 62-52 oC (-1 in every cycle) for 11 cycles and 54 oC for 24 cycles for LAF2, LAF10, LAF11, LAF15, LAF18, LAF20, LAF21 and LINT12, and (c). Both programs were employed for LAF3, LAF7, LAF12, LAF14, LAL3, LINT1 and LINT11. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014).42 Table 2.5 Validation of 31 EST-SSR markers in L. angustifolia and L. x intermedia. EST-SSRs L. angustifolia (n= 8) L. x intermedia (n=7) Alleles (bp) Na Pa He PIC D R Na Pa He PIC D R LAF1 6 6 0.47 0.36 0.61 2.89 6 6 0.43 0.34 0.91 2.86 240-400 LAF2 4 4 0.49 0.37 0.83 2.00 2 2 0.46 0.35 0.60 1.14 240-350 LAF3 - - - - - - - - - - - - - LAF4 4 4 0.40 0.32 0.93 2.00 3 2 0.47 0.36 0.87 0.86 135-170 LAF5 4 4 0.50 0.37 0.73 2.00 4 4 0.41 0.32 0.50 2.29 225-320 LAF6 3 0 0 0 0 0 5 1 0.20 0.18 0.22 0.86 150-325 LAF7 - - - - - - - - - - - - - LAF8 2 2 0.31 0.26 0.36 1.56 1 1 0.49 0.37 0.71 0.86 275-350 LAF9 4 4 0.49 0.37 0.81 2.89 3 3 0.49 0.37 0.83 2.29 140-200 LAF10 2 2 0.50 0.38 0.76 0.89 2 2 0.50 0.38 0.77 1.14 175-450 LAF11 4 4 0.49 0.37 0.70 2.67 4 4 0.48 0.36 0.85 2.57 180-380 LAF12 - - - - - - - - - - - - - LAF13 7 4 0.48 0.37 0.66 1.56 7 7 0.49 0.37 0.68 4.29 150-500 LAF14 - - - - - - - - - - - - - LAF15 5 4 0.50 0.37 0.72 2.67 5 5 0.47 0.36 0.61 2.86 145-200 LAF16 6 6 0.48 0.36 0.85 2.44 1 1 0.41 0.32 0.52 0.57 130-160 LAF18 3 3 0.31 0.26 0.36 1.56 4 4 0.41 0.32 0.50 1.43 200-300 LAF19 5 5 0.49 0.37 0.70 3.11 5 5 0.48 0.36 0.85 2.57 145-200 LAF20 5 5 0.50 0.37 0.77 3.78 4 4 0.50 0.37 0.72 2.86 165-260 LAF21 4 3 0.49 0.37 0.70 2.22 2 1 0.13 0.12 0.14 0.29 140-400 LAL2 7 7 0.48 0.37 0.83 3.56 7 6 0.49 0.37 0.80 2.57 170-420 LAL3 - - - - - - - - - - - - - LAL4 10 8 0.49 0.37 0.68 4.22 9 5 0.42 0.33 0.52 3.71 160-365 Subtotal 85 75 74 63 Mean 4.7 4.2 0.44 0.33 0.67 2.33 4.1 3.5 0.43 0.33 0.64 2.00 LINT1 - - - - - - - - - - - - - LINT4 6 5 0.42 0.33 0.51 2.00 7 5 0.48 0.37 0.65 2.86 130-200 LINT5 1 1 0.20 0.18 1.00 0.22 2 2 0.41 0.32 0.51 1.14 250-275 LINT6 2 2 0.35 0.29 0.42 0.44 3 2 0.49 0.37 0.69 1.14 165-350 LINT10 3 2 0.44 0.35 0.58 0.67 3 2 0.41 0.32 0.51 0.86 135-150 LINT11 - - - - - - - - - - - - - LINT12 2 2 0.40 0.32 0.49 1.11 2 2 0.49 0.37 0.69 0.86 175-265 LINT14 4 2 0.38 0.30 0.44 0.44 7 6 0.48 0.37 0.65 2.86 330-550 Subtotal 18 14 24 19 Mean 3.0 2.3 0.36 0.29 0.57 0.81 4 3.2 0.46 0.35 0.62 1.62 Total 103 89 98 82 Mean 4.3 3.7 0.42 0.32 0.64 1.95 4.1 3.4 0.44 0.34 0.64 1.90 Max 10 8 0.50 0.38 1.00 4.22 9 7 0.50 0.38 0.91 4.29 43 EST-SSRs L. angustifolia (n= 8) L. x intermedia (n=7) Alleles (bp) Na Pa He PIC D R Na Pa He PIC D R Min 1 0 0 0 0 0 1 1 0.13 0.12 0.14 0.29 n= number of cultivars; Na= number of bands (alleles); Pa= polymorphic bands; He = expected heterozygosity; PIC= Polymorphic information content; D = discriminating power; R = resolving power; "-" represents no results from markers that failed to amplify detectable products. (Adapted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.3.4 Cross-species transferability of EST-SSR markers in Lavandula As shown in Table 2.5, L. angustifolia SSRs showed 100 % cross-species amplification with comparable polymorphism level in L. x intermedia. In fact, the loci with the highest He and PIC values (LAF10) and the highest D (LAF1) and R (LAF13) values in L. x intermedia were identified from L. angustifolia (Table 2.5). Similarly, 100 % transferability rate with comparable polymorphism level was also obtained for SSRs identified from L. x intermedia in L. angustifolia, although the number of alleles, D and R detected was considerably lower in both species. L. angustifolia EST-SSRs amplified a total of 85 alleles with 88 % polymorphism in L. angustifolia and 74 alleles with 85 % polymorphism in L. x intermedia. On the other hand, the L. x intermedia EST-SSRs detected a total of 24 alleles with 79 % polymorphism in L. x intermedia and 18 alleles with 78 % polymorphism in L. angustifolia. The number of alleles detected by L. x intermedia SSRs was low because only six of the 24 markers were from L. x intermedia. This considerably higher cross-species marker transferability and polymorphism level was expected since the two species are genetically related, L. x intermedia is a natural half progeny of L. angustifolia. We also tested the cross-species transferability of these markers in six other species identified in different sections of the genus Lavandula (Table 2.1). The markers showed 100 % transferability rate in L. latifolia species followed by rates ranging from 50 % in L. buchii, L. lusitanica and L. stoechas to 83.3 % in L. x ginginsii, a natural inter-section hybrid between L. 44 latifolia and L. dentata (Table 2.6). LAF5, LAF9, LAF21 and LAL4 loci showed 100 % cross-species transferability in tested species (Table 2.6). Sizes of the alleles amplified across species were within the ranges detected in the donor species for each locus, suggesting that the products were likely derived from the same loci and that the primer binding sites of the alleles were highly conserved. Table 2.6 Cross-species transferability of 18 EST-SSR markers in six Lavandula species. Loci name L. latifolia L. buchii L. lusitanica L. dentata L. x ginginsii L stoechas Alleles (bp) LAF2 + + - + + + 240-350 LAF4 + - - - + + 135-170 LAF5 + + + + + + 225-320 LAF6 + + + + + - 150-325 LAF8 + - - - + - 275-350 LAF9 + + + + + + 140-200 LAF11 + - + + + + 180-380 LAF13 + - - + + - 150-500 LAF15 + + - - + - 145-200 LAF18 + - + + + + 200-300 LAF19 + - + + - - 145-200 LAF20 + - - + - - 165-260 LAF21 + + + + + + 140-400 LAL2 + - - - + - 170-420 LAL4 + + + + + + 160-365 LINT4 + + - + + - 130-200 LINT6 + - - + + + 165-350 LINT14 + + + + - - 330-550 TR ( %) 100 50 50 77.7 83.3 50 TR= cross-species transferability rate. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.3.5 EST-SSR-based genetic relationship analysis Eighteen cross-species amplified EST-SSRs were used to analyze genetic similarity levels among eight different species (including donor species) identified in the different sections of Lavandula according to previous taxonomic classifications (Upson and Andrew, 2004). The genetic similarity index, as explained by the Jaccard Similarity Coefficient, among the eight 45 species ranged between 0.11 – 0.6 (Figure 2.5a). The SSRs were effective in categorizing the different species into their respective sections as described previously using morphological, ITS and a few barcoding markers (Upson and Andrew 2004; Moja et al. 2016; Hind et al. 2018). It is, however, worth noting that although L. x intermedia was grouped between its parental lines in the deduced tree, it was rooted closer to L. angustifolia than L. latifolia. The most likely explanation for this is the fact that most of the polymorphic SSRs were derived from L. angustifolia unigenes and also those derived from L. x intermedia showed greater polymorphism in L. angustifolia than L. latifolia. A similar trend was also observed in L. x ginginsii – an inter-section hybrid of L. latifolia and L. dentata – where it was more closely rooted with L. latifolia than with L. dentata. This was also likely because the selected markers showed 100 % cross-species transferability with a higher number of alleles in L. latifolia as opposed to the 77.8 % rate seen in L. dentata. Similarly, 24 SSRs successfully discriminated fifteen cultivars belonging to L. angustifolia and L. x intermedia according to their species with Jaccard's similarity coefficient ranging from 0.4 – 0.74. In the deduced dendrogram, the cultivars were grouped in their respective species (Figure 2.5b). The maximum similarity level (74 % similarity) was detected between L. angustifolia cv. Folgate and Sachet, suggesting that the markers were also effective in discriminating the cultivars from each other. Figure 2.6a showed LAL4 amplification in L. angustifolia and L. x intermedia cultivars, while the amplification of LAL4 and LAL5 markers from the eight species was presented in Figure 2.6b and c, respectively. 46 Figure 2.5 The phylogenic relationship among Lavandula species and cultivars deduced from similarity index results of the EST-SSRs. UPGMA dendrograms showing the genetic relationship among (a) eight Lavandula species based on 18 EST-SSRs markers; and (b) among L. angustifolia and L. x intermedia cultivars using 24 EST-SSRs markers. L. x ginginsii is an inter-sectional hybrid between L. lanata (Lavandula subsection) and L. dentata (dentatae section). The scale bar indicates the level of Jaccard's similarity coefficient between samples. Bootstrap values after 2000 replicates are shown if ≥ 35 %. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). b) a) 47 Figure 2.6 PCR amplification of SSR markers in selected Lavandula species and cultivars. a) LAL4 amplified from genomic DNAs of L. angustifolia (Lane 1-8) and L. x intermedia (Lane 9-15) cultivars, b) LAL4 amplified from genomic DNAs of eight Lavandula species and c) LAF5 amplified from genomic DNAs of eight Lavandula species. Lane descriptions a) M: 50 bp DNA ladder (NEB, Ipswich, MA), 1) L. a. cv Tucker's early purple, 2) L. a. cv Betty's Blue, 3) L. a. cv Folgate, 4) L. a. cv Sachet, 5) L. a. cv Sharon Robert, 6) L. a. cv Royal velvet, 7) L. a. Subsp. angustifolia, 8) L. a. cv Maillette, 9) L. x i. cv Hidcote Giant, 10) L. x i. cv Grosso, 11) L. x i. cv Provence, 12) L. x i. cv Supper, 13) L. x i. cv Fred Boutin, 14) L. x i. cv Seal and 15) L. x i. cv Abrialli; b and c) 50 bp DNA ladder (NEB, NE), 1) L. angustifolia, 2) L. latifolia, 3) L. x intermedia, 4) L. buchii, 5) L. dentata, 6) L. lusitanica, 7) L. x ginginsii and 8) L. stoechas. Polyacrylamide gel (6 %) stained with SYBR safe were used for resolution. (Reprinted by permission from Springer Nature: Planta 241(4): 987-1004, Identification, validation and cross-species transferability of novel Lavandula EST-SSRs by Ayelign M. Adal, Zerihun A. Demissie and Soheil S. Mahmoud, © 2014). 2.4 Discussion 2.4.1 Lavandula EST-SSRs identification and characterization Of the 39 species and their numerous inter-species and inter-section hybrids, L. angustifolia, L. latifolia and their natural hybrid L. x intermedia are the major sources of essential oils sold worldwide. The market value and bioactivities of these oils are mainly determined by their monoterpene profiles, which in turn depend on the species and/or cultivars used and a) b) c) 48 environmental conditions (Cavanagh and Wilkinson 2002). Thus, in order to ensure genetic purity of species or cultivars used, a marker that can transcend environmental effects and phenotypic overlaps among genetically related species is required. However, to our knowledge, such markers have not yet been reported in Lavandula. SSRs are polymorphic DNA sequences with proven potential to successfully distinguish both intra-species and inter-species diversities in both model and non-model plants (Varshney et al. 2005; Sharma et al. 2007). Given that genomic sequences have not yet been ready for Lavandula, we identified 3,459 EST-SSR motifs from 13,625 Lavandula unigenes (Lane et al. 2010; Demissie et al. 2012; Sarker et al. 2012), and validated 31 of these SSRs for their polymorphism and discrimination power among cultivars and species. Approximately 18.8 % of the unigenes contained at least one SSR locus. Although this ratio was higher compared to previous reports, such as 7 – 10 % in cereals (Varshney et al. 2002), 7.7 % in soybean (Xin et al. 2012), 9.3 % in sugarcane (Singh et al. 2013) etc, it was less than the 35 % ratio reported for citrus EST-SSR (Palmieri et al. 2007). The 2.81 kb per unigene SSR density obtained in this study was also higher than that reported in rice (3.4 kb), wheat (5.4 kb) and soybean (7.4 kb), but comparable with the 2.4 kb per locus density reported for D. versipellis (Guo et al. 2014). These variations in the frequency, distribution and abundance of SSRs identified from different species and databases are commonly associated with the use of different search criteria, size of the database and database-mining tools employed (Sharma et al. 2009). In fact, Peng and Lapitan (2005) identified 36,520 (7.41 %) EST-SSRs from a wheat database that contains 492,832 ESTs compared to the 22,290 ESTs (13,625 unigenes) we had in our library. Of the 1,812 EST-SSRs containing PFR, only category I EST-SSRs (252 of them or ~14 %) were considered for further characterization. This was because previous research had confirmed that the polymorphic capacity of SSR is directly proportional to its length (Temnykh et 49 al. 2001; Singh et al. 2009). More than 49 % of the category I EST-SSRs were dinucleotide motifs, which was contrary to the overall SSR motif distribution where trinucleotides were dominant. This result was obtained despite using 18 bp length as the minimum requirement for trinucleotide repeat classification under this category while that of dinucleotide was set at 20 bp. Nonetheless, similar types of results have been reported in other crops (Lagercrantz et al. 1993; Grover et al. 2007). Interestingly, only ~33 % of the dinucleotides were located in the CDS of the unigenes compared to ~81 % of the trinucleotides and 83 % of the hexanucleotides. These results were also consistent with previous reports. For example, only 19 % of the dinucleotides in wheat were located in CDS, while 74 % of their trinucleotides were located in CDS (Yu et al. 2004). This tendency of organisms to tolerate tri- and hexanucleotides in their translated genomic region as opposed to di-, tetra- or pentanucleotides likely stems from the fact that the later motifs are vulnerable for frame-shift mutations (Duran et al. 2009; Guichoux et al. 2011). It is also worth noting that EST-SSRs located in the 3’ UTR are generally less abundant across the three Lavandula unigene databases, 8.73 % in flower, and 1.98 % in leaf and oil gland. This was likely because our EST databases were developed by sequencing the 5’ end of our cDNA library (Lane et al. 2010; Demissie et al. 2012; Sarker et al. 2012), which favored the enrichment of the 5’ end sequences in the database. 2.4.2 EST-SSR polymorphism EST-SSR polymorphism is generally lower than that of genomic-SSRs. However, previous research has shown that EST-SSRs with longer repeat size had sufficient level of polymorphism to distinguish closely related species (Yu et al. 2000; Singh et al. 2009; Dutta et al. 2011). This is particularly true for plants with complex polyploidy structure where their genome complexity favors polymorphism for a given locus. For example, the number of alleles per locus in sugarcane, a complex polyploid plant, ranged from 2 – 22 alleles with 65.5 % polymorphism (Oliveira et al. 2009). In addition, Pinto et al. (2006) compared 51 EST-SSRs with 50 50 genomic-SSRs and found that their mean discrimination power among 18 sugarcane varieties was non-significant. The authors also reported that dendrograms developed using the two SSR types were in agreement with documented pedigree information. Most markers in Lavandula, like sugarcane, were multi-allelic (with 2 – 10 alleles per marker) and 85 % of them were polymorphic. The higher allele number recorded in sugarcane could be due to its superior complex polyploidy and genome size (10 Gb) (Souza et al. 2011) compared to the estimated 900 Mb size of L. x intermedia (Urwin et al. 2007; Urwin 2014) and 870 Mb size of L. angustifolia (Malli et al. 2019). However, the markers identified from Lavandula were more polymorphic likely due to the heterogeneity of source materials we used compared to the clonally propagated sugarcane varieties used by Pinto et al. (2006). All EST-SSRs, except three, also produced amplicons within the expected size ranges, implying that most markers and their primer binding sites were highly conserved between the two species. This was not surprising considering the high genetic similarity shared between L. angustifolia and L. x intermedia. Similar high levels of marker conservation with sporadic unexpected amplicon sizes have also been reported in other crops, for example in soybean and sugarcane (Pinto et al. 2004; Xin et al. 2012). The main reason for this variation is differences in number and sizes of introns among alleles (Varshney et al. 2006). One of the major advantages of EST-SSRs is their uniqueness in revealing alterations in expressed structural and regulatory genes. Thus, the fact that putative function could be assigned to 1,933 (83.3 %) of the ESTs containing SSRs prompted us to study the localization of highly polymorphic loci and correlate that with their assigned function. We were particularly interested in polymorphic loci located in CDS of genes whose alteration is likely to be accompanied by phenotypic consequences. Despite the fact that SSRs located in CDS are less polymorphic than those in UTRs (Li et al. 2004; Dutta et al. 2011) and EST-SSRs of closely related species are mostly conserved owing to their genic nature (Li et al. 2004), polymorphic 51 loci located in CDS of functionally important lavender genes were identified. For instance, the locus LAF20 – with PIC and D values of 0.37 and 0.77 in L. angustifolia and 0.37 and 0.72 in L. x intermedia, respectively – was located within the coding region of a RHOMBOID-like serine protease homolog. The locus LAF18 – with PIC and D values of 0.26 and 0.36 in L. angustifolia and 0.32 and 0.50 in L. x intermedia, respectively – was located in the coding region of an ABA-inducible bHLH-type transcription factor homolog. These proteins play a key role in plant development and ecological interactions. Plant RHOMBOID-like serine proteases are believed to play a role in correct root growth, floral development, fertility and photoprotection (Thompson et al. 2012), while bHLH-type transcription factors are involved in stress-induced signal transduction pathway regulation (Nakata et al. 2013). A polymorphic locus LINT12 was also located in the CDS of a gene our group previously characterized as 1,8-cineole synthase, an enzyme catalyzing the synthesis of one of the major Lavandula EO constituents (Demissie et al. 2012). This result was not surprising as we previously reported slight variations among the genomic sequences of 1,8-cineole synthases of L. x intermedia, L. angustifolia and L. latifolia (Demissie et al. 2012). In addition, most of the markers that produced the highest heterozygous alleles, with greater discrimination and resolving powers in L. angustifolia and L. x intermedia were located in CDS of pollen-specific proteins, filament-like plant proteins and other hypothetical proteins. This implies the potential use of EST-SSR markers in gaining insights into functional diversification, including alterations in the monoterpene profile, in Lavandula. 2.4.3 Cross-species transferability and application in genetic relationship analysis Prior sequence information (genomic or ESTs) is a prerequisite for SSR marker development. However, since flanking sequences of SSRs are highly conserved in related species – sometimes even across genera – SSR primers obtained from well-studied species are often successfully used to cross amplify polymorphic loci from non-model species (Gupta and Varshney 2000; Pierantoni et al. 2004; Varshney et al. 2005). For example, Singh et al. (2013) 52 reported 87 – 93 % inter-species and 80 – 87 % inter-genera cross transferability for sugarcane EST-SSRs. A similar trend has been reported for other EST-SSRs as well (Sharma et al. 2009; Gong and Deng 2010; Dutta et al. 2011). In this study, L. angustifolia EST-SSRs showed 100 % transferability and 85 % polymorphism in L. x intermedia while those from L. x intermedia showed 100 % transferability and 78 % polymorphism in L. angustifolia. Therefore, we decided to test the cross-species transferability of EST-SSRs in six other Lavandula species that have different levels of genetic relationships with the donor species. Owing to the fact that L. latifolia belongs to the same section with the donor species (Upson and Andrew 2004), it was expected to find 100 % transferability. The transferability rate recorded for other species (50 – 83.3 %) was also consistent with the genetic distance of the species from the donor species. For instance, 50 % of the loci were amplified in L. buchii that shared only 11 % similarity with L. angustifolia and L. x intermedia. However, even this lowest transferability rate (50 %) is much higher than the only other SSR marker transferability study reported for Lavandula to date (Karaca et al. 2013). In fact, Karaca et al. (2013) tested cross-genera transferability of EST-SSRs originated from Salvia officinalis on L. x intermedia and found that only 0.1 % of these markers were transferable. Although the bulk of lavender oils sold worldwide are derived from L. angustifolia and L. x intermedia, their market value and application is largely determined by the species/cultivars used. Hence, obtaining a marker that has the power to discriminate these two species and their cultivars is of paramount interest both from a quality maintenance point of view and genetic diversity studies aimed at improving the quality and quantity of oil production. As presented in Figure 2.5, the loci reported here delineated the two species and their cultivars into their respective group in separate clades. The markers were also able to discriminate all the cultivars from each other; the maximum similarity level recorded among all cultivars used in this study was the 74 % similarity level between L. angustifolia cv. Folgate and Sachet. This together with 53 some loci produced novel fingerprints with high marker efficiency imply the likely chance of identifying species / cultivar-specific markers through further detailed analysis of the markers reported here and by validating more EST-SSRs identified from our EST libraries. 54 Chapter 3: Cloning and functional characterization of short-chain trans-isoprenyl diphosphate synthases involved in the biosynthesis of precursors for monoterpenes in lavender (Lavandula) 3.1 Overview Lavenders produce essential oils (EOs) composed of terpenoids, mainly monoterpenes and a lesser amount of sesquiterpenes. Terpenoid precursors are formed from isoprene units by isoprenyl diphosphate synthases (IDSs) and used as substrates for terpene synthases (TPSs). The level of terpenoids in the EOs can be regulated by TPS expression and by precursor partitioning. Previous studies have mainly focused on isolation and functional characterization of genes encoding TPSs, and on the only short-chain cis-IDS that was involved in irregular monoterpene precursor (C10) biosynthesis in Lavandula. However, the biosynthesis of major terpenoid precursors and their regulatory roles in the levels of terpenoids have not been determined. Here, this chapter aims to clone and functionally characterize genes encoding short-chain trans-IDSs involved in the biosynthesis of regular monoterpene precursors (C10) in Lavandula. Using EST and transcriptome databases of Lavandula, we identified and cloned cDNAs corresponding to the genes encoding geranyl diphosphate synthase (LiGPPS) large and small subunits, geranylgeranyl diphosphate synthase (LiGGPPS) and farnesyl diphosphate synthase (LiFPPS). The constructs of these genes were (co-)expressed in E. coli and the recombinant proteins were (co-)purified with nickel–nitrilotriacetic acid (Ni-NTA) agarose affinity chromatography. The recombinant protein of each GPPS subunit by itself was inactive as GPPS when assayed with substrates (IPP and DMAPP). Co-purified recombinant proteins of LiGPPS large subunit and small subunit 1 (LiGPPS.LSU and LiGPPS.SSU1) or LiGGPPS and LiGPPS.SSU1 from E. coli were active as heteromeric GPPS to produce GPP from the same substrates. When co-expressing with LiGGPPS in E. coli and overexpressing in N. benthamiana, LiGPPS.SSU1 served as a 'modifier' (not as an 'accelerator') of GGPPS activity 55 into GPPS. The transcript levels of large and small subunits of GPPS corresponded to the levels of total monoterpenes in EO-producing tissues, with more transcripts and monoterpenes in flowers than in leaves. The recombinant protein of LiFPPS catalyzed IPP with DMAPP, or IPP with GPP, to produce FPP. The levels of LiFPPS transcripts also corresponded to the amount of total sesquiterpenes produced in both tissues examined, with more transcripts and sesquiterpene accumulation in leaves than those in flowers. Taken together, identified heteromeric LiGPPS and homomeric LiFPPS controlled the biosynthesis of monoterpene and sesquiterpene precursors, respectively. 3.2 Materials and Methods 3.2.1 Plant growth conditions and chemicals L. x intermedia (cv. Grosso) (wild-type) and Nicotiana benthamiana (wild-type and transgenic) plants were grown in potting Pro-Mix high porosity (HP) soil in a growth room under 16 h light /8 h dark cycles at 25 °C. The light intensity was controlled at approximately 200 μmol m-1 s-1 of light using cool-white fluorescent bulbs. Irrigation was applied once every other day. Authentic standards of isoprenoids (e.g., geraniol, farnesol) were obtained from Sigma (http://www.sigmaaldrich.com/), while prenyl pyrophosphates (precursors), including isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), neryl pyrophosphate (NPP) and farnesyl pyrophosphate (FPP) were purchased from Echelon Biosciences (http://www.echelon-inc.com/). Unless otherwise mentioned, all other assay chemicals were obtained from Sigma. 3.2.2 Analysis of trans-IDS sequences The candidate short-chain trans-IDSs were identified from Lavandula ESTs and transcriptome databases using previously known short-chain trans-IDSs from different plants, including Mentha piperita, Antirrhinum majus, Catharanthus roseus and Humulus lupulus (Burke 56 et al. 1999; Tholl et al. 2004; Wang and Dixon 2009; Rai et al. 2013). The relative frequency of each amino acid in the key conserved motifs was determined with Weblogo, a sequence logo web server (Crooks et al. 2004). The full-length amino acid sequences encoding these candidate genes were aligned by using ClustalW, and a maximum likelihood phylogenetic tree was constructed by using MEGA7 (Kumar et al. 2016) with default settings. Multiple sequence alignments of each gene subfamily along with closely related genes from other plants were made using T-Coffee (http://tcoffee.crg.cat/apps/tcoffee/do:regular). BOXSHADE 3.21 (www.ch.embnet.org/software/BOX_form.html) was used for shading amino acid sequence alignments. The GenBank accession numbers of the proteins used in the phylogenetic analysis, including proteins used in this study are listed in Table D.1. 3.2.3 Subcellular localization studies in N. benthamiana protoplasts The open reading frames (ORFs) for candidate trans-IDSs were analyzed for the presence of potential transit peptides using different bioinformatics tools: TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/), ChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/), PWoLF PSORT (https://wolfpsort.hgc.jp/), SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and iPSORT (http://ipsort.hgc.jp/). To determine their localization experimentally, we used a p326::sGFP construct (contains a synthetic green fluorescent protein (GFP)) (Figure E.1) that was a gift from Dr I. Hwang, Pohang University of Science and Technology, Korea. The full-length ORF cDNAs encoding proteins of LiGPPS subunits and LiGGPPS were fused at the 5'-end of GFP in this vector for expression of GFP reporter construct in N. benthamiana protoplasts. The resulting positive clones carrying constructs – designated as LiGPPS.SSU1::GFP, LiGPPS.SSU2::GFP, LiGPPS.LSU::GFP and LiGGPPS::GFP – were confirmed by restriction digestion and Sanger sequencing. The cloning primers used in this study are listed in Table 3.1. 57 Nicotiana benthamiana protoplasts were isolated from young leaves of one to two week old seedlings following a modified version of the procedure described by Yoo et al., (2007). In brief, young leaves cut into approximately 0.5 mm strips were soaked in the freshly prepared enzyme solution (1 % cellulase R10 (Gold Biotechnology), 0.1 % macerozyme R10 (Gold Biotechnology), 0.25 mM polyvinylpyrrolidone (PVP)-10, 0.5 M sucrose and 4 mM CaCl2, pH 5.7) and incubated overnight in the dark at room temperature. After very gently swirling, the protoplast solution was filtered and mixed with a one-tenth volume of W5 solution (6 mM glucose, 150 mM NaCl, 125 mM CaCl2 and 5 mM KCl, pH 5.8-6). After centrifuging at 1494 rpm for 10 min, the protoplasts (in the top layer) were resuspended in fresh W5 solution to remove the remaining cellulase and spun as described above. Pelleted protoplasts were again resuspended with ~5 mL of W5 solution, incubated at 4 oC for 30 min and counted using a hematocytometer (VWR). For polyethylene glycol (PEG)-mediated transfection, protoplasts were spun as described above, and pellets were resuspended in MaCa solution (0.5 M mannitol, 0.02 M CaCl2 and 0.1 % MES, pH 7.5) to obtain a final concentration of ~ 4-5 x 106 protoplasts mL-1. Ten microlitres of plasmid DNA (1 μg μL-1) were mixed with 300 μL of ice-cold protoplasts, followed by addition of 300 μL of freshly prepared PEG solution (40 % PEG4000 and 10 % DMSO). The transfection mixture was incubated at 4 oC for 15 min in the dark after adding Minimum solution (0.7 M mannitol, 10 mM CaCl2, 1 mM KNO3, 1 mM MgSO4 x 7H2O, 0.2 mM KH2PO4) to recover protoplasts. Protoplasts were pelleted as described above and resuspended with 4 mL of Minimum solution. Finally, the protoplasts were cultured at room temperature for 24 h in the dark and transiently expressed GFP-fused proteins were visualized using an inverted fluorescence microscope (Zeiss Axiovert 200M). GFP fluorescent signals were visualized with excitation and emission wavelengths of 488 nm and 509 nm, respectively. Chlorophyll 58 autofluorescence (red signal) was observed using an excitation of 488 nm and an emission of 650 nm. The empty vector (p326::sGFP) was used as a negative control. 3.2.4 Cloning, expression and recombinant protein purification For enzymatic assays, ORFs of LiGPPS.LSU and LiGGPPS genes without transit peptides were cloned upstream and in-frame of the His-tag sequences in pET32a (containing ampicillin resistant marker gene; Figure E.2) (Novagen) to express tagged LiGPPS.LSU and LiGGPPS recombinant proteins. The resulting constructs were designated as pET32a::LiGPPS.LSU-His and pET32a::LiGGPPS-His. LiGPPS.SSU1 and LiGPPS.SSU2 cDNAs, lacking the putative transit peptides, were also cloned upstream and in-frame of the His-tag sequences in pET41b(+) (containing Kanamycin resistant marker gene; Figure E.1) (Novagen) to express tagged LiGPPS.SSU1 and LiGPPS.SSU2 recombinant proteins. The constructs were hereafter designated as pET41b::LiGPPS.SSU1-His and pET41b::LiGPPS.SSU2-His. For co-expression with tagged GPPS.SSUs (pET41b::LiGPPS.SSU1-His and pET41b::LiGPPS.SSU2-His), truncated LiGPPS.LSU and LiGGPPS with their stop codons were cloned into pET32a to generate untagged constructs, pET32a::LiGPPS.LSU and pET32a::LiGGPPS. In addition, the full-length of LiFPPS ORF was cloned upstream and in-frame of the His-tag sequences in a pET41b+ vector, yielding pET41b::LiFPPS-His. Positive clones carrying target constructs were selected and confirmed by restriction digestion and Sanger sequencing. Cloning restriction sites and primers used in this Chapter are mentioned in Table 3.2. The resulting tagged constructs were transformed into E. coli Rosetta (DE3)pLysS cells (Appendix F) (Novagen) individually, and two constructs (tagged and untagged) were co-transformed together in a single transformation event. A single positive colony (after restriction enzyme digestion and PCR detection) was grown in Luria-Bertani (LB) medium supplemented with 34 mg L-1 of chloramphenicol and 30 mg L-1 of kanamycin or 100 mg L-1 of ampicillin or 59 both at 37 °C to an optical density at 600 nm (OD600) of 0.5-0.6, followed by induction with 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG), and then grown overnight at 18 °C. Induced cells were harvested by centrifugation and frozen overnight at -80 °C, and the recombinant His-tagged proteins were then purified using PerfectPro nickel-nitrilotriacetic acid (Ni-NTA) Agarose chromatography (5Prime GmbH, Germany) according to the manufacturer’s instruction. The protein concentration was determined through the Bradford assay (Bio-Rad). 3.2.5 Enzymatic assay and product identification To determine the in vitro activity of Lavandula short-chain trans-IDSs using gas chromatography-mass spectrometry (GC-MS), the enzymatic activity assays were performed in a final volume of 250 μL containing assay buffer (25 mM MOPSO, pH 7.0, 10 % (v/v) glycerol, 2 mM DTT, and 10 mM MgCl2), 40 μM IPP and 40 μM co-substrate (DMAPP or GPP) and 25 μg of partially purified recombinant proteins. The assay mixtures were incubated for 3 h at 30 °C, followed by addition of calf intestine alkaline phosphatase (CIP) (New England Biolabs) (10 U) to dephosphorylate the products, and was overlaid with 400 μl of pentane and incubated at 30 °C overnight. The assays were frozen at -80 oC, and then the products were extracted with pentane according to previous reports (Demissie et al. 2013). As a control, heat-denatured purified proteins and purified proteins from E. coli lysate cells harboring empty pET41b(+) vector were assayed with substrates under similar conditions. Dephosphorylated assay products (the nearest prenyl alcohols) and authentic standards were identified by gas chromatography-mass spectrometry (GC-MS) using a Varian GC 3800 Gas Chromatographer (Varian Inc, USA) coupled with a Saturn 2200 Ion Trap mass detector following the methods previously described (Demissie et al. 2011; Demissie et al. 2013). In brief, the samples (1- 2 µl volume) were injected with the cold on-column mode, at which the injector temperature was set to 40 °C. The oven temperature was set at 40 °C for 3 min, and 60 increased to 130 °C at a rate of 10 °C min-1 and held for 2 min. The temperature was further ramped to 230 °C at a rate of 50 °C min-1 and held for 8 min. The flow rate of the carrier gas (helium) was set to 1 mL min-1. The product identity was confirmed by comparing their retention times and mass spectrum to that of corresponding authentic standard. 3.2.6 Bacterial genetic complementation assay The construct pACCAR25ΔcrtE (Kainou et al. 1999) contains all gene clusters encoding carotenoid biosynthesis enzymes, except crtE gene encoding GGPPS. The constructs described in this Chapter section 3.2.4 were also used here for GGPPS complement by co-expression with pACCAR25ΔcrtE in E. coli BL21(DE3) cells (Novagen). A pair of constructs, such as [pET41b::LiGPPS.SSU1 and pET32a::LiGPPS.LSU], [pET41b::LiGPPS.SSU1 and pET32a::LiGGPPS], [pET41b::LiGPPS.SSU2 and pET32a::LiGPPS.LSU], [pET41b::LiGPPS.SSU1 and pET32a:LiGGPPS], and [pET41b::LiGPPS.SSU1 and pET32a::LiGPPS.SSU2] were co-transformed together with pACCAR25ΔcrtE into E. coli BL21(DE3) cells to assess heteromeric GGPPS activity. Positive co-transformed colonies were inoculated in 5 mL of LB medium containing 34 μg mL-1 of chloramphenicol and 100 μg mL-1 of ampicillin or 30 μg mL-1 of kanamycin for the co-expression of each construct with pACCAR25ΔcrtE, and the three antibiotics for the co-expression of two constructs with pACCAR25ΔcrtE. The overnight culture (100 μL) was transferred to 50 mL of the same medium and continued to grow at 37 °C to reach an OD600 of 0.3. The cells were then induced by supplementing 0.2 mM IPTG and grown overnight at 18 °C. Bacterial cells were harvested by centrifugation, and pelleted cells were used for comparison of different constructs. For carotenoid quantification, the same wet weight pellet was re-suspended in 80 % (v/v) acetone to extract the pigments and the absorbance at 450 nm was measured using Ultrospec 2100 pro UV/ Visible Spectrophotometer (Biochrome Ltd, Cambridge, UK), as previously described 61 (Chang et al. 2010). Human GGPPS (Kainou et al. 1999) and the pET41b+ empty vector were used as positive and negative controls, respectively. 3.2.7 Generation of binary vectors and Agrobacterium preparation For overexpression studies in N. benthamiana, the full-length of LiGPPS.SSU1 or LiGPPS.SSU2 ORF was cloned into the pGAdekG/NIb.L vector that was derived from pGA482 (named hereafter as pGA) carrying the cauliflower mosaic virus (CaMV 35S) promoter, neomycin phosphotransferase (NPTII; kanamycin resistance gene), and NOS (nopaline synthase) terminator (An 1986) between the EcorI and KpnI sites (see vector map in Figure E.4). Primers used for cloning are detailed in Table 3.1. The resulting plasmids were transformed into Agrobacterium tumefaciens strain GV3101 (Appendix F) using the freeze-thaw method (Jyothishwaran et al. 2007) and plated on LB agar medium supplemented with 50 mg L-1 of kanamycin and 25 mg L-1 of rifampicin. After confirming by PCR, the positive colony was grown in 5 mL of LB with antibiotics as above at 28 oC shaking at 200 rpm overnight in dark. Then, 1 mL of overnight culture was transferred into fresh 100 mL of LB with the same antibiotics and grown at 28 oC overnight at 100 rpm. The next day, OD600 was measured using Ultrospec 2100 pro UV/ Visible Spectrophotometer, and when OD600 reached to ~0.4-0.5, the cells were spun at 3000 rpm for 10 min and resuspended with equal volume of co-cultivation medium (Appendix G) supplemented with 100 µM acetosyringone (PhytoTechnology laboratories) and incubated at 28 oC shaking at 50 rpm for 3 h. Then, the cells were spun again as above and resuspended with 5-10 mL of co-cultivation medium for leaf disc infiltration. 3.2.8 N. benthamiana leaf transformation and regeneration For leaf disc transformation, young green leaves of N. benthamiana were washed with running water and soap, and then soaked in 10 % (v/v) commercial bleach with a single drop of Triton X-100 for 10 min. The tissues were washed with sterile water three times for 5 min each. 62 Sterilized leaves were cut into ~1 cm2 using a surgical blade and kept in co-cultivation liquid medium until Agrobacterium infiltration. Leaf discs were transformed by A. tumefaciens GV3101 carrying pGA::LiGPPS.SSUs or pGA::GUS (control) as previously described (Duan et al. 2016). The overview of regeneration of independent plants from leaf disc, followed by rooting and hardening is shown in Figure 3.1. Briefly, leaf discs were soaked in Agrobacterium solution with gentle shaking for 5 min and transferred to sterile Whatman filter paper for drying. The dried leaf discs were then transferred to co-cultivation solid medium, and the cultures were wrapped with aluminum foil and incubated at 28 oC. After four days of co-cultivation, the leaf discs with overgrown bacteria were washed with sterile water with 500 mg L-1 of TIMENTIN® (ticarcillin disodium and clavulanate potassium) (Gold Biotechnology), followed by air drying on sterile filter paper. Then, the discs were transferred to regeneration medium with antibiotics (200 mg L-1 of cefotaxime + 125 mg L-1 of TIMENTIN to kill the remaining Agrobacterium, and 100 mg L-1 of kanamycin for transgenic plant selection). The cultures were then incubated in a growth chamber in the dark for a week and moved to light conditions until new shoots generated. The new shoots were detached and transferred to rooting medium supplemented with antibiotics. Well-rooted plants were transferred to pots for further PCR detection using LiGPPS.SSUs and NPTII -specific primers, and then to produce T0 seeds. All regeneration and rooting media compositions are described in Appendix G. The positive T0 and T1 seedlings were grown on Murashige and Skoog (MS) medium containing 3 g L-1 of gellan gum (PhytoTechnology Laboratories) and 200 mg L-1 of kanamycin (PhytoTechnology Laboratories) for selection. Wild-type N. benthamiana plants were used as a control for selection. All cultures were grown at 25 ̊C under a 16 /8 h day/night photoperiod with cool white fluorescent bulbs. Selected T1 N. benthamiana transgenic plants were grown in pots in a growth room for further analysis. 63 Figure 3.1 An overview of shoot regeneration from leaf discs N. benthamiana. The leaf discs were infiltrated and co-cultivated with Agrobacterium tumefaciens carrying GPPS.SSU constructs, followed by shoot regeneration, rooting and hardening of regenerated plants. A. tumefaciens carrying GUS constract was used for regenerating control transgenic plants. Images © Ayelign M. Adal. 3.2.9 RNA isolation and qPCR analysis Young leaves from sample plants were collected and flash-frozen in liquid nitrogen and stored at -80 °C until use. Total RNA was extracted from the leaf samples using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. The total RNA (1 μg) was then reverse transcribed with oligo d(T) and random hexamer primers and M-MuLV Reverse Transcriptase (New England Biolabs) following the manufacturer's protocol. The quantitative PCR (qPCR) was performed using SYBR Premix (Applied Bioscience) in the StepOne Plus Real-Time PCR system (Applied Bioscience), according to the manufacturer’s instructions. Primer sets used in qPCR are listed in Table 3.1. qPCR was run with a final reaction volume of 10 µL, consisting of 5 µL of SYBR premix, 0.6 µM of each primer and ~150 ng of cDNA 64 template. The amplification conditions were set with initial stage at 50 °C for 2 min, and 95 °C for 2 min, followed by 50 cycles at 95 °C for 3 s and 60 °C for 30 s, as well as a final melting curve stage at 95 °C for 15 s and 60 °C for 1 min. For relative quantification among EO-producing tissues of L. x intermedia, the primer efficiency of every primer set used in this study was calculated in every qPCR run using LinRegPCR (Ruijter et al. 2009). The generated data were analyzed using the Livak method (Livak and Schmittgen 2001), expressed as 2−ΔΔCT of a gene of interest. β-actin was used as a reference gene. For absolute quantification in N. benthamiana transgenic plants, LiGPPS.SSU1 transcripts were determined based on standard curve derived from a known PCR product of LiGPPS.SSU1 amplified from a plasmid carrying full-length cDNA of LiGPPS.SSU1 gene. Standard samples were run along with unknown samples in every qPCR run to construct the standard curve under the same conditions. Each data point represents three independent biological samples, with three technical replicates. Table 3.1 List of primers used in this study. Genes Primer name sequences (5' ==> 3') Vectors For cloning: LiGPPS.SSU1 gSSU1_NdeI_F ATCTATCATATGGCCCTCAATCTCTCT pET41b(+) with His-tag gSSU1_EcoRI_R ATTATAGAATTCCAAGCCGGACAAAGG LiGPPS.SSU2 gSSU2_NdeI_F TTATATCATATGCGCCGCACAATCCG pET41b(+) with His-tag gSSU2_EcoRI_R ACTATAGAATTCGACCTCTCAGCAGCA LiGPPS.LSU glsu_NdeI_F1 TATGGCACTTCTGATCAAGGAGGA pET32a(+) with His-tag glsu_NdeI_F2 TGGCACTTCTGATCAAGGAGGA gpps_lsu_KpnI_R2 TACTAAGGTACCGAATTATCTCTGTGGG glsu_NdeI_F1 TATGGCACTTCTGATCAAGGAGGA pET32a(+) without His-tag glsu_NdeI_F2 TGGCACTTCTGATCAAGGAGGA glsu_stopKpnI_R2 TACTGAGGTACCTCAATTATCTCTGTGG LiGGPPS ggpps_NdeI_F TATATCATATGGTCCTGACCGGCGAG pET32a(+) with His-tag ggpps_SacI_R TATATGAGCTCGAGTTCTGCCGGTGAG 65 Genes Primer name sequences (5' ==> 3') Vectors ggpps_NdeI_F TATATCATATGGTCCTGACCGGCGAG pET32a(+) without His-tag ggpps_SacIstop_R TATATGAGCTCTTAGTTCTGCCGGTGAG LiFPPS fpps_NdeI_F AAGTATCATATGGCGAATATGAACGGA pET41b(+) with His-tag fpps_KpnI_R CTGTATGGTACCTTTCTGCCTCTTGTA For subcellular localization: LiGPPS.SSU1 gssu1_XhoI_F CATCTCGAGATGGCCCTCAATCTC p326::sGFP gssu1_KpnI_R TATAGGTACCAGCCGGACAAAGGAT LiGPPS.SSU2 gssu2_XhoI_F TATCTCGAGATGGTATTCGCTCCAGT p326::sGFP gssu2_KpnI_R ATAGGTACCCCTATCAGCAGCATAATC LiGPPS.LSU glsu_XhoI_F CGTCTCGAGATGAGTATCCTAATTGCC p326::sGFP glsu_KpnI_R ATAGGTACCATTATCTCTGTGGGCAATG LiGGPPS ggpps_XhoI_F CGTCTCGAGATGAGGTCTATGAATCTG p326::sGFP ggpps_KpnI_R ATATAGGTACCGTTCTGCCGGTGAG For N. benthamiana transformation and PCR detection: LiGPPS.SSU1 gssu1_EcoRI_F CATGAATTCATGGCCCTCAATCTC pGA248 gssu1_KpnI_R ATAGGTACCTCAAGCCGGACAAAG LiGPPS.SSU2 gssu2_EcoRI_F TTATGAATTCATGGTATTCGCTCCAGT pGA248 gssu2_KpnI_R ATAGGTACCTTACCTATCAGCAGCAT NPTII (KanR) Nptii_c_F AGAGGCTATTCGGCTATGACTG Nptii_c_R TCAGAAGAACTCGTCAAGAAGGC For qPCR analysis: LiGPPS.SSU1 gssu1_F TCGCAGGGATTGGTAGAT gssu1_R CGTGCATCTCACCGTATTT LiGPPS.SSU2 gssu2_F GACGTATTGGACGTCTCTTATT gssu2_R AGAATAGCGCATAGCTTCATAG LiGPPS.LSU glsu_F CGATCCAGATCCAGTTTCTTC glsu_R TCGGATTGTCCTCCTTGAT LiGGPPS ggpps_F GGTCTATGAATCTGGGTGTTC ggpps_R GATGGGCTTCCGTGATTT LiFPPS fpps_F CCACTACGGAAAGGAAGATAAGG fpps_R CGTAGCTCTTGCTCTCAAACT 66 Genes Primer name sequences (5' ==> 3') Vectors ß-actin actin_F TGTGGATTGCCAAGGCAGAGT actin_R AATGAGCAGGCAGCAACAGCA Note: Underlined sequences indicate the respective restriction site. 3.2.10 Analysis of pigments, GPPS activity and phenotypes in transgenic plants To determine the contents of GGPP-derived terpenoids in transgenic N. benthamiana plants expressing LiGPPS.SSU1, 1 g of leaf tissue was frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle. After homogenizing, the pigments were then extracted from 25 mg leaf powder with 1 mL of 80 % acetone overnight at 4oC. After centrifugation, the absorbance values of the supernatant were measured at 470, 646 and 663 nm using an Ultrospec 2100 pro UV/ Visible Spectrophotometer as previously described (Lichtenthaler and Wellburn 1976; Brix 1987). The resulting absorbance values were used to calculate the concentration of total carotenoids, chlorophyll a (Ch a), chlorophyll b (Ch b) and total chlorophylls (Ch a + Ch b) in µg per mg of plant extracts using the following formula (Lichtenthaler and Wellburn 1976).  Ch a = (12.21*A663) - (2.81*A646)  Ch b= (20.13*A646) - (5.03*A663)  Cx+c= [(1000*A470) - (3.27*Ch a) - (104*Ch b)]/229 Where, Ch a = chlorophyll a; Ch b = chlorophyll b; Cx+c = total carotenoids; Ch a +Ch b = total chlorophylls. The total soluble protein was extracted from N. benthamiana leaves expressing LiGPPS.SSU1 according to protocols described previously (Orlova et al. 2009; Yin et al. 2016). Briefly, 1 g of leaf sample was ground in liquid N2 using mortar and pestle and transferred to a 15-mL tube. One millilitre of extraction buffer (25 mM MOPSO, pH 7.0, 10 mM MgCl2, 1 mM Dithiothreitol (DTT), 1 % (w/v) polyvinylpyrrolidone (PVP‐40), 1 mM methanesulfonyl fluoride 67 and 10 % (v/v) glycerol) was added to these tubes and mixed completely by vortexing. After centrifugation at 4000 rpm for 10 min at 4 oC, the supernatant was transferred to a 2-mL centrifuge tube, followed by centrifugation at 12,000 rpm for 10 min under the same conditions. The supernatant (total soluble protein) was then transferred into a new 1.5‐mL tube and the concentration was quantified through Bradford assay. Using 50 μg of the total soluble protein isolated from samples of each transgenic line, enzymatic activity assays were performed and the resulting products were detected based on the procedures described in this Chapter section 3.2.5. Plant growth patterns in terms of plant height (cm), total leaf number, leaf area (cm2) and days to flowering were compared between transgenic (LiGPPS.SSU1) and control (GUS) plants. The first three growth parameters were measured at 45 days after planting, while the flowering days were recorded until all plants produced flowers. Plant height was a measure of the total above-ground height of the plants, while the leaf area was derived from the width and length of leaves that were randomly selected between the 3rd and 5th pair of leaves from the bottom. Two leaves per sample plant were measured. For all statistical analysis, we employed the Student's t-test with a significant difference at P < 0.05 and P < 0.01 using SigmaPlot v 12.5 (Systat Software, Germany) to determine statistical significance between transgenic and control plants. 3.3 Results 3.3.1 Identification of cDNAs encoding short-chain trans-IDSs To identify short-chain trans-IDSs involved in Lavandula terpenoid biosynthesis, a homology search for candidate GPPS, FPPS and GGPPS sequences was carried out against Lavandula EST (Lane et al. 2010; Demissie et al. 2012) and transcriptome databases (Adal et al. 2019) using previously reported related sequences from M. piperita and A. thaliana (Burke et 68 al. 1999; Chen et al. 2015). The full-length cDNA sequences of GPPS.SSU1 and FPPS were identified from L. x intermedia EST databases that are hereafter referred to as LiGPPS.SSU1 and LiFPPS. A total of 10 ESTs represented FPPS, whereas two ESTs were detected for LiGPPS.SSU1. However, a thorough search for sequences similar to GPPS.LSU, GGPPS, and GPPS.SSU2 in EST databases was not successful. A homology blast against the Lavandula transcriptome database resulted in sequences similar to GPPS.LSU, GGPPS, and GPPS.SSU2. Amino acid sequences of candidate trans-IDSs were compared to homologs from different plants within a short-chain trans-IDS subfamily. The results showed that LiGPPS.SSU1 had the highest amino acid identity to GPPS.SSU1 of M. piperita (67.35 %), A. majus (56.84 %) and C. roseus (54.86 %) (Figure H.1). The candidate LiGPPS.SSU2 was 69.81 and 54.11 % homologous to A. thaliana GPPS.SSU2 and O. sativa GGPPS recruited protein, respectively (Figure H.2). LiGPPS.LSU had over 71 % amino acid identity with GGPPS from L. x intermedia, and 62.2 - 65.2 % with previously reported GPPS.LSUs from M. piperita, A. majus, C. roseus and H. lupulus (Figure H.3). In addition, LiFPPS shared 69 - 92 % amino acid identity with FPPSs from different plants, for example, S. miltiorrhiza (92 %) and A. thaliana (78 %) (Figure H.4). Multiple sequence alignment analysis revealed the presence of most catalytically important conserved motifs in most of the identified sequences (Table 3.2 and Figure 3.2). The two common aspartate-rich motifs, the first (DD(x)2-4D) and the second (DD(x)2D), are present in sequences of LiGPPS.LSU, LiGGPPS, LiGPPS.SSU2, and LiFPPS, whereas both motifs are absent in LiGPPS.SSU1. In addition, the two conserved cysteine-rich (CxxxC) motifs are in LiGPPS.SSU1 & LiGPPS.SSU2, and LiGPPS.LSU & LiGGPPS have one such conserved motif. LiFPPS lacks any of these cysteine-rich motifs. 69 Table 3.2 The Lavandula short-chain trans-isoprenyl diphosphate synthases (IDSs) with their conserved amino acid motifs. Gene name Isomer type Amino acids Mw (kDa) pI 'CxxxC' Aspartate motifs First Second FARM SARM GPPS.SSU1 trans 295 32.35 6.80 + + - - GPPS.SSU2 trans 323 35.46 5.73 + + + + GPPS.LSU trans 353 37.85 5.61 + - + + GGPPS trans 352 37.95 5.69 + - + + FPPS trans 349 40.14 5.43 - - + + 'trans' – the carbon chain of products generated by IDSs contain the functional groups on 'opposing sides'. 'short chain' refers to the C10 - C25 products generated by IDSs. Mw – predicted average molecular weight of the full-llength translated; pI (isoelectric point)– the calculated average net charge of the entire protein peptides (as function of the pH). 'CxxxC' – cysteine-rich motif functions in physical interaction of subunits. 'FARM' – first aspartate rich motif, and 'SARM' – second aspartate-rich motif, both of which have roles in substrate binding and catalysis. '+': present; '–': absent. Figure 3.2 Conserved motifs in L. x intermedia GPPS subunits, GGPPS and FPPS. a) IDSs with and without transit peptide, and the key conserved motif residues, b) WebLogos showing the relative frequency of the conserved motif residues found in each subfamily of short-chain trans-IDS. The first and second aspartate-rich motifs are designated as FARM and SARM, respectively. 70 3.3.2 Phylogenetic relationship of short-chain trans-IDSs To determine the phylogenetic relationships among identified L. x intermedia and other selected plant short-chain trans-IDSs, we constructed a maximum likelihood phylogenetic tree based on their amino acid sequences. The resulting tree clustered the short-chain trans-IDSs into recently described catalytically distinct subfamilies (Jia and Chen 2015); trans-IDS-a (FPPS), trans-IDS-d (GPPS.LSU/GGPPS) and trans-IDS-e (GPPS.SSU1 and GPPS.SSU2) (Figure 3.3). Each identified L. x intermedia IDS was placed within one of these subfamilies, except LiGPPS.LSU and LiGGPPS, which were clustered together under the same trans-IDS-d subfamily. LiGPPS.LSU and LiGGPPS were closely related, with high amino acid identity as compared to the rest of IDS found in L. x intermedia, and more evolutionarily close to GPPS.LSU/GGPPS from H. lupulus. Although GPPS small subunits have been grouped under the same subfamily (trans-IDS-e), GPPS.SSU1 and candidate GPPS.SSU2 sequences were separately placed into GPPS.SSU1 and GPPS.SSU2 clades. 71 Figure 3.3 A phylogenetic tree of the short-chain IDSs from Lavandula and other selected plants based on their amino acid sequences. Scale bar indicates the number of amino acid substitutions per site. The GenBank accession numbers of these genes used here are listed in Table D.1. 72 3.3.3 Subcellular localization studies Analysis of amino acid sequences of identified LiGPPS subunits using in silico subcellular localization prediction programs predicted that four proteins are localized in plastids, and one in the cytosol. For those proteins predicted to be localized in plastids, we further determined their subcellular localization experimentally. The full-length ORFs of these genes were fused to the 5'-end of green fluorescent protein (GFP) gene and transiently expressed in N. benthamiana leaf protoplasts. The signal of GFP was detected in chloroplasts for all of the tested proteins encoded by LiGPPS.LSU, LiGPPS.SSU1, LiGPPS.SSU2 and LiGGPPS (Figure 3.4). The transient expression of these proteins in plastids confirming the presence of transit peptides that target the plastids. 73 Figure 3.4 Transiently expressed full-length cDNAs of GPPS subunits – fused separately to GFP – in N. benthamiana protoplasts. The GFP expression was analyzed by inverted fluorescent microscopy. The "Green" column shows GFP fluorescence detected in the green channel; the "Red" column shows chlorophyll autofluorescence detected in the red channel, and the "Merged" column shows combined green and red channels. p326::sGFP showed cytosolic GFP signals, while others had plastidial-based GFP signals, confirming the presence of transit peptides that target the chlorophyll. The scale bar = 20 µm. Images © Ayelign M. Adal. 74 3.3.4 The trans-IDSs expression and terpenoid composition in L. x intermedia To test whether the expression of short-chain trans-IDSs is tissue-specific, and corresponds to their respective terpenoid accumulation in the specific tissues, we performed qPCR analysis of flower and leaf tissues from the source species, L. x intermedia (Figure 3.5a). Among the LiGPPS subunits, LiGPPS.SSU1 and LiGPPS.LSU transcripts were 9.5 and 5.8-fold more abundant, respectively, in flower compared to leaf tissues. However, the candidate LiGPPS.SSU2 was constitutively expressed in both tissues. The relative expression patterns of LiFPPS and LiGGPPS were also examined in these tissues (Figure 3.5a). Unlike the two LiGPPS subunits, the transcript levels of LiFPPS were slightly higher in leaves (~7.5 fold) than in flowers, while LiGGPPS was constitutively expressed in both tissues tested. Samples of EO-producing tissues used for spatial expression study were used to determine the corresponding total terpenoids that can be derived from either GPP or FPP as substrates (Figure 3.5b). Most of lavender EO components detected in flowers and leaves were monoterpenes (mostly derived from GPP by specific TPSs) ranging from approximately 73 % in leaf oil to 86 % in floral oil. Compared to monoterpenes, levels of total sesquiterpenes were lower in both tissues. Sesquiterpene levels in oil from leaves were much higher (~27 %) than from flowers (~14 %). 75 Figure 3.5 Relative expression of short-chain trans-IDSs and the composition of the corresponding total mono- and sesquiterpenes in L. x intermedia. a) Transcript levels of short-chain trans-IDSs in flowers relative to young leaf tissues determined by qPCR; and b) accumulation of total monoterpenes and sesquiterpenes in EO of these tissues quantified by GC-MS. Data represent mean value of replicate plants ± standard errors (n=3), **P ≤ 0.01. The control for relative expression (leaf) was assigned the arbitrary value of 1.0. LiGPPS.SSU1LiGPPS.LSULiGGPPSLiGPPS.SSU2LiFPPSNormalized relative expressi on024681012LeafFlower** ** Monoterpenes SesquiterpenesTotal terpenoids in L. x intermedia oi ls (%)020406080100LeafFlower** ** a) b) 76 3.3.5 Heterologous expression of LiGPPS and LiFPPS cDNAs in E. coli To examine the functions of identified GPPS subunits, truncated cDNAs (lacking transit peptides) encoding LiGPPS.SSU1 and LiGPPS.SSU2 with a C-terminal (His)8-tag in pET41b(+) were expressed in Rosetta E. coli expression cells. Truncated LiGPPS.LSU cDNA with (His)8-tag in pET32a was also expressed in cells of the same strain of E. coli. The partially purified recombinant protein of each subunit was inactive when assayed with IPP and any of the two potential co-substrates, DMAPP or GPP. When co-expressing tagged LiGPPS.SSU1 with untagged LiGPPS.LSU in E. coli, partially purified heteromeric protein was active with IPP and DMAPP to produce GPP as a sole product (Figure 3.6). However, the recombinant protein that was partially purified from E. coli cells co-expressing tagged LiGPPS.SSU2 with untagged LiGPPS.LSU had no detectable activities with IPP and DMAPP. In addition, to determine whether LiGPPS.SSUs are able to interact with LiGGPPS leading to the formation of active heteromers, we co-expressed untagged LiGGPPS with tagged LiGPPS.SSUs in E. coli. LiGGPPS was co-purified with tagged LiGPPS.SSU1 and the active heteromers produced GPP when assayed with IPP and DMAPP (Figure 3.6). However, no detectable GPP was obtained when recombinant protein purified from E. coli cells co-expressing untagged LiGGPPS with tagged LiGPPS.SSU2 was assayed with IPP and DMAPP. As a control, the lysates from E. coli expressing empty vector, or heat-denatured purified recombinant protein for each subunit or co-expressed subunits were assayed with IPP and the potential co-substrates DMAPP or GPP. None of the control reactions had detectable IDS activities. 77 Figure 3.6 GC-MS analysis depicting the catalytic activity of recombinant Lavandula GPPS enzymes expressed in E. coli. The left panel shows the chromatography of co-expressed GPPS subunits (LiGPPS.LSU/LiGPPS.SSU1 and LiGGPPS /LiGPPS.SSU1 recombinant proteins assayed with 40 µM IPP and 40 µM DMAPP, and authentic geraniol standard. The right panel indicates the mass spectrum of the detected products. Reaction products were (enzymatically) hydrolysis using calf intestine alkaline phosphatase (CIP), and the resulting prenyl alcohol (geraniol) was detected by GC-MS. A single peak very close to geraniol was detected from all assay products, including negative control, which could arise from the solvent or charcoal treatment of the assay products. 78 To investigate the functions of FPPS from L. x intermedia, the full-length cDNA encoding LiFPPS protein with His-tag in pET41b(+) was expressed in Rosetta E. coli expression cells. The purified recombinant LiFPPS protein produced FPP when assayed with IPP and each of the two potential co-substrates, DMAPP or GPP (Figure 3.7). Heat-inactivated recombinant LiFPPS or lysate from cells expressing empty vector was assayed with IPP and DMAPP or GPP as co-substrate with the same conditions mentioned above. None of these control reactions produced detectable FPP. Figure 3.7 The catalytic activity of recombinant Lavandula FPPS enzyme expressed in E. coli as detected by GC-MS. The left panel shows the chromatography and the right panel indicates the mass spectrum of detected products. Assays of recombinant LiFPPS were run with IPP and DMAPP, as well as IPP and GPP, followed by an overnight enzymatic hydrolysis using CIP to generate farnesol. Recombinant LiFPPS catalyzes IPP and DMAPP as well as IPP and GPP to FPP. 79 3.3.6 In vivo genetic complementation assay The transcript level of heteromeric GPPS subunits highly correlates with the monoterpene biosynthesis in some plants, for example, in M. piperita and A. majus (Mcconkey et al. 2000; Tholl et al. 2004; Croteau et al. 2005). It is also possible that the heteromeric GPPS can be a bifunctional enzyme (i.e., as GPPS and GGPPS), similar to the heteromeric GPPS/GGPPS from H. lupulus (Wang and Dixon, 2009). Thus, to investigate whether LiGPPS possesses GGPPS activity, we used a genetic complementation assay by substituting crtE gene encoded GGPPS in the crt gene cluster of yellow pigmented bacteria (Pantoea ananatis) used for carotenoid biosynthesis (Figure 3.8a) (Misawa et al., 1990; Zhu et al., 1997; Kainou et al., 1999; Engprasert et al., 2004; Ye et al., 2007). Since E. coli lacks an endogenous GGPPS, E. coli cells incorporating pACCAR25ΔcrtE, which carries all crt cluster genes, except crtE (GGPPS), are unable to accumulate carotenoids. Co-expression of the pACCAR25ΔcrtE with human GGPPS (as a positive control) showed a notable carotenoid pigment, indicating the functional substitution of crtE, whereas the pACCAR25ΔcrtE with the empty vectors (as negative controls) did not generate such pigments (Figure 3.8b). Co-expression of the pACCAR25ΔcrtE with either LiGPPS.SSU1 or LiGPPS.SSU2 had no GGPPS activity. Although the LiGPPS.LSU shared over 70 % amino acids with LiGGPPS, only LiGGPPS showed GGPPS activity when co-expressed with pACCAR25ΔcrtE. Co-transformation of GPPS subunits (LiGPPS.LSU with LiGPPS.SSU1 or LiGPPS.LSU with LiGPPS.SSU2) in E. coli carrying pACCAR25ΔcrtE resulted in no detectable yellow pigmentation (Figure 3.8b), confirming their inactivity as GGPPS. To investigate whether LiGPPS.SSUs function as 'modifiers' and/or 'accelerators' of GGPPS activity in vivo, the LiGGPPS that functions as GGPPS (Figure 3.8b) was co-transformed with each of LiGPPS.SSU1 and LiGPPS.SSU2 in E. coli carrying pACCAR25ΔcrtE. The data revealed that LiGPPS.SSU1 strongly inhibited the activity of LiGGPPS resulting in a 80 lack of yellow pigment accumulation, whereas LiGPPS.SSU2 co-expression resulted in a partial reduction of LiGGPPS activity (Figure 3.8b). Figure 3.8 Detection of GGPPS activity using in vivo genetic complementation assays. a) Diagram for the carotenoid biosynthesis pathway starting from the formation of GGPP and other subsequent intermediate genes within a pACCAR25ΔcrtE construct (Kainou et al. 1999) that lacks crtE (GGPPS). b) Genetic complementation assay detecting carotenoid pigments expressed and extracted from E. coli co-expressing pACCAR25ΔcrtE and expression vectors harboring candidate genes. pET41b(+) vector was used as a negative control. pBH (human GGPPS) construct was also used as a positive control. The assay showed that all candidate genes tested, except LiGGPPS and co-expression of LiGGPPS and LiGPPS.SSU2 did not complement the GGPPS and generate yellow pigments. LSU – LiGPPS.LSU; SSU1 – LiGPPS.SSU1; SSU2 – LiGPPS.SSU2. 3.3.7 Expression of LiGPPS.SSU1 in N. benthamina To determine the 'modifier' roles of LiGPPS.SSU1 in plant cells using endogenous GGPPSs as heteromeric partners, and examine the flux of regular monoterpene precursor (GPP), we expressed LiGPPS.SSU1 full-length cDNA in N. benthamiana under the control of CaMV 35S promoter (Figure 3.9a & b). We generated independent transgenic N. benthamiana plants expressing LiGPPS.SSU1. With the aid of the selection marker and gene-specific PCR screening, four independent transgenic lines were identified and grown to produce viable seeds. 81 Transgenic plants expressing GUS were used as negative controls. T1 progenies were used for further analysis. Using qPCR, we determined the transcriptional expression of LiGPPS.SSU1 in the transgenic N. benthamiana plants. The transcript levels of LiGPPS.SSU1 were high in the plants tested, ranging from 4.1 – 4.6 (log copies ng-1 of total RNA) (Figure 3.9c). When using LiGPPS.SSU1-specific primers, no qPCR amplification signal was detected from cDNA templates synthesized from total RNA of GUS transgenics. Furthermore, we extracted total soluble protein from the corresponding transgenic lines and assayed with IPP and DMAPP as substrates to assess the impact of LiGPPS.SSU1 on the formation of GPP. The assay products were dephosphorylated to prenyl alcohols (geraniol) and used to compare their GPPS activity. The results showed that expression of LiGPPS.SSU1 enhanced GPPS activity in these transgenic lines to produce GPP (Figure 3.9d). LiGPPS.SSU1 transgenic lines had distinct levels of GPPS activities, with a significant increase in all lines examined as compared to the control. Maximum and minimum GPPS activities were obtained from transgenic plants of SSU1 5-1 (0.52 pkat mg-1 total soluble protein) and SSU1 16 (0.37 pkat mg-1 total soluble protein), respectively. The results suggest that transcript levels of LiGPPS.SSU1 correlate positively with GPPS activity in all LIGPPS.SSU1 transgenic lines examined. 82 Figure 3.9 Impact of LiGPPS.SSU1 expression on GPPS activity and phenotypes of N. benthamiana plants. a) T-DNA maps showing the construct of full-length LiGPPS.SSU1 or GUS cDNA, b) LiGPPS.SSU1 and GUS (control) N. benthamiana T1 generation plants 6 weeks after germination, c) transcript levels of LiGPPS.SSU1 in transgenic lines determined by qPCR and d) GPPS activity in LiGPPS.SSU1 transgenic lines quantified by GC-MS using total protein isolated from these transgenic lines and assayed with IPP and DMAPP. The LiGPPS.SSU1 transgenic lines exhibiting high transcripts of LiGPPS.SSU1 and phenotype changes showed a significant increase in GPPS activity. KanR – kanamycin resistant gene; 35S – CaMV 35S promoter; nos-T – nopaline synthase terminator; RB – right border; LB – left border; SSU1 lines – independent transgenic N. benthamiana lines expressing LiGPPS.SSU1 full-length cDNA. ** P < 0.01, n= 3 plants per line. Images © Ayelign M. Adal. 3.3.8 Impacts of LiGPPS.SSU1 on GGPP-derived terpenoids and plant phenotypes Despite its role in GGPPS modification role in vitro and in E. coli, the regulatory functions of GPPS.SSU1 in plants can be varied, depending on the plant species from which GPPS.SSU1 cDNA is isolated. For example, in previous reports, GPPS.SSU1 modified GGPPS activity to 83 GPPS leading to the development of dwarf and variegated phenotypes with reduced chlorophyll and carotenoid contents in transgenic N. tabacum expressing A. majus GPPS.SSU1 (Orlova et al. 2009). On the contrary, M. piperita GPPS.SSU1 enhanced GGPPS and GPPS activities without any phenotypic alterations of N. tabacum and N. benthamiana transgenic plants expressing this gene (Yin et al. 2016). Here, we examined the N. benthamiana transgenic plants expressing LiGPPS.SSU1 to assess whether LiGPPS.SSU1 is involved in the regulation of GGPP-derived terpenoids and the corresponding plant phenotypes. These transgenic lines exhibited visible phenotypic aberrations, including leaf variegation, late flowering, delayed growth, and stunting compared to the control plants (Figure 3.10). A significant reduction of the total carotenoid levels was observed in these tested transgenic plants, with minimum and maximum reductions in SSU 5-1 (45 %) and SSU1 16 (77 %) lines, respectively, compared to the control (Figure 3.10a). Likewise, the total chlorophyll contents were considerably reduced in transgenic plants, with the lowest and the highest reduction in SSU1 5-1 (~49.5 %) and in SSU1 16 (73 %), respectively, relative to control plants (Figure 3.10b). In addition, flowering time was delayed by 15 to 35 days in transgenic lines compared to control plants (Figure 3.10c). Overall growth (in terms of leaf area, plant height, and leaf number) was also much lower in the transgenic plants than the control (Figure 3.10d and Figure I.1). Thus, the results suggest that expression of LiGPPS.SSU1 in plants can cause chain-length modification of NbGGPPS activity, which subsequently results in reduction of GGPP-derived metabolite levels and plant growth. 84 Figure 3.10 Effects of LiGPPS.SSU1 expression on GGPP-derived terpenoids and plant growth in N. benthamiana transgenic lines. a) Levels of total carotenoids, b) levels of chlorophylls, c) days of flowering, and d) growth of the transgenic lines relative to the respective control plants. The transgenic lines exhibited a significant reduction in the total chlorophyll and carotenoid contents, leaf area, plant height and the number of leaves compared to the control plants. Plant growth parameters from control transgenic plants were assigned the arbitrary value of 1.0. The transgenic plants had flowers 15-35 days after the control plants. ** significant level at P< 0.01 for LiGPPS.SSU1 transgenic lines vs control (GUS transgenic). n= 3 plants per transgenic line, with 3 replications per plant for pigments and 2 leaves per plant for leaf area. Dwarfing has been correlated to limited availability of active gibberellin (GA) in transgenic N. tabacum lines expressing A. majus GPPS.SSU (Orlova et al. 2009). Given that, we conducted a growth complementation assay by treating the dwarf transgenic N. benthamiana plants expressing LiGPPS.SSU1 with 30 µM of exogenous GA3 to complement the plant growth in MS medium. The resulting growth revealed that exogenous GA3 improved plant height in transgenic lines, with insignificant differences compared to control plants (Figure 3.11). 85 Figure 3.11 Dwarf phenotype complementation assay using exogenous GA3 in transgenic N. benthamiana expressing LiGPPS.SSU1. a) phenotypes of transgenic lines compared with control (GUS) plants grown on MS media supplemented with 0 or 30 µM GA3 for two weeks, and b) plant height of the transgenic lines with and without exogenous GA3 relative to the control plants, which were assigned the arbitrary value of 1.0. Exogenous GA3 improved the height of LiGPPS.SSU1 transgenic plants. ** P< 0.01, n= 6 plants per transgenic line. ns = non-significant. Images © Ayelign M. Adal. 3.3.9 Expression of LiGPPS.SSU2 in N. benthamiana We have previously shown with a genetic complementation assay in E. coli, that LiGPPS.SSU2 partially inhibits total carotenoid accumulation when co-expressed with LiGGPPS (Figure 3.8). To test this inhibitory effect in plants, we expressed LiGPPS.SSU2 in N. benthamiana. The results revealed that none of the transgenic plants expressing LiGPPS.SSU2 were impacted by this gene, particularly the GGPP-derived metabolites, GPPS activity and overall phenotypes, compared to control plants (Figure 3.12 and Table I.1 & I.2). These results were consistent with transcript levels of LiGPPS.SSU2 in L. x intermedia tissues (Figure 3.5a) and in vitro activities of LiGPPS.SSU2. 86 Figure 3.12 Transgenic N. benthamiana plants expressing LiGPPS.SSU2. None of the GGPP-derived terpenoid-based phenotypes were affected by expression of LiGPPS.SSU2. The detailed results about GGPP-derived terpenoids and phenotypes are shown in Table I.1 & I.2. Images © Ayelign M. Adal. 3.4 Discussion 3.4.1 Analysis of Lavandula short-chain trans-IDSs sequences Terpenoid metabolism can be regulated by the levels of TPS expression and amount of supplied precursors in the plants (Dudareva et al. 2000; Orlova et al. 2009). Several genes and enzymes responsible for the formation of terpenes and their precursors have been reported in different plants (Tholl et al. 2004; Wang and Dixon 2009; Rai et al. 2013). However, over the last few years, most of Lavandula terpenoid metabolism research has largely focused on isolation and functional characterization of genes and the corresponding enzymes of TPSs (Demissie et al. 2012; Sarker et al. 2012; Sarker et al. 2013; Demissie et al. 2013; Jullien et al. 2014; Benabdelkader et al. 2015; Despinasse et al. 2017; Adal et al. 2019). While monoterpenes are the dominant constituents of Lavandula oils (Aprotosoaie et al. 2017), the biosynthesis of their precursor prenyl diphosphates has not yet been determined. To date, 87 LPPS – an irregular monoterpene precursor (LPP) synthase – has been reported from Lavandula (Demissie et al. 2013). Regular monoterpenes are derived from GPP/NPP (formed from IPP and DMAPP) in a reaction catalyzed by GPPS/NPPS depending on the plant species (Burke et al. 1999; Schilmiller et al. 2009). In this study, we have identified five candidates of short-chain trans-IDSs from EST and transcriptome sequences that were developed from EO-producing tissues, including flowers, leaves and floral based glandular trichomes of L. x intermedia (Lane et al. 2010; Demissie et al. 2012; Adal et al. 2019). Two of these (i.e., LiGPPS.SSU1 and LiFPPS) were detected from both EST and transcriptome sequences, while the other three (i.e., LiGPPS.LSU, LiGPPS.SSU2, and LiGGPPS) were only found in transcriptome sequences (Adal et al. 2019). Isolated short-chain trans-IDSs in this study were evolutionarily distantly related and clustered separately into previously reported plant trans-IDS subfamilies, which were classified based on their catalytic functions as FPPS, GPPS.LSU/GGPPS, and GPPS.SSU (Jia and Chen 2015). Four of these IDSs contained FARM (DD(x)4D) and SARM (DD(x)2D) motifs that are essential for substrate binding and catalytic functions (Kellogg and Poulter 1997). However, both motifs are absent in LiGPPS.SSU1 amino acids. This has also been observed with GPPS.SSU1 from M. piperita and A. majus, among others (Burke et al. 1999; Tholl et al. 2004). Interestingly, SARM is present in LiGPPS.SSU2, in contrast to most GPPS.SSU2 subfamilies that lack SARM (particularly the last ‘D’) in plants, for example in Arabidopsis (Wang and Dixon 2009; Chen et al. 2015) and in rice (Zhou et al. 2017). Furthermore, as expected, the two 'CxxxC' conserved motifs – useful for the physical interaction of the two subunits to form a heteromer complex (Wang and Dixon 2009) – were detected in LiGPPS.SSU1 and LiGPPS.SSU2 and one such motif is present in LiGPPS.LSU and LiGGPPS. 88 3.4.2 GPP is synthesized by heteromeric LiGPPS Despite the bulk of regular monoterpenes in Lavandula EOs, GPPS responsible for the formation of GPP – a universal precursor of regular monoterpenes – has not been known in the genus Lavandula. Until recently, two structurally different GPPSs (i.e., homomeric and heteromeric GPPSs) were identified and functionally characterized from diverse angiosperm and gymnosperm species (Bouvier et al. 2000; Burke and Croteau 2002; Van Schie et al. 2007a; Schmidt and Gershenzon 2007; Hsiao et al. 2008; Schmidt and Gershenzon 2008; Rai et al. 2013). The functions of such GPPS enzymes and the mechanisms that regulate the flux toward GPP have been reported in vitro and in plants (Burke and Croteau 2002; Orlova et al. 2009; Yin et al. 2016). However, the type of GPPS (homomeric or heteromeric), its regulation on the flux of GPP and subsequent roles on the biosynthesis of monoterpenes have not been determined in Lavandula. In this study, we cloned cDNAs encoding LiGPPS.SSU1, LiGPPS.SSU2 and LiGPPS.LSU proteins and functionally characterized them in E. coli. The resulting recombinant protein of each subunit was not active by itself when assayed with IPP and DMAPP to produce GPP, as it was previously reported for both M. piperita GPPS subunits (Burke et al. 1999) and Arabidopsis GPPS.SSU2 (Wang and Dixon 2009). When LiGPPS.SSU1 was co-expressed with LiGPPS.LSU in E. coli, the resulting recombinant heteromeric protein was active as GPPS with IPP and DMAPP to produce GPP. However, when assayed with IPP and DMAPP, the recombinant protein of co-expressed LiGPPS.SSU2 and LiGPPS.LSU had no detectable products. Despite amino acid sequence similarity, the catalytic function of LiGPPS.SSU2 was different from previously reported GPPS.SSU2 orthologues from Arabidopsis and rice. GPPS.SSU2 from Arabidopsis was found to be a modifier of GPPS.LSU/ GGPPS to enhance the production of GPP (Wang and Dixon 2009; Chen et al. 2015). A similar GPPS.SSU2 (in terms of amino acid sequences) from rice also accelerated GGPPS activity to increase 89 chlorophyll biosynthesis (Zhou et al. 2017). Thus, the result suggests that LiGPPS.SSU2 might have a limited role in the biosynthesis of GPP as a small subunit. Moreover, GPP was formed by heteromeric GPPS purified from E. coli cells co-expressing LiGGPPS with LiGPPS.SSU1. This implies that LiGPPS.SSU1 can recruit and modify the chain length specificity of distantly related GGPPSs in vitro, as observed previously by GPPS.SSU1 from different plants (Burke and Croteau 2002; Tholl et al. 2004; Wang and Dixon 2009). 3.4.3 Transcripts of LiGPPS.SSU1 and LiGPPS.LSU correspond to the accumulation of monoterpenes Members of the genus Lavandula produce most of their monoterpene-rich EOs in flower tissue. It has been reported that most genes encoding regular monoterpene synthases are highly expressed in Lavandula flowers (Landmann et al. 2007; Demissie et al. 2012; Adal et al. 2019). Here, we showed that LiGPPS.SSU1 and LiGPPS.LSU were constitutively expressed in flowers and leaves, but the transcripts for both genes were abundant in flowers. A similar scenario was reported in A. majus, where GPPS.SSU1 transcripts were found to be floral-specific (Tholl et al. 2004). Our results indicate that the levels of LiGPPS.SSU1 and LiGPPS.LSU expression could control the formation of GPP, which eventually serves as a precursor substrate for regular monoterpenes in Lavandula EOs. 3.4.4 LiGPPS.SSU1 can recruit GGPPS to enhance GPP formation Regulatory roles have been previously proposed for the small subunits of heteromeric GPPS, and for the homomeric GPPS in the biosynthesis of regular monoterpenes (Tholl et al. 2004; Hsiao et al. 2008; Wang and Dixon 2009; Schmidt et al. 2010; Chen et al. 2015). GPPS.SSUs can recruit and modify the chain length specificity of distantly related GGPPSs in vitro (Burke and Croteau 2002; Tholl et al. 2004; Wang and Dixon 2009) and in tobacco to regulate the formation of GPP (Orlova et al. 2009; Yin et al. 2016). For example, GPPS.SSU1 90 from M. piperita enhanced accumulation of GPP and other metabolites without any inhibitory effects on GGPP-derived primary metabolites in N. tabacum and N. benthamiana (Yin et al. 2016). Further, A. majus GPPS.SSU1 increased GPP and the corresponding monoterpenes at the expense of GGPP-derived terpenoids, including pigments and gibberellins that are useful for plant growth and development in N. tabacum (Orlova et al. 2009). In this context, our results –LiGPPS.SSU1 recruited LiGGPPS as GPPS.LSU when co-expressed with LiGGPPS in E. coli and formed an active heteromeric GPPS with limited GGPPS activity – are in agreement with the roles of GPPS.SSU1 from other plants (Burke and Croteau 2002; Wang and Dixon 2009). It appears that the outcome of the interaction of GPPS.SSU1 with GGPPS is not consistent in all plants, particularly when overexpressing GPPS.SSU1 in plants. For example, GPPS.SSU1 from M. piperita and A. majus served as an 'accelerator' (Yin et al. 2016) and a 'modifier' (Orlova et al. 2009), respectively, of endogenous GGPPSs in tobacco plants. Lavandula LiGPPS.SSU1 played a 'modifier' role in N. benthamiana plants, exhibiting reduced GGPP-derived terpenoids and altered plant phenotypes after overexpressing LiGPPS.SSU1. This may be due to modification of the chain length specificity of endogenous GGPPSs by LiGPPS.SSU1, which subsequently redirects the flux toward GPP at the expense of GGPP-derived metabolites. These in planta LiGPPS.SSU1-derived responses were different from those by M. piperita GPPS.SSU1. Although both L. x intermedia and M. piperita belong to the mint family and contain cDNAs encoding GPPS.SSU1 with similar sequences, both GPPS.SSU1 regulated GGPPS differently in tobacco, suggesting that these genes are not conserved functionally. The difference in these responses may be associated with the presence of multiple GGPPS and GGPPS-like genes in plants. Some of these GGPPS genes could be specific and serve as an alternative GPPS.LSU if they are located in the same compartment with GPPS.SSUs as for example, in Arabidopsis (Chen et al. 2015). Given that, the proteins of 91 single or multiple LiGGPPS genes may serve as LiGPPS.LSU of GPPS, leading potentially to a high GPPS activity in Lavandula. Previously, two GPPS.SSU2-like genes, belonging to GPPS.SSU2 clade under GPPS.SSU subfamily (trans-IDS-e), have been isolated and functionally characterized from A. thaliana and rice (Wang and Dixon 2009; Zhou et al. 2017). These subunits were involved in the formation of a heteromeric complex with either GPPS.LSU or GGPPS or both, and have different functions. A. thaliana GPPS.SSU2 redirects the flux toward GPP and then enhances monoterpene biosynthesis (Wang and Dixon 2009; Chen et al. 2015). In contrast, rice GPPS.SSU2-like protein, designated as a GGPPS recruiting protein, formed a heteromeric complexes with GGPPS and other downstream proteins to enhance the production of GGPP, and resulted in increased chlorophyll biosynthesis (Zhou et al. 2017). Lavandula GPPS.SSU2 had high amino acid sequence similarity (~70 %) to GPPS.SSU2 from A. thaliana, and was clustered under the same trans-IDS-e subfamily. However, LiGPPS.SSU2 did not show any GPPS activities, and its transcripts did not parallel the total monoterpene levels from the corresponding plants. This implies that LiGPPS.SSU2 may not be involved in GPP biosynthesis at all, or it may have very low activity under specific conditions. In this context, LiGPPS.SSU2 did not affect GPPS activity and plant phenotypes in transgenic N. benthamiana plants. However, it partially inhibited the carotenoid pigment accumulation in a genetic complementation assay in E. coli. This result provides support for the postulate that LiGPPS.SSU2 may not control GGPP at all, or may have an inhibitory effect on this protein only under certain cercumstances. Clearly, additional investigations are needed to elucidate the exact role of LiGPPS.SSU2 in terpenoid metabolisms. 92 3.4.5 LiFPPS catalyzed substrates to produce FPP Several genes encoding FPPS catalyzing IPP with DMAPP or with GPP to produce FPP have been reported in different plant species (Schmidt and Gershenzon 2007; Richter et al. 2015). In Lavandula, sesquiterpenes that are derived from FPP are the second most abundant constituents of EOs. So far, a number of sesquiterpene synthases that catalyze FPP into specific sesquiterpenes have been isolated and characterized from different species of Lavandula (Landmann et al. 2007; Sarker et al. 2013; Jullien et al. 2014; Benabdelkader et al. 2015). However, the gene and enzyme involved in the biosynthesis of the sesquiterpene precursor (FPP) in this genus have not been described. Using EST and transcriptome databases developed from EO-producing tissues (Lane et al. 2010; Demissie et al. 2012; Adal et al. 2019), we identified a cDNA encoding LiFPPS protein that catalyzes the reaction of IPP and DMAPP / GPP to FPP. As expected, LiFPPS was also constitutively expressed in both tissues examined, but had more transcripts in leaves than flowers, and was parallel to the levels of sesquiterpenes in these tissues. This suggests that the level of LiFPPS expression controls the formation of FPP, and thus ultimately regulates the biosynthesis of sesquiterpenes in Lavandula. 93 Chapter 4: Cloning and functional characterization of two monoterpene synthases from Lavandula x intermedia 4.1 Overview Lavenders produce essential oils (EOs) consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene (3-carene) and scent-determining oil constituents (S)-(+)-linalool and (R)-(-)-linalool (S- & R-linalool). Although R-linalool dominates the EOs, varying amounts (depending on the species) of the S-linalool enantiomer can also be found in lavenders. Despite its relatively low abundance, S-linalool contributes a sweet and pleasant scent to lavender EOs. While several terpene synthase genes, including R-linalool synthase, have been cloned from lavenders, many important terpene synthases, including 3-carene and S-linalool synthases, have not been described from these plants. Thus, with the work described in this Chapter, I aimed to clone and functionally characterize 3-carene synthase (Li3CARS) and S-linalool synthase (LiS-LINS) from L. x intermedia using ESTs, RNA-Seq and genomic sequencing data of Lavandula. Phylogenetic analysis based on deduced amino acid sequences revealed that Li3CARS is a member of TPS-b, while LiS-LINS belongs to the TPS-g subfamily. The open reading frame, excluding transit peptide, of each gene was cloned into an expression vector, expressed in E. coli and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as a major product and catalyzed NPP to produce multiple products along with a small amount of 3-carene. Li3CARS used both GPP and NPP as substrates to produce 3-carene, although it had high catalytic efficiency with GPP. Formation of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves relative to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene 94 production and was up-regulated with MeJA 8 - 72 h post-treatment. The result suggests that Li3CARS may have defensive roles in Lavandula. The recombinant LiS-LINS catalyzed the conversion of both GPP and NPP to S-linalool as the sole product. LiS-LINS exhibited very low (~30 %) sequence similarity to other Lavandula terpene synthases, including R-linalool synthase (LiR-LINS). LiS-LINS transcripts were detected in flowers, but were much less abundant than those corresponding to LiR-LINS, paralleling enantiomeric composition of S-linalool in L. x intermedia oils. These data indicate that production of S-linalool is at least partially controlled at the level of transcription from LiS-LINS. 4.2 Materials and Methods 4.2.1 Plant growth condition and chemicals L. x intermedia (cv. Grosso) plants were propagated by cuttings and were grown in potting Pro-Mix HP in a growth room as described in Chapter 3, section 3.2.1. The plants were watered every other day with the addition of fertilizer (Miracle-Gro Fertilizers) once a week. Authentic terpenoid standards (e.g., 3-carene) were purchased from Sigma (http://www.sigmaaldrich.com/) and substrates for enzyme assays from Echelon Biosciences (http://www.echelon-inc.com/). An authentic standard of S-linalool (75 % linalool extracted from orange) for assay product identification was purchased from TREATT, Lakeland, Florida, USA. Unless otherwise mentioned, all other assay chemicals were obtained from Sigma. 4.2.2 Identification and cloning of monoterpene synthases To clone the full-length Li3CARS cDNA, amino acid sequence similarity-based screening of monoterpene synthases (mTPSs) from L. x intermedia EST library was done based on the sequences of L. angustifolia limonene synthase and S. officinalis bornyl pyrophosphate synthase genes. The selected cDNA clone from the corresponding cDNA library was used for Sanger sequencing. The generated full-length sequence of Li3CARS (GenBank: KX024762) 95 was analyzed for potential targeting sequences using TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) and ChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/), and found to encode a chloroplast transit peptide at the N-terminus. Similarly, known mTPSs from lavender species and S-linalool synthases from different plant species were used to BLAST against the Lavandula database, followed by manual analysis targeting the common mTPS conserved motifs. The ORF for the selected candidate (LiS-LINS, GenBank: MG870571) was further analyzed for the presence of potential transit peptide using different bioinformatics tools, including TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/), ChloroP 1.1 (http://www.cbs.dtu.dk/services/ChloroP/), PWoLF PSORT (https://wolfpsort.hgc.jp/), SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and iPSORT (http://ipsort.hgc.jp/). These tools were, however, inconsistent in predicting the sequence of the N-terminus plastids-targeting transit peptide. To confirm the localization further experimentally, a subcellular localization study was conducted in protoplasts as described in detail separately in this Chapter, section 4.2.3. For heterologous expression in E. coli, the putative N-terminal transit peptides were removed prior to designing the gene-specific cloning primer set. All primers used in this study are listed in Table 4.1. The ORFs, excluding the transit peptides, and stop codons, of Li3CARS and LiS-LINS genes were cloned into the NdeI/KpnI and NdeI/EcoRI sites, respectively, of pET41b(+) by sticky-end cloning (Zeng 1998). The resulting constructs – designated hereafter as pET41b::Li3CRAS and pET41b::LiS-LINS – were further confirmed by restriction digestion and sequencing. Using appropriate primers the full-length genomic DNA for Li3CARS (GenBank: KX024761) was amplified from L. x intermedia and subsequently cloned into pGEM-T vector (Figure E.5) (Promega) using TA cloning (Bielefeldt-Ohmann and Fitzpatrick 1997) and sequenced by Sanger sequencing. Genomic organization of Li3CARS from L. x intermedia was 96 predicted using the NCBI Spidey genomic DNA-mRNA aligner (http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/spideyweb.cgi), targeting intron-exon number and size as well as intron position in the sequences. The intron-exon structure of Li3CARS was then compared with those of four previously reported TPSs from Lavandula. Additionally, since the genomic DNA of S-LINS was analyzed from genomic sequences of L. angustifolia – a half parental species of L. x intermedia – by Malli et al. (2019), analysis of LiS-LINS genomic organization was not included in this study. 4.2.3 Subcellular localization of LiS-LINS Due to the ambiguous prediction of the plastid-targeting transit peptide using online bioinformatics tools, we tested the subcellular localization of LiS-LINS (GenBank: MG870571) experimentally. Briefly, the first 50 amino acids of LiS-LINS, designated hereafter as LiS-LINS-TP(50aa), were fused upstream of green flourescent protein (GFP) in the cloning sites XbaI and SalI of the p326::GFP vector harboring CaMV 35S promoter (a gift from Dr I. Hwang, Pohang University of Science and Technology, Korea). R-linalool synthase from our L. x intermedia (LiR-LINS, GenBank: MH375883) EST database shared about 95 % amino acid identity with the previously cloned R-linalool synthase from L. angustifolia (Landmann et al. 2007). Thus, the first 50 amino acids of R-linalool synthase from L. x intermedia (LiR-LINS-TP(50aa)) were also fused upstream of GFP in the cloning sites XbaI and SalI of the p326::GFP. All primers used for the cloning are listed in Table 4.1. The integrity of the resulting constructs was then confirmed by Sanger sequencing prior to transfecting to N. benthamiana protoplasts. N. benthamiana protoplast isolation, transfection and visualization were performed as described in Chapter 3, Section 3.2.3. 97 4.2.4 Phylogenetic analysis Multiple sequence alignments of deduced amino acids of Li3CARS or LiS-LINS along with the corresponding closely related TPSs from other plants were generated using T-Coffee (http://tcoffee.crg.cat/apps/tcoffee/do:regular), and shaded using BOXSHADE 3.21 (www.ch.embnet.org/software/BOX_form.html) software. A maximum likelihood phylogenetic tree was constructed based on deduced amino acids of mono- and sesquiterpene synthases, including Li3CARS, using the default parameters of CLC Genomics Workbench 8.0.3 (https://www.qiagenbioinformatics.com/). TPSs were clustered into previously defined distinct TPS subfamilies (Bohlmann et al. 1998; Chen et al. 2011). For LiS-LINS, phylogenetic trees were separately constructed based on deduced amino acids of TPSs identified from the genus Lavandula, and other selected plant TPS-g subfamily members available in public databases with 1000 bootstrap replicates in MEGA7 (Kumar et al. 2016). The GenBank accession numbers of related genes from selected plant species, including lavenders used in phylogenetic analysis of Li3CARS and LiS-LINS are listed in Table D.1 & D.2, respectively. 4.2.5 Recombinant protein expression and purification The pET41b::Li3CARS and pET41b::LiS-LINS constructs were separately expressed in E. coli RosettaTM (DE3)plysS cells (Novagen). E. coli cells carrying constructs were grown in LB medium supplemented with 30 mg L-1 of kanamycin and 34 mg L-1 of chloramphenicol at 37 °C to an OD600 of 0.5-0.7, followed by induction with 0.4 - 1 mM IPTG at 20 °C overnight. After harvesting, the pelleted cells were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole, pH 8.0) and were further disrupted by sonication. The crude protein was then purified using PerfectPro Ni-NTA Agarose chromatography (5Prime Gmbh, Germany), in which His-tagged recombinant proteins were bound to Ni-NTA agarose according to the 98 manufacturer's instructions. Partially affinity-purified proteins were then quantified using the Bradford assay prior to enzyme assays. 4.2.6 Enzyme assays and product identification The activity of partially purified recombinant proteins for each of Li3CARS and LiS-LINS was assayed with each of GPP, NPP and FPP as substrates. For Li3CARS, the assay reactions were prepared in a final volume of 500 µl, containing an enzyme reaction buffer (2 mM dithioerythritol, 12.5 % (v/v) glycerol, 1 mM MgCl2 and 1 mM MnCl2), pH buffer, purified enzyme (22 µg) and substrates (0.1 - 100 µM). The optimal assay pH was determined using MES (50 mM, pH 5.5-6.5) and MOPS (50 mM, pH 7.0-8.0) buffers, and the appropriate temperature was optimized between 27 oC and 40 oC. The assays were overlaid with 400 µl of pentane (Fisher Scientific) and were incubated for 10 - 90 min. The products were then extracted with pentane after freezing the samples at -80 oC, according to the previous reports (Demissie et al. 2011; Demissie et al. 2012). Purified proteins from E. coli lysate cells harboring the empty pET41b(+) vector were also assayed with substrates as a negative control. Assay product identification and quantification were carried out using GC-MS as described in Chapter 3, section 3.2.5. Authentic standards of monoterpenes were also run on GC-MS under similar conditions to identify the products. Michaelis-Menten enzymatic saturation curves were generated using GraphPad Prism 6 (GraphPad Software, Inc.). Km and Vmax values were estimated using the linear regression method of double-reciprocal plots (or Lineweaver Burk) using the same software. For LiS-LINS, the assay reactions were prepared in a final volume of 500 µl, containing an enzyme reaction buffer (2 mM dithioerythritol, 12.5 % (v/v) glycerol, 1 mM MgCl2 and 1 mM MnCl2), MOPS (pH 6.5) buffer, purified enzyme (25 µg) and substrates (40 µM), and were overlaid with 400 µl of pentane and incubated at 30 oC for 30 min. The assays were frozen at -80 oC and then the products were extracted with pentane as described above. As controls, heat-denatured LiS-LINS enzyme and purified proteins from E. coli lysate cells harboring the 99 pET41b(+) lacking LiS-LINS were assayed with substrates with similar conditions. Assay products were identified using GC-MS as described in Chapter 3, section 3.2.4. Linalool enantiomers were analyzed using a GC-flame ionization detector (GC-FID) with a 30 m × 0.25 mm fused silica capillary column coated with Chirasil-DEX (cyclodextrin directly bonded to dimethylpolysiloxane) (Agilent Technologies Canada Inc.) according to a previous report (Galata et al. 2014). The assay products were injected at 60 °C, with the oven temperature of 60 °C for 3 min. The oven temperature subsequently increased to 150 °C (at a rate of 10 °C min-1 and 3 min holding time) and 280 °C (at a rate of 20 °C min-1 and 3 min holding time). The flow rate of the carrier gas (helium) was set to 1 mL min-1. Authentic standards of linalool isomers were also run on GC-MS and GC-FID under similar conditions to identify the assay products. 4.2.7 Induction of Li3CARS and 3-carene by methyl jasmonate Approximately three month-old L. x intermedia (cv. Grosso) plants were used for methyl jasmonate (MeJA) induction experiments. The plants were sprayed with a 5 mM aqueous solution of MeJA (95 % solution, Sigma-Aldrich) in water with 0.1 % Triton X-100 and water (mock treatment, control) supplemented with 0.1 % Triton X-100. The treated plants were then covered with Ziploc plastic bags for two hours and allowed to dry in open air for an hour before transferring them into a growth chamber. EO was extracted from fresh young leaves harvested at 0, 24, 48 and 72 h post-treatments, and was used to determine EO compositions. Likewise, fresh young leaves at 0, 8, 24, 48 and 72 h post-treatments were harvested and flash-frozen in liquid nitrogen and kept at - 80 oC for RNA extraction. 4.2.8 Relative expression qPCR analysis was performed to determine the constitutive (from flower and leaf tissues) and induced (leaves induced by MeJA) mRNA abundance for Li3CARS and transcript levels of LiS-LINS in EO-producing tissues. qPCR was performed using the StepOne Plus Real-100 Time PCR system (Applied Bioscience). Briefly, total RNA was extracted from each collected sample using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. The RNA samples were then reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad). Expression levels of the gene of interest were normalized using β-actin and 18S rRNA as internal reference genes. The relative qPCR reactions were prepared, run and analyzed using procedures mentioned in Chapter 3, section 3.2.8. The statistical analysis was made with Student's t-test using SigmaPlot v 12.5 (Systat Software, Germany). The primers used in this study are listed in Table 4.1. Table 4.1 List of all primer sets used in this study. Gene Name Primer name Primer sequences (5'==> 3') Purpose Li3CARS NdeI-Forward CAGGATCATATGTCGACACAACTAGAC Cloning into pET41b(+) cDNA EcoRI-Reverse ATTATAGAATTCGAAGCATACGGCTCG LiS-LINS Forward CTGGCATATGAAGCAGTGTCGTATCAAC cDNA Reverse ATATAGAATTCAAGCTCTGAATCAGGTTTGGC Li3CARS Forward ATGTGTAGCATTATCATGCATGTGG Genomic DNA (cloning into pGEM-T) gDNA Reverse TATAATCTCGAGCTAAGCATACGGCTC Li3CARS Forward CCAATCAACACGGCTAGAA qPCR quantification qPCR Reverse GACCCGATCCCTTACAAATG LiS-LINS Forward CAATCCCATCAACTCCCTAAA qPCR Reverse GCTCTACTTCGTCTCTGAATTT LiR-LINS Forward ACACGCACGACAATTTGCCA qPCR Reverse AGCCCTCCAATGAAGTGGGAT LiS-LINS-TP(50aa) Forward ATCTCTAGATGGCACTTCCATGCAATATC Reverse AATAGTCGACGTTGATACGACACTGCT Subcellular localization (cloning into p326::GFP) LiR-LINS-TP(50aa) Forward GCCGTCTAGATGTCGATCAATATCAACA Reverse GCCAGTCGACCTTATACTGAGAATTGAG β-actin Forward TGTGGATTGCCAAGGCAGAGT Reference genes for qPCR normalization Reverse AATGAGCAGGCAGCAACAGCA 18s rRNA Forward GTGACGGGTGACGGAGAA 101 Gene Name Primer name Primer sequences (5'==> 3') Purpose Reverse GACTCAATGAGCCCGGTA Note: underlined and bold sequences are restriction sites. aa – amino acids 4.2.9 EO extraction and GC-MS analysis Approximately 0.5 g of MeJA-treated or mock (control) leaf samples was harvested and immediately placed separately in 25-mL test tubes containing 6 mL of pentane and 160 μg of thymol as an internal standard. This was followed by sonication on ice for 30 min using Ultrasonic water bath sonicator (VWR). Pentane-containing EO from each sample was then analyzed using a Varian CP 3800 GC coupled to a Saturn 2200 MS/MS, following the procedures detailed in Chapter 3, section 3.2.5. The concentrations of EO constituents in the MeJA-treated and control samples were calculated based on an internal standard (thymol) as previously described (Erland, 2015). Statistical analysis (Student's t-test) was performed using SigmaPlot v 12.5. 4.3 Results 4.3.1 Characterization of 3-carene synthase from L. x intermedia 4.3.1.1 Sequence analysis Using homology-based screening, we detected partial sequences of Li3CARS from ten ESTs from the L. x intermedia flower glandular trichome-derived EST library (Demissie et al. 2012). The full-length cDNA was then obtained from the corresponding cDNA library. The complete ORF of Li3CARS cDNA (GenBank: KX024762) encoded a protein of 599 amino acids with a 45 amino acid plastid targeting sequence. The encoded protein was predicted to have a molecular mass of 69.6 kDa and a pI of 5.08. The deduced amino acid sequence was further characterized for the conserved motifs against orthologous TPSs across the genus Lavandula and other angiosperms. As with other plant TPSs, the key motifs that are crucial for prenyl diphosphate ionization (RRx8W), and for divalent metal ion binding (DDxxD and 102 (N,D)D(L,I,V)x(S,T)x3E) (DDLxTx3E was in our case) were well conserved in Li3CARS (Figure 3.2). Additionally, Li3CARS exhibited high amino acid identity with L. angustifolia limonene synthase (75 %) and S. officinalis bornyl pyrophosphate synthase (SoBPPS) (65 %). Finally, Li3CARS was found to be less homologous to 3-carene synthases from S. stenophylla (~50 %) and P. abies (~29 %) (Figure 4.1). Li3CARS 1 MCSIIMHVGVLNKPAAI-HLPNLDTRASKPLS-----ISSTPATS---RLRVSCSTQ-LD SoBPPS 1 MSIISMNVSILSKPLN--CLHNLERRPSKALL-----VPCTAPTA---RLRASCSSK-LQ LaLIMS 1 MSIISMHVGILNRPAAYNHLRNLDRRASKPRH-----VSSTAAAT---RLRVSCATQ-LE Ss3CARS 1 MAFPRNPTKLLHKPH---------NKSSKLIS-----NSRISSYG---HLPLRCSSQQLP Pa3CARS 1 MSVISI-LPLASKSC----LYKSLMSSTHELKALCRPIATLGMCRRGKSVMASKSTS-LT Li3CARS 51 IK-PVADETRRSGNYQPSAWDFDYIQSLNNPYKEESYLTRHAELTSQVKMLWDEEM---- SoBPPS 50 ----EAHQIRRSGNYQPALWDSNYIQSLNTPYTEERHLDRKAELIVQVRILLKEKM---- LaLIMS 52 IK--SVDETRRSGNYNPTAWDFNYIQSLDNQYKKERYSTRHAELTVQVKKLLEEEM---- Ss3CARS 44 TD--EFQVERRSGNYSPSKWDVDYIQSLHSDYKEERHTRRASELIMEVKKLLEKEP---- Pa3CARS 55 TAVSDDGVQRRIGDHHSNLWDDNFIQSLSSPYGASSYGERAERLIGEVKEIFNSLSRTDG Li3CARS 106 -------EAVQQLELIEDLKNLGISYLFKDKIKQILSLIYDD---NKCFQNNEV--EARD SoBPPS 102 -------EPVQQLELIHDLKYLGLSDFFQDEIKEILGVIYNE---HKCFHNNEV--EKMD LaLIMS 106 -------EAVQKLELIEDLKNLGISYPFKDNIQQILNQIYNE---HKCCHNSEV--EEKD Ss3CARS 98 -------NPTRQLELIDDLQKLGLSDHFNNEFKEILNSVYLD---NKYYRNGAMKEVERD Pa3CARS 115 ELVSHVDDLLQHLSMVDNVERLGIDRHFQTEIKVSLDYVYSYWSEKGIGSGRDI--VCTD Li3CARS 154 LYFTALGFRLLRQHGFEVSQEIFDCFKKENGTDFKQT---LADDTKGLLQLYEASFLLRE SoBPPS 150 LYFTALGFRLLRQHGFNISQDVFNCFKNEKGIDFKAS---LAQDTKGMLQLYEASFLLRK LaLIMS 154 LYFTALRFRLLRQQGFEVSQEVFDHFKNEKGTDFKPN---LADDTKGLLQLYEASFLLRE Ss3CARS 148 LYSTALAFRLLRQHGFQVAQDVLECFKNTKG-EFEPS---LSDDTRGLLQLYEASFLLTE Pa3CARS 173 LNTTALGFRILRLHGYTVFPDVFEHFKDQMGR-IACSDNHTERQISSILNLFRASLIAFP Li3CARS 211 GEDTLELARQFSTKLLQKKVDD---HEIED-NLLLWIRHSLELPLHWRVQRLEGRWFLDA SoBPPS 207 GEDTLELAREFATKCLQKKLDEG-GNEIDE-NLLLWIRHSLDLPLHWRIQSVEARWFIDA LaLIMS 211 AEDTLELARQFSTKLLQKKVDENGDDKIED-NLLLWIRRSLELPLHWRVQRLEARGFLDA Ss3CARS 204 GENTLELARDFTTKILEEKLRN---DEIDDINLVTWIRHSLEIPIHWRIDRVNTSVWIDV Pa3CARS 232 GEKVMEEAEIFSATYLKEALQT---IPV-S-SLSQEIQYVLQYRWHSNLPRLEARTYIDI Li3CARS 267 YSTRP-----DMN-PIIFELAKLEFNISQSTRLEEMKDVSRWWNSLCLIDQLPFVRDRVV SoBPPS 265 YARRP-----DMN-PLIFELAKLNFNIIQATHQQELKDLSRWWSRLCFPEKLPFVRDRLV LaLIMS 270 YVRRP-----DMN-PIVFELAKLDFNITQATQQEELKDLSRWWNSTGLAEKLPFARDRVV Ss3CARS 261 YKRRP-----DMN-PIVLELAVLDSNIVQAQYQEELKLDLQWWRNTCLAEKLPFARDRLV Pa3CARS 287 LQENTKNQMLDVNTKKVLELAKLEFNIFHSLQQNELKSVSRWWKESGFPD-LNFIRHRHV Li3CARS 321 EAQFWAIGALEPHQYGYQRKLIAKIITFVTVIDDIYDVYGTLEELEAFTDIIRRWDTESI SoBPPS 319 ESFFWAVGMFEPHQHGYQRKMAATIIVLATVIDDIYDVYGTLDELELFTDTFKRWDTESI LaLIMS 324 ESYFWAMGTFEPHQYGYQRELVAKIIALATVVDDVYDVYGTLEELELFTDAIRRWDRESI Ss3CARS 315 ESYFWGVGVVQPRQHGIARMAVDRSIALITVIDDVYDVYGTLEELEQFTEAIRRWDISSI RR -(x8)- W DDxxD 103 Pa3CARS 346 EFYTLVSGIDMEPKHCTFRLSFVKMCHLITVLDDMYDTFGTIDELRLFTAAVKRWDPSTT Li3CARS 381 NQLPQYMQVCFLALHNFVSELAYNILRDKGFNCLSHIQRSWIDLVEANLEEAKWYYSGYT SoBPPS 379 TRLPYYMQLCYWGVHNYISDAAYDILKEHGFFCLQYLRKSVVDLVEAYFHEAKWYHSGYT LaLIMS 384 DQLPYYMQLCFLTVNNFVFELAHDVLKDKSFNCLPHLQRSWLDLAEAYLVEAKWYHSRYT Ss3CARS 375 DQLPSYMQLCFLALDNFINDIAYDVLKEQGFNIIPYLRKSWTDMIEGFLLEAKWYHNGHK Pa3CARS 406 ECLPEYMKGVYTVLYETVNEMAQEAQKSQGRDTLSYVRQALEAYIGAYHKEAEWISSGYL Li3CARS 441 PSLEQYICSSRISISCPTIISQIYFTLET-SIEKSA---IESMYKYHDILYLSGMLLRLP SoBPPS 439 PSLDEYLNIAKISVASPAIISPTYFTFANASHDTAV---IDSLYQYHDILCLAGIILRLP LaLIMS 444 PSLEEYLNIARVSVTCPTIVSQMYFALPI-PIEKPV---IEIMYKYHDILYLSGMLLRLP Ss3CARS 435 PKLEEYLENGWRSIGSTVVLTHAFFGVTH-SLTKEN---IDQFFGYHEIVRLSSMLLRLA Pa3CARS 466 PTFDEYFENGKVSSGHRIATLQPTFMLDI-PFPHHVLQEIDFPSKFND---FACSILRLR Li3CARS 497 DDLGTSTFELKRGDVPKAIQCYMKEK-NIPEEEARDHVKFLIREAWKQMNTA-IEAGSPF SoBPPS 496 DDLGTSYFELARGDVPKTIQCYMKET-NASEEEAVEHVKFLIREAWKDMNTA-IAAGYPF LaLIMS 500 DDLGTASFELKRGDVQKAVQCYMKER-NVPENEAREHVKFLIREASKQINTA-MATDCPF Ss3CARS 491 DDLGTSTDEVSRGDVPKAIQCYMNDNIGASEAEAREHVKWCIWETWKKMNKVRVARDTPF Pa3CARS 522 GDTRCYQADRARGEEASCISCYMKDNPGSTQEDALNHINNMIEETIKKLNWELLKPDNNV Li3CARS 555 TYDLVVAAANLGRVANFVYVDGDGFGVQHSKIIQQIAGLMFEPY--A SoBPPS 554 PDGMVAGAANIGRVAQFIYLHGDGFGVQHSKTYEHIAGLLFEPY--A LaLIMS 558 TEDFAVAAANLGRVANFVYVDGDGFGVQHSKIYEQIGTLMFEPY--P Ss3CARS 551 SQDFIVCAMGMGRMGQYMYHYGDGHGIQHSIIHQQMSTCLFHPSSSN Pa3CARS 582 PISSKKHAFDINRGLHHFYNYRDGYTVASNETKNLVIKTVLEPVPM- Figure 4.1 Sequence alignment of Li3CARS (KX024762) with SoBPPS (AAC26017.1), LaLIMS (ABB73044.1), Ss3CARS (AF527416.1) and Pa3CARS (AAO73863.1). The predicted transit peptide is located a few amino acids before RR(x)8W motif, and the main TPS conserved motifs are labeled accordingly. (Adapted from Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud (2017) Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia. Plant Molecular Biology, 93: 641-657. By permission from Springer Nature). 4.3.1.2 Genomic DNA organization and phylogenetic analysis The genomic sequence of the Li3CARS was amplified from genomic DNA extracted from L. x intermedia leaves and characterized to understand its evolutionary relationship with TPSs from Lavandula. The complete genomic sequence of Li3CARS (GenBank: KX024761) was 2782 bp long and contained a typical intron-exon structure of Lavandula and other angiosperm TPS-a and TPS-b subfamily genes, bearing 7 exons interrupted with 6 introns (Figure 4.2a). Every exon and intron junction of Li3CARS showed an identical pattern of ∼NNN▾GT at 3′ and AG▾NNN~ at 5′, in which 'NNN' represents sequences of exons. A comparison with previously reported genomic DNA of Lavandula TPSs showed that Li3CARS DDLxTxxxE 104 was highly homologous to these TPSs in terms of exon size (Figure 4.2a). The length of exon4 remained conserved in all of the five TPSs, whereas Li3CARS bore unique lengths at exon3 and exon7. Most of the exons, such as exon1, exon4, exon6 and exon7 contained the common TPS conserved motifs at the expected positions. Intron sizes were the most variable genomic structures among the five Lavandula TPSs (Figure 4.2b). The shortest and longest introns in the five Lavandula TPSs were intron-4 (58 bp) in LiCINS and intron-1 (926 bp) in LaLINS, respectively. Those introns were replaced by a small size intron-2 (70 bp) and a large size intron-3 (430 bp) in Li3CARS. Figure 4.2 Schematic presentation of Li3CARS genomic DNA organization and intron length comparison. a) L. x intermedia (cv Grosso) 3CARS genomic DNA organization in comparison with genomic DNAs of previously reported Lavandula TPSs. b) Comparison of intron lengths among Lavandula TPSs. Exons with rectangular boxes are connected with lines of introns (1 - 6). Every exon length is measured by amino acids (shown in the box). The approximate locations of the main conserved motifs are indicated by arrows at Exon1, Exon4, Exon6 and Exon7. The three TPSs, such as LaLIMS- limonene synthase, LaLINS- linalool synthase and LaBERS- trans-α-bergamotene synthase from L. angustifolia (Landmann et al. 2007), while 1,8-cineole synthase (LiCINS) was from L. x intermedia (Demissie et al. 2012). (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). a) b) 105 Based on their amino acid sequences, we constructed a neighbor-joining phylogenetic tree for Li3CARS along with some TPSs subfamilies of angiosperms and gymnosperms (Figure 4.3). The tree showed that Li3CARS was clustered into the TPS-b subfamily. The TPS-b subfamily members were further divided into three subclades (I, II & III). The two angiosperm 3-carene synthases – S. stenophylla 3-carene synthase and L. x intermedia Li3CARS – were grouped separately under subclade II and III, respectively. More specifically, Li3CARS had a very close evolutionary relationship to that of L. angustifolia limonene synthase under the same subclade III. Most angiosperm sesqui-TPSs and gymnosperm TPSs, including P. abies 3-carene synthase, were categorized into TPS-a and TPS-d subfamilies, respectively. 106 Figure 4.3 Phylogenetic analysis of Lavandula and other selected plant monoterpenes and sesquiterpene synthases. a) Lavandula and some plant monoterpenes and sesquiterpene synthases. b) Lavandula TPSs that are identified and functionally characterized so far, except α -pinene synthase from Lavandula viridis (Lv_a-pinene_S), are further compared separately. Numbers at each node represents a bootstrap percentage. The deduced amino acid sequences are used for phylogenetic comparison. GenBank accession numbers b) a) 107 of those genes/proteins used in this phylogenetic analysis are listed in Table D.2. (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). 4.3.1.3 Functional characterization of Li3CARS Expression of the Li3CARS ORF in E. coli RosettaTM (DE3) pLysS strain using the pET41b(+) expression vector produced a soluble protein (64.9 kDa) that could be purified using Ni-NTA-agarose affinity chromatography. The partially purified protein was assayed for activity with GPP, NPP and FPP. The products of the reactions were analyzed by GC-MS. Different products were detected from assays of Li3CARS with GPP and NPP (Figure 4.4). The activity of Li3CARS incubated with GPP (0.5 - 100 μM) extended from 10-90 min with optimum pH (7.0 - 7.5) (Figure 4.5a) and temperature (30oC) conditions. Li3CARS converted GPP to 3-carene (~76 %) and a few other minor products (24 %) (Table 4.2). Michaelis-Menten enzymatic saturation curve was generated using GPP (Figure 4.5b), and the corresponding double-reciprocal plot revealed the Km and Vmax values of 3.69 ± 1.17 µM and 128 ± 7.84 pkat/mg, respectively. The calculated values of turnover (kcat) and catalytic efficiency (kcat/Km) were 2.01 s-1 and 0.56 µM-1s-1, respectively. 108 Figure 4.4 GC-MS analysis of monoterpenes generated by recombinant Li3CARS from NPP and GPP substrates. Assay products of Li3CARS from NPP (a) and GPP (b), as well as authentic standards of 3-carene and limonene (c). Detected monoterpenes from both substrates are α -thujene, β-pinene, α-phellandrene, 3-carene, limonene, 2-carene and terpinolene. Camphor (200 ng mL-1) was used as an internal standard. Detector responses = product concentration (Kcount). (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). c) b) a) 109 Substrate preference of Li3CARS was examined by assaying the affinity-purified protein with NPP (the cisoid isomer of GPP) in assay conditions similar as used for GPP. GC-MS analysis of the reaction mixture revealed multiple products including limonene (33 %), terpinolene (29 %), α-thujene (20 %), α-phellandrene (8.8 %) and 3-carene (8.5 %) (Table 4.2). To investigate the catalytic efficiency of Li3CARS with this particular substrate, we determined the kinetic properties using NPP over the concentration range of 1-150 μM. The enzyme activity showed typical Michaelis-Menten kinetics for all of these detected products (Figure 4.5b-c & Appendix J). The corresponding double reciprocal plots indicated that Li3CARS had apparent Km values of 14 to 36 μM and Vmax values ranging from 50 to 203 pkat mg-1 (Figure 4.5d-e & Appendix J). The turnover (kcat) of Li3CARS varied from 0.636 to 2.59 s -1, depending on the distribution of the products (Table 4.3). However, it had a comparable catalytic efficiency among the products with kcat/Km values ranging from 0.044 to 0.088 µM-1s-1. As a control, affinity-purified extracts from an empty vector transformed E. coli RosettaTM (DE3) pLysS, and boiled affinity-purified Li3CARS protein was assayed with GPP and NPP. The results showed that none of the aforementioned products from both substrates were detected in the control reactions. Table 4.2 Distribution of products from in vitro enzymatic assay of recombinant Li3CARS using GPP and NPP as substrates. Compounds RT GPP NPP % ± SE % ± SE α-Thujene 5.895 - 20.41 ± 0.27 β-Pinene 6.765 5.87 ± 0.17 - α-Phellandrene 7.162 - 8.86 ± 0.20 3-Carene 7.257 76.02 ± 0.54 8.51 ± 0.15 Limonene 7.598 9. 34 ± 0.24 33.02 ± 0.22 2-Carene 8.827 8.77 ± 0.64 - Terpinolene 8.955 - 29.20 ± 0.20 The resulting assay products were quantified based on known internal standard. Means in peak areas and standard errors are calculated from four independent experiments. The major product from GPP is shown in bold. RT = retention time; SE= standard error (n=4). (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). 110 Figure 4.5 Kinetic assay of affinity-purified recombinant Li3CARS with GPP and NPP. Li3CARS activity across a range of pH values (a). Velocity of Li3CARS activity at increasing concentrations of GPP (0.5 - 100 µM) (b) and NPP (1 -150 µM) (c) to produce 3-carene. Lineweaver–Burk (Double-reciprocal) plots for determining the Km and Vmax of Li3CARS from GPP (d) and NPP (e). Detailed Michaelis-Menten kinetic parameters from GPP and NPP are described in Table 4.3. (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). a) b) d) c) e) 111 Table 4.3 Comparison of the kinetic properties of recombinant Li3CARS using GPP and NPP as substrates. Parameters GPP NPP 3-carene 3-carene α-thujene limonene α-terpinolene Km (µM) 3.69 ± 1.17 14.3 ± 2.56 19.19 ± 3.14 23.21 ± 3.34 36.23 ± 4.33 Vmax (pkat/mg) 128 ± 7.84 49.9 ± 2.37 105.7 ± 4.99 164.6 ± 7.27 203.5 ± 8.64 k cat (s-1) 2.070 0.636 1.349 2.117 2.596 k cat/Km (µM-1s-1) 0.561 0.044 0.070 0.088 0.072 (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). 4.3.1.4 Relative expression and level of the corresponding monoterpene Owing to the low EST copy numbers of the candidates from flower glandular trichomes, we predicted that Li3CARS is either differentially expressed or induced by other factors. To test this hypothesis, we performed qPCR analysis of L. x intermedia leaf and flower tissues. The results showed that Li3CARS transcripts were 16-fold more abundant in leaves compared to flowers. The expression pattern of Li3CARS was also examined in response to MeJA application. MeJA induced Li3CARS transcript levels in leaves (Figure 4.6) significantly (2.5-3.8 fold) (P ≤ 0.05) as early as 8 h post-treatment. Not surprisingly, this up-regulation declined after 24 h post-treatment and at 72 h reached a transcript level that was comparable to that in the corresponding control leaves. 112 Figure 4.6 qPCR analysis for temporal expression of Li3CARS from L. x intermedia foliage. Induced expression of Li3CARS from young plant leaves in response to MeJA (5 mM) was comparable to mock-treated controls. Mock samples (controls) were treated with aqueous 0.1 % Triton X-100. The control was assigned the arbitrary value of 1.0. Li3CARS transcript levels were normalized to β-actin and 18S rRNA genes. Results represent mean values of biological replicates ± standard errors (n=3), **P ≤ 0.01. (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). Monoterpene content was quantified in the flowers and leaves of L. x intermedia plants in order to determine the in planta contribution of Li3CARS to EO metabolism. Despite variations in quantity, most of the products obtained from the Li3CARS in vitro assay were detected in leaf tissue (Appendix K), where the gene is strongly expressed. Further, MeJA resulted in a significant increase (P ≤ 0.05) in the levels of 3-carene and limonene at 24 and 48 h post-treatment (Figure 4.7). The 3-carene content reached a maximum of 200 ng mg-1 higher than its control at 48 h post-treatment. Limonene concentration also increased by nearly 95 ng mg-1 of fresh leaf tissue over its control at 24 h post-treatment. The tissue levels of both 3-carene and limonene did not change after 48 h post-treatment. The accumulation of α-thujene – Li3CARS in vitro assay product from NPP – were not affected by 5 mM MeJA in all tested exposure time courses (Figure 4.7). ** ** ** 113 Figure 4.7 Accumulation of monoterpenes in young L. x intermedia plant foliage in response to MeJA. a) 3-Carene, b) α-thujene and c) limonene. Mock samples were treated with water supplemented with 0.1 % Triton X-100. Monoterpene results (ng mg-1 fresh weight) are shown as mean ± standard errors. Asterisk (*) indicates significant differences (* = P< 0.05) between MeJA (5 mM) treated and mock (control) in Student's t-test. Bars show standard errors (n=4). (Adapted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). a) * * b) * * c) 114 4.3.2 Characterization of S-linalool synthase from L. x intermedia 4.3.2.1 Analysis of sequences and phylogenetic relationship Several TPS genes have been cloned from various Lavandula species and functionally characterized. Several partial TPS-like sequences also exist in our previously described EST databases (Lane et al. 2010; Demissie et al. 2012), and in our recently sequenced draft L. angustifolia genome (Malli et al. 2019). Using a sequence similarity-based approach, we screened the assembled transcriptomes reported here for TPS-related sequences. The search demonstrated that the database contained sequences for all TPS genes previously cloned from the three lavender species and several unique TPS-like sequences that have not yet been characterized. Among them, there was a sequence corresponding to a unique TPS-like gene with six exons (unlike the previously reported Lavandula TPSs with seven exons) that was identified in the L. angustifolia draft genome (Malli et al. 2019). This gene, which was later shown to be S-linalool synthase (LiS-LINS), is distantly related to other Lavandula TPSs and lacks one of the common conserved motifs (Figure 4.8). Transcripts for this gene were not present in any of our previously described EST databases including one derived from floral-based glandular trichomes of L. x intermedia (Demissie et al. 2012), indicating that it is not very strongly expressed, or that its mRNA is not very stable. The complete ORF of LiS-LINS (GenBank: MG870571) encoded a protein of 571 amino acids with a predicted molecular mass of 66.14 kDa and calculated pI of 5.16. Comparison of the deduced amino acids to orthologous linalool synthases revealed a high degree of sequence similarity within the conserved motifs. As with most plant TPSs, the key conserved motifs including DDxxD and (N,D)D(L,I,V)x(S,T)x3E) that are crucial for divalent metal ion binding are well conserved in LiS-LINS (Figure 4.8). However, like other S-linalool synthases from different plants, LiS-LINS lacked the N-terminus RR(x)8W motif that is important for substrate ionization and protein stability (Figure 4.8). 115 LiS-LINS 1 MALP-CNIFSPILRSTTT--------------SIFVTEPKYFAKTNLVRIAILLSPNIPK AmNES/LINS1 1 MS------------------------------NLHVKKTDRIAS--GFDVAYLSAANKTH AmNES/LINS2 1 MAFYIGNNLSGVPHACSLNTNITKFAKFGNMSNLHVKKTDRIAS--GFDVAYLSAANKTH LiS-LINS 46 QCRI-NTINSKPYRL-QEYQLDVVSQVSI--------IDERVEKIRELVEWRDEEDPMEG AmNES/LINS1 29 QWSIADDIASTPGSPRLHYPMDFNGQSTRTPDNFSMEYDKKIEEIKNLLRSKREEEPIDR AmNES/LINS2 59 QWSIADDIASTPGSPRLHYPMDLNGQSTRTPDNISLEYDKKIEEIKNLLQSKREEEPIDR LiS-LINS 96 LMFVDAIQRLGVAHHYEKEIEMIMEQHYLNKE---NGIKSLRDVALSFRLLRQRGYHISA AmNES/LINS1 89 LMFVDAIQRLGVNHNFEELIETILRNYYESTSANICGFHTLHDVSLFFRLMRQHGYDISS AmNES/LINS2 119 LMFVDAIQRLGVNHNFEEMIETILRNYYESTSANICGFHTLHDVSLFFCLMRQHGYDISS LiS-LINS 153 DVFNSFKGKDGKFRDEVEQDIWGLVELYEATQLSFEGEDILEEAQDFSSQILQQHLARDP AmNES/LINS1 149 DVFNKFKGDDGRFRGELQRDTRGLMELYEASQLRFEGEYTLDEAESFSSQNLNKYLA-DM AmNES/LINS2 179 NVFNKFKGDDGRFRGELQRDTRGLMELYEASQLRFEGEYTLDEAESFSSQNLNKYLA-DM LiS-LINS 213 KFKGSNIIQMRLRYPYHTTITNFTR--DVGGMNYRWITTLTELSEQQFLIGRDVHKDELL AmNES/LINS1 208 DCSSCRLVTNKLQHPYRKSIGRLTTRYDFRGKN-QWGKTLHELAAMDLRMRKSEYQKELF AmNES/LINS2 238 DCSSRRLVTNKLQHPYRKSIGRLTTRYDFRGKN-QWGKTLQELAAMDLRMRKSEYQKELF LiS-LINS 271 QISKWWSGLNFKRDLKLVRNQPVKWYTWSMAMLADDNTLSAERVQLTKSIVFIYLIDDVF AmNES/LINS1 267 QVSEWWKELRIAENLSLARNQPLKWYTCSMAILIDDITLSEQRIELTKSITFIYLIDDIF AmNES/LINS2 297 QVSEWWKALRIAENLSLARNQPLKWYTWSMAILIDDITLSEQRIELTKFITFIYLLDDIF LiS-LINS 331 DLYGTPQDLTVFTEAVNKWEYAAIDTLPDYMKLCYKSLLDITNQIGSSISEKHGYNPINS AmNES/LINS1 327 DVYGSPEELVIFAEAVSKWDYAAVETLPDYMKLCYKSLLDTTNEIGYKIYEKYGYNPIDS AmNES/LINS2 357 DVYGSPEELVILAEAVSKWDYAAVETLPDYMKLCYKSLLDTTNEIGYKIYEKYGYNPIDS LiS-LINS 391 LKQTWVSLCNAFLVEAQWLASRDLPRAEEYLANGKVSSGVHVVLVHLFFLLGLD-----A AmNES/LINS1 387 LKTTWASLCNAFLEEAKWFASGNLPNATKYLENGKVSSGVYVVMVHLFFLLGLGGTCGSA AmNES/LINS2 417 LKTTWASLCNAFLEEAKWFASGNLPNATKYLENGKVSSGVYVVMVHLFFLFGLGGTCGSA LiS-LINS 446 ANVDDISSLVSCVAAILRLWDDLGSAQDEHQEGRDGSYIECYMRDNPESTFEQARDHVLD AmNES/LINS1 447 IHLNDTSKLMSSVATILRLWDDLGSAKDEHQDGKDGSYIECYMKEHISLSTEQAQQHAID AmNES/LINS2 477 IHLNDTSKLMSSVATILRLWDDLGSAKDEHQDGKDGSYIECYMKEHTSLSLEQAQQHAID LiS-LINS 506 RIGMEWKRLNEECFNLSNPSSSALQFKEAALNMARMISLMYNYDKNQAFPALEKYVQIML AmNES/LINS1 507 LISSEWKLLNKECFNLNHVSTSSI--KKAALNTARMVPLMYSYDENQGLPILEEYVKIML AmNES/LINS2 537 LISSEWKLLNKECFSLNHVSTSSL--KKAALNTAKIVPLMYSYDENQRLPILEEYVKFML LiS-LINS 566 PNLIQS AmNES/LINS1 565 FD---- AmNES/LINS2 595 FD---- Figure 4.8 Multiple sequence alignment of LiS-LINS (MG870571) with its closest plant homologs AmNES/LINS1 (EF433761) and AmNES/LINS2 (EF433762). LiS-LINS shares over 50 % amino acid sequence identity with both AmNES/LINS1 and AmNES/LINS2. As in most angiosperm mTPSs, LiS-LINS also has two aspartate-rich motifs, but lacks RR(x)8W motif. The main TPS conserved motifs are labeled accordingly. (Adapted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining (DDLxSx3E DDxx D (N,D)D(L,I,V)x(S,T)x3E) *** * * ** * * Putative transit peptide 116 monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). Phylogenetic analysis indicated that LiS-LINS belongs to the TPS-g subfamily that lacks RR(x)8W (Figure 4.9), although it exhibited a relatively low amino acid identity with TPSs within this group. LiS-LINS has less sequence similarity to the rest of known TPSs of the genus Lavandula, with maximum amino acid sequence identities with germacrene D synthase (28.74 %) and R-linalool synthase (28.47 %) of L. angustifolia (Figure 4.9a). The highest amino acid identity of LiS-LINS was 53.27 % and 50.57 % with nerolidol synthase (AmNES/LINS-2) and S-linalool synthase (AmNES/LINS-1) of Antirrhinum majus, respectively (Figure 4.9b). Also, this protein is distantly related to known S-linalool synthases of Coriandrum sativum (29.14 % identity) (TPS-b) and C. breweri (22.64 % identity) (TPS-f). 117 Figure 4.9 Phylogenetic analysis of LiS-LINS and selected plant TPSs based on deduced amino acid sequences. Comparison of LiS-LINS amino acid sequence identity with (a) most of previously known Lavandula TPSs, and with (b) selected plant TPS-g subfamily members. The tree shows that LiS-LINS clusters with Li_3-carene_S La_Limonene_S La_(R)-Linalool_S Li_Cineole_S La_beta-phellandrene_S La_Bornyl_diphosphate_S La_alpha-bergamotene_S Li_epi-caryophyllene_STPS-b La_Germacrene_D_S Ls_Germacrene_A_S La_cadinol_STPS-aTPS-g L. x intermedia_(S)-LINS10010010066991001009880 O.brachyantha_(S)-LINS O.sativa_(S)-LINS B.napus_(S)-LINS B.x.oleracea_(S)-LINS R.sativus_(S)-LINS B.rapa_(S)-LINS C.sativa_(S)-LINS A.thaliana_(S)-LINS T.hassleriana_(S)-LINS A.majus_NES/LINS2 A.majus_NES/LINS1 L.x.intermedia_(S)-LINS A.polygama_(S)-LINS A.arguta_(S)-LINS V.vinifera_(S)-LINS/NES/GLINS M.domestica_NES F.ananassa_NES2 F.ananassa_NES199987973459885999999469996567161TPS-b TPS-a a) A –––– 0.10 b) 118 TPS-g, which is different from the TPS-a and TPS-b subfamilies that include all of previously reported Lavandula sesqui-TPS and mono-TPS, respectively. The trees were generated using the Phylogeny Analysis MEGA7.0 program (Kumar et al., 2016). The resulting trees were bootstrap analyzed with 1, 000 replicates, and bootstrap values > 50 % are shown on the trees. The taxon in the rectangular box indicates the L. x intermedia S-LINS sequence identified in this study. The clades of TPS subfamilies were designated as TPS-a, TPS-b and TPS-g according to Bohlmann et al. (1998) and Chen et al. (2011). The scale bar shows the number of amino acid substitution per site. The Genbank accession numbers of TPSs used in the phylogenetic analysis are mentioned in Table D.3. (Reprinted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). 4.3.2.2 Subcellular localization studies Selected bioinformatics tools were used to predict the presence of targeting transit peptides in N-terminus of LiS-LINS protein sequence. Among all tested prediction tools (detailed in Materials and Methods, section 4.2.3), three programs predicted a plastid-targeting N-terminal transit peptide in the LiS-LINS protein. Among these 'ChloroP' and 'TargetP' tools predicted very short plastid-targeting N-terminus transit peptides with a length of four amino acids, while 'iPSORT' detected a 30 amino acid long targeting sequence at N-terminus. To experimentally determine the subcellular localization of LiS-LINS protein, we fused the transit peptide – the first 50 amino acids – of this protein to the N-terminus of GFP reporter gene and expressed the fusion protein transiently in N. benthamiana protoplasts. We analyzed the transient expression of GFP fused constructs in protoplasts by fluorescent microscopy (Figure 4.10). Protoplasts expressing LiS-LINS-TP(50 aa) produced GFP fluorescence in discrete patterns associated with plastids (Figure 4.10d-f). The pattern of the LiS-LINS-TP(50 aa)::GFP accumulation in plastids resembled that of GFP fused to TP(50 aa) of R-linalool synthase (transit peptide), a plastidial monoterpene synthase (Figure 4.10g–i), and was entirely different from empty vector (GFP) that was localized in the cytosol (Figure 4.10a-c). These results clearly showed that the LiS-LINS protein contains a transit peptide that targets it to the plastids. 119 Figure 4.10 Transiently expressed LiS-LINS-TP(50 aa)::GFP fusion in N. benthamiana protoplasts analyzed by fluorescent microscopy. TP – transit peptide, 50 aa – the first 50 amino acids of the full ORF. The "Green" column shows GFP fluorescence detected in the green channel; the "Red" column shows chlorophyll autofluorescence detected in red channel, and the "Merged" column shows combined green and red channels. p326::sGFP (cytosolic) and LiR-LINS-TP(50 aa)::GFP (plastidial targeting transit peptide) are used as controls. The figure shows the plastidial localization of LiS-LINS-TP(50 aa)::GFP similar to the positive control LiR-LINS-TP(50 aa)::GFP, confirming its plastidial targeting enzyme. (Reprinted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). 4.3.2.3 Expression and assay of the recombinant LiS-LINS We expressed the LiS-LINS ORF, excluding the transit peptide, in E. coli RosettaTM (DE3) pLysS strain using the pET41b(+) expression vector, and purified the recombinant protein using Ni-NTA-agarose affinity chromatography. The partially purified recombinant protein was 120 assayed for activity with GPP, NPP, FPP, DMAPP as well as a combined IPP and DMAPP as substrate. The products of each of the reactions were analyzed along with the authentic standards by GC-MS. Linalool was detected as a single product from both assays of partially purified LiS-LINS with GPP and NPP (Figure 4.11a-f). However, the recombinant LiS-LINS protein was catalytically inactive when supplied with FPP, DMAPP, and a mix of IPP and DMAPP. As a control, affinity-purified extracts from an empty vector transformed E. coli, or heat-denatured purified recombinant LiS-LINS proteins were assayed with GPP or NPP. None of the control reactions produced detectable linalool. The assay products of the recombinant LiS-LINS protein supplemented with each of GPP and NPP were further analyzed with GC-FID using a chiral column. The results revealed the presence of enantiomerically pure S-linalool from GPP or NPP supplemented assay reactions (Figure 4.12b-d). 121 Figure 4.11 GC-MS analysis of reaction product generated by the recombinant L. x intermedia S-linalool synthase from GPP, and monoterpene standards. a – f) Gas chromatogram and Mass spectrum of standard S-linalool (a & b), the racemic mixture (50/50 %) of S- and R-linalool (c & d), and LiS-LINS assay product (e & f). (Reprinted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). 122 Figure 4.12 Linalool enantiomer biosynthesis by recombinant linalool synthases from Lavandula, and chiral analysis using GC-FID. a) biosynthesis of linalool enantiomers from GPP/NPP; b) racemic mixture (50/50 %) of R- and S- linalool standard; c) S-linalool from assay product; and d) S-linalool standard. Previously reported recombinant LaLINS catalyzed the conversion of GPP to R-linalool from L. angustifolia (Landmann et al. 2007). The recombinant LiS-LINS assay produced S-linalool as a single product from GPP/NPP (this study). (Reprinted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). 4.3.2.4 Relative expression of the two linalool synthase genes To examine the spatial expression patterns of the two linalool isomer synthases, we quantified the levels of LiS-LINS and LiR-LINS transcripts in L. x intermedia flower, leaf and root tissues using qPCR (Figure 4.13). The results showed that although both genes are expressed 123 in floral tissue, they have distinct expression patterns. In flowers, LiR-LINS transcripts were much more abundant than those of LiS-LINS. On the other hand, transcript levels for LiS-LINS were substantially (~ 7 fold) more abundant than those of LiR-LINS in leaves. In this context, transcript levels for LiR-LINS and LiS-LINS paralleled the concentration of the corresponding linalool enantiomer (R-linalool – 89.8 % and S-linalool – 10.2 %) in L. x intermedia, cv. grosso floral oil (Renaud et al. 2001). Figure 4.13 qPCR analysis for the spatial expression of L. x intermedia linalool synthases (LiS-LINS and LiR-LINS). Transcript levels of LiS-LINS and LiR-LINS in flower, leaf and root tissues of L. x intermedia, cv Grosso. The transcripts were normalized to β-actin gene. The control (in this case, root tissues) was assigned the arbitrary value of 1.0. Bars represent mean values of biological replications ± standard errors (n=3). (Adapted by permission from Springer Nature: Planta, 249(1):271–290, RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula) by Ayelign M. Adal, Lukman S. Sarker, Radesh PN Malli, Ping Liang and Soheil S. Mahmoud, © 2018). 124 4.4 Discussion 4.4.1 L. x intermedia 3-Carene synthase 4.4.1.1 Li3CARS sequence has typical mTPS conserved motifs Availability of public sequence databases facilitates the isolation and functional study of genes using homology-based cloning. In this study, we isolated Li3CARS from L. x intermedia glandular trichomes (Demissie et al. 2012) that encoded 599 amino acids consisting of major conserved motifs present in typical plant TPSs (Figure 4.1). Most of the conserved motifs have been well-characterized in TPSs, among which the first motif located at the very beginning of the N-terminus of the protein is RR(x8)W required in mTPSs for cyclization (Williams et al. 1998), or protein stabilization (Hyatt et al. 2007). The second and third motifs presented at the C-terminus were DDxxD and (N,D)D(L,I,V)x(S,T)xxxE, both of which are responsible for substrate binding and coordination of divalent metal ion cofactors (Degenhardt et al. 2009). Also, alterations of the common residues are often observed in these motifs from sesqui-TPSs. For example, cadinol and germacrene D synthases from L. angustifolia contain a deletion and/or substitution of one arginine (R) in the RR(x8)W motif, whereas one aspartate (D) is substituted by glutamate (E) at DDxxD of germacrene D synthases (Jullien et al. 2014). 4.4.1.2 The evolutionary inference from Lavandula TPS genes As evidenced by the protein analysis data, Li3CARS is highly homologous to the L. angustifolia limonene synthase (LaLIMS), and is clustered with this gene under the angiosperm mTPSs (TPS-b) subfamily (Bohlmann et al. 1998). However, Li3CARS has low homology to limonene synthases and 3-carene synthases from other species. This indicates that LaLIMS and L. x intermedia Li3CARS originated from the same ancestor or one has evolved from the other through gene duplication, which could fuel divergent functional evolution leading to sub-functionalization and/or neofunctionalization of mTPSs. It is not 125 surprising that Li3CARS is more homologous to other Lavandula TPSs rather than to 3-carene synthases from other species, as this phenomenon (that TPSs genes with different functions are more homologous to each other within a given species rather that TPSs with the same function in a different species) has been observed in several cases before. For example, in Sitka spruce, (+)-3-carene synthase family genes share 89-92 % nucleotides identity with (-)-sabinene synthase, but a single amino acid at a specific position determined the plasticity and evolution of both enzymes functions (Roach et al. 2014). The genomic structure of Li3CARS contained typical features of the Class III TPSs, bearing seven exons and six introns (Chen et al. 2011). It also exhibited similar patterns of exon-intron structures to TPSs in most of the angiosperms. 4.4.1.3 Li3CARS has high affinity to GPP The genes and encoding enzymes for the biosynthesis of some of the major Lavandula monoterpenes have been identified and functionally characterized in vitro from economically important species, including L. angustifolia and L. x intermedia (Landmann et al. 2007; Demissie et al. 2011; Demissie et al. 2012). The clone Li3CARS was found to encode an mTPS that catalyzes the conversion of GPP, the universal precursor of the monoterpenes, to 3-carene, and a few other minor products. Generation of major and minor monoterpenes by Li3CARS activity from GPP was not a surprise, as multi-product mTPS from both angiosperms and gymnosperms are well known (Wise et al. 1998; Fäldt et al. 2003; Demissie et al. 2012). The distribution of 3-carene (76 %) produced in vitro by Li3CARS is in close range to 3-carene generated by 3-carene synthases from S. stenophylla (Hoelscher et al. 2003) and from P. abies (Fäldt et al. 2003), accounting for 73 % and 78 %, respectively. However, there are species-specific minor products with distinct distributions, such as myrcene (6 %), 4-carene (4 %) and β-phellandrene (1 %) from S. stenophylla (Hoelscher et al. 2003); myrcene (3 %), 126 terpinolene (11 %), limonene (0.4 %) and β-phellandrene (0.7 %) from P. abies (Fäldt et al. 2003); and β-pinene (5.8 %), limonene (9.3 %) and 2-carene (8.7 %) from L. x intermedia. Variations among minor products with discrete distribution would be expected, as the Li3CARS enzyme is not identical to others, in that it shares only 50 % and 28 % amino acid homology to 3-carene synthase from S. stenophylla and P. abies, respectively. Although the kinetic properties of 3-carene synthase have not yet been studied in Lamiaceae, such investigations have been conducted in coniferous plants (Savage and Croteau, 1993; Hall et al. 2011). The optimum assay conditions for Li3CARS activity with GPP were found to be similar to most of lavender mTPSs (Demissie et al. 2011; Demissie et al. 2012) and 3-carene synthases of S. stenophylla and P. abies ( Savage and Croteau, 1993; Hoelscher et al. 2003). The high catalytic efficiency of Li3CARS was apparent with Km= 3.692 ± 1.17 µM and kcat = 2.07 s-1, which appear to be in close range of the Km (1.47 - 8.09 µM) and kcat (0.06-2.25 s-1) values of coniferous 3-carene synthases (Savage and Croteau, 1993; Hall et al. 2011). However, Li3CARS displayed a lower Km (3.692 ± 1.17 µM) value than that of the most common mTPSs from Lavandula, including β-phellandrene synthase (6.55 ± 1.01 µM), 1,8-cineole synthase (5.75 ± 0.91 µM), linalool synthase (47.7 ± 4.6 µM) and limonene synthase (47.4 ± 3.8 µM) (Landmann et al. 2007; Demissie et al. 2011; Demissie et al. 2012). This low Km value indicates that Li3CARS has a strong affinity to GPP compared to the rest of the previously identified mTPSs from Lavandula. Most Lavandula mTPSs accept both GPP and NPP as substrates, producing comparable amounts of the same end products (Demissie et al. 2011; Demissie et al. 2012). However, the recombinant Li3CARS converted NPP to a different set of compounds than those it derived from GPP. While GPP was converted mainly to 3-carene, NPP was transformed into limonene (33 %), terpinolene (29 %) and α-thujene (20 %) as main products, and minor amounts of a few other monoterpenes including 3-carene (8.5 %). The catalytic efficiency 127 (kcat/Km) of Li3CARS to produce 3-carene was nearly 13-fold lower when NPP, rather than GPP, was used as a substrate. However, Li3CARS displayed a higher catalytic efficiency for producing limonene and terpinolene from NPP than GPP. Taken together, these results indicate that substrate availability may play a key role in the production of certain monoterpenes in plants. It must be noted that the gene and encoding enzyme for NPP biosynthesis have not yet been identified in Lavandula. Therefore, what is observed in in vitro assays may not occur in plants. This argument is supported by the fact that products derived from NPP by recombinant Li3CARS may also be obtained from GPP by various mTPSs in lavenders. For example, L. angustifolia limonene synthase (LaLIMS) converts GPP to limonene (Landmann et al. 2007). EO extracted from L. x intermedia leaves is also abundantly composed of the monoterpenes that are mainly the products of recombinant Li3CARS from GPP (Figure 3.5b). Although recombinant Li3CARS catalyzed both GPP and NPP, the native Li3CARS enzyme more likely uses GPP as a substrate in lavender plant cells to produce the major product of 3-carene and other minor monoterpenes. 4.4.1.4 Li3CARS is differentially expressed To gain insight into the potential biological role of 3-carene in lavenders, we examined the tissue-specific transcriptional expression of Li3CARS by qPCR. The results indicated that Li3CARS is more strongly expressed in leaves than in flowers of L. x intermedia plants. The high transcript levels for this gene in leaves correlated well with the accumulation of 3-carene in leaf tissue, indicating that production of 3-carene is regulated (at least in part) through the transcriptional regulation of Li3CARS. In this context, a high concentration of 3-carene has been reported in peltate glandular trichomes of S. stenophylla (Hoelscher et al. 2003), and shoots and foliage of P. abies (Fäldt et al. 2003), where the corresponding 3-carene synthase genes are highly expressed. 128 The transcriptional expression of Li3CARS was induced by MeJA. Transcript levels for the gene were temporarily elevated upon MeJA application to a maximum at 24 h post-treatment, which positively correlated to the leaf 3-carene concentrations. This outcome is not surprising as MeJA is a known inducer of TPS gene expression in plants. For example, MeJA increased the expression of genes involved in the volatile compound formation 12 h and 24 h post-treatment (Shi et al. 2015). Similarly, several mTPSs and sesquiTPSs were induced with MeJA treatment in P. abies, lima bean, tomato and A. sinensis (Martin et al. 2002; Van Schie et al. 2007b; Menzel et al. 2014; Xu et al. 2016). The up-regulation of Li3CARS transcripts by MeJA treatment points to a potential defensive role for this gene in Lavandula, in particular in young leaves (where 3-carene is mainly produced) that are more prone to insect and pathogen attack. This postulate is supported by several lines of evidence. For example, previous findings demonstrated that several monoterpenes are toxic to insects and pathogens (Fäldt et al. 2003; Fujimoto et al. 2011; War et al. 2011). In this context, EOs of various plants (including lavenders) have been used in controlling important insects and microbes in vitro (Moon et al. 2006; De Rapper et al. 2013; Erland et al. 2015). This is also supported by reports that production of certain defensive monoterpenes, including 3-carene, myrcene, α-pinene, and limonene, in various plants, is induced in response to MeJA, mechanical wounding, pathogen infection and herbivore attack (Martin et al. 2002; Robert et al. 2010). Further, the expression of several mTPS genes, including (+)-3-carene synthase and linalool synthase, is regulated by MeJA, and by pathogens and insects in tomato, rice, lima beans, Sitka spruce and pine trees (Van Schie et al. 2007b; Moreira et al. 2009; Robert et al. 2010; Menzel et al. 2014; Taniguchi et al. 2014). Despite the above arguments, it should be noted that direct evidence for a defensive role for 3-carene in Lavandula is missing at this point. However, in the light of our result, this area is worth exploring. 129 4.4.2 L. x intermedia S-linalool synthase 4.4.2.1 LiS-LINS belongs to the TPS-g subfamily The genomes of most studied plants contain a wide range of TPSs (20-150 genes), which have been classified into 7 families – TPS-a to TPS-h – based on the structure and function of the encoded enzymes (Bohlmann et al. 1998; Chen et al. 2011). While all of the TPS genes reported previously from Lavandula have been assigned to one of the above classifications, scarcely expressed genes have not been reported before. In this study, we cloned and characterized one of these genes (S-LINS) (GenBank: MG870571). The putative function of this gene as a TPS was initially predicted based on the presence of TPS conserved motifs as well as structural homology to known TPSs from Lavandula and other plants. For example, LiS-LINS protein contained both DDxxD and (N,D)D(L,I,V)x(S,T)x3E) motifs that are responsible for substrate binding and coordination of divalent metal ion cofactors (Degenhardt et al. 2009). However, unlike most TPSs, LiS-LINS lacked RR(x)8W – a motif required for product cyclization in Class III TPS proteins (Whittington et al. 2002; Hyatt et al. 2007) – suggesting that this gene potentially encoded acyclic TPS. Based on phylogenetic analysis using deduced amino acids, LiS-LINS was placed in the TPS-g subfamily. The TPS-g subfamily is defined as a group of mTPSs that are responsible for the synthesis of acyclic monoterpenes, including myrcene and ocimene in A. majus, linalool in A. thaliana and V. vinifera (Dudareva et al. 2003; Chen et al. 2003; Martin et al. 2010). Within the TPS-g subfamily, LiS-LINS was clustered with the bifunctional linalool/nerolidol synthase of A. majus (Nagegowda et al. 2008) and Fragaria spp (Aharoni et al. 2004). It is worth noting that unlike LiS-LINS, most of previously known Lavandula TPSs are grouped to TPS-a and -b subfamilies (Demissie et al. 2011; Demissie et al. 2012; Sarker et al. 2013; Jullien et al. 2014). 130 4.4.2.2 Recombinant LiS-LINS catalyzes S-linalool formation from GPP & NPP Several Lavandula EO-related genes and the related encoding enzymes have been previously described (Landmann et al. 2007; Demissie et al. 2011; Demissie et al. 2012; Sarker et al. 2012; Sarker et al. 2013; Demissie et al. 2013; Jullien et al. 2014; Benabdelkader et al. 2015; Despinasse et al. 2017). The R-linalool synthase was first cloned from L. angustifolia, and its recombinant protein catalyzed the conversion of GPP into R-linalool (Landmann et al. 2007). However, the gene responsible for the production of S-linalool in Lavandula has not been previously reported. In this study, we cloned the S-linalool synthase from L. x intermedia (LiS-LINS) through deep transcriptome sequencing. The recombinant LiS-LINS converted GPP – the universal precursor of regular monoterpenes – into a single product, S-linalool. Although most TPSs are multi-products, mono-product TPSs are also common in plants. For example, S-linalool synthase of A. thaliana (Chen et al. 2003) and R-linalool synthase of L. angustifolia (Landmann et al. 2007) are both uni-product enzymes. The recombinant LiS-LINS also accepted NPP (cis isomer of GPP) as a substrate and transformed it to S-linalool. The acceptance of an alternative but closely related substrate (in this case NPP) is not surprising, and has been reported for other plant TPSs before (Demissie et al. 2011; Demissie et al. 2012). 4.4.2.3 LiS-LINS is differentially regulated qPCR analysis showed that LiS-LINS is strongly expressed in flowers – the major EO-producing tissue – compared to other tissues examined. However, transcripts for this gene were much less abundant than those of LiR-LINS. Given that most of the linalool found in lavender flowers are of the R-type, this finding was not surprising. In this context, the abundances of R- and S-LINS transcripts paralleled the concentrations of the corresponding products [R- and S- linalool, respectively] in floral tissue of L. x intermedia cv. grosso reported previously (Renaud et al. 2001). The low expression of LiS-LINS is probably due to a weak promoter activity, or negative regulation of the gene at transcription and /or translation levels. Furthermore, LiS-LINS 131 expression levels could be impacted by the stage of floral development as has been observed with transcript levels of linalool synthase and linalool emissions in Jasminum grandiflorum (Pragadheesh et al. 2017). 4.4.2.4 The implication of S-linalool biosynthesis in Lavandula oil quality Although the EOs of lavenders contain several monoterpenes, linalool and linalyl acetate are considered to be the most important aroma-determining EO constituents in these plants. S-linalool is of particular importance as it imparts a sweet scent to these oils (Renaud et al. 2001; Flores et al. 2005; Özek et al. 2010). In lavenders, the natural enantiomeric distribution of R-linalool and S-linalool varies from 80 - 93 % and 5 - 20 % in L. angustifolia, and from 87 - 97 % and 3 - 13 % in L. x intermedia oils, respectively (Renaud et al. 2001; Baser et al. 2005; Özek et al. 2010; Satyal and Pappas 2016). The possible addition of exogenous S-linalool to enhance the scent and market value of lavender oils has been reported (Renaud et al. 2001; Flores et al. 2005; Aprotosoaie et al. 2014; Do et al. 2015). In this context, some of the commercially marketed lavender EOs contain up to 32 % of S-linalool (Renaud et al. 2001). There is an interest in enhancing S-linalool content in lavenders through metabolic engineering. For example, a recent study reported enhancing S-linalool production by overexpressing the C. breweri S-linalool synthase gene in spike lavender (Mendoza-Poudereux et al. 2014). The cloning of S-LINS from L. x intermedia provides a new tool for such studies. The overexpression of the homologous S-LINS may result in enhanced S-linalool production in lavenders. However, increased S-linalool composition in lavender oils may need further fine-tuning since the chirality of linalool influences the sensory profile and biological activity of lavender oils (Aprotosoaie et al. 2014). 132 Chapter 5: General conclusion Several members of the genus Lavandula are cultivated worldwide for essential oil (EO) production and other horticultural purposes. Most lavender species and their cultivars are phenotypically closely related but, with certain variations in developmental growth stages, demanding high-resolution molecular markers that are able to detect their genetic variations. Differences in species and cultivars also contribute to variations in quality and yield of EOs, in which the quality of EO is determined by a complex mixture of terpenoids, mainly monoterpenes and a lesser amount of sesquiterpenes. Terpenoid metabolism, in turn, can vary across different plant species and cultivars, and it can be regulated by the amount of precursors (substrates) and the expression of genes encoding terpene synthases (TPSs) (Dudareva et al. 2000; Orlova et al. 2009). Although few genes encoding TPSs have been identified from Lavandula and studied in vitro using advanced molecular techniques and genomic resources, the biosynthesis of key monoterpenes and their precursor, geranyl pyrophosphate (GPP), has not yet been determined in these plants. Additionally, despite promising progress on understanding terpenoid metabolism, little attempt has been made to exploit advanced molecular tools and genomic resources to develop diagnostic markers for use in various applications, including fingerprinting and genetic variation studies in Lavandula. Hence, using genomic resources of Lavandula developed in Dr. Soheil Mahmoud's lab, experiments reported in this thesis aimed to develop diagnostic molecular markers, and to identify and functionally characterize cDNAs encoding genes involved in the biosynthesis of monoterpenes and their precursor. Specific objectives included the development of expressed sequence tag (EST)-derived simple sequence repeat (SSRs) (EST-SSRs) markers, and functional characterization of genes responsible for the formation of GPP and two monoterpenes (3-carene and S-linalool). 133 5.1 Development of EST-SSR markers The first objective of this thesis was set to identify and characterize SSRs from Lavandula EST databases. This was achieved by identifying and characterizing 3,459 EST-SSR markers (≥ 12 bp length) from Lavandula EST databases (>13,000 unigenes) (Chapter 2). Most of the identified markers were di- and trinucleotide repeats, and over 250 had a length of ≥ 18 bp. Of these long EST-SSRs, thirty-one were validated (as a proof of concept) across donor species and related species to test whether SSR markers would delineate various cultivars of L. angustifolia and L. x intermedia, and if they applied to related lavender species. The results showed that EST-SSRs had a high polymorphism, with a strong discrimination power in donor species and a considerable transferability into related species. Therefore, these markers could be useful for the identification of economically important lavender species and their cultivars. They might also have applications in quality control, marker assisted breeding, association mapping and assessing the genetic diversity and subsequent conservation of Lavandula. In addition, since the markers showed a remarkable cross-species transferability rate and polymorphism, they might be useful in fingerprinting and genetic studies of related lavender species. Identified Lavandula EST-SSRs can be useful for various applications in donor species as well as in related species that lack initial sequence information to develop species-specific markers. However, detection of SSR markers in plants with unknown ploidy level has been challenging (Albertini et al. 2003). Lavender species are complex polyploid plants with unknown ploidy level (Upson and Andrew 2004). Therefore, an unequal number of SSR loci may exist across different species, which makes it difficult to use SSRs as co-dominant markers (Albertini et al. 2003). Another limitation of this study was the development of only genic-derived SSRs, which may be less effective for detection of variations within very closely related cultivars or species, because most transcribed sequences of a gene are conserved across these plants (Li 134 et al. 2004; Varshney et al. 2005). These challenges could be addressed in the future using different advanced molecular approaches, for example, SSR-Seq (Šarhanová et al. 2018) and genome-derived SSRs (Conesa et al. 2005; Chabane et al. 2005; Liu et al. 2013). SSR-Seq was a recently developed tool to distinguish targeted samples by sequencing of SSR amplicons. It provides clear variations among amplicons within a sample and among samples regardless of ploidy levels. Genome-derived SSRs have been shown to be highly polymorphic markers within and among very closely related species due to the presence of highly variable regions in the genome, e.g., introns, transposable elements, etc. (Chabane et al. 2005; Grover et al. 2007; Tehrani et al. 2008; Liu et al. 2013). Thus, the use of genomic-derived SSR-Seq would be a powerful option to alleviate the challenges encountered with distinguishing very closely related cultivars or species of Lavandula. 5.2 Biosynthesis of precursor (GPP) for regular monoterpenes The second objective of this thesis was to identify and characterize cDNAs encoding short-chain trans-isoprenyl diphosphate synthases involved in the biosynthesis of precursor for regular monoterpenes. This was accomplished by identifying and characterizing L. x intermedia geranyl diphosphate synthase (GPPS) and farnesyl diphosphate synthase (FPP), which produce precursors for monoterpenes and sesquiterpenes, respectively (Chapter 3). While in lavenders FPPS is a homomeric protein, GPPS is made up of a large subunit (LiGPPS.LSU) and a small subunit which has two variants (LiGPPS.SSU1 and LiGPPS.SSU2). Although there are two isoforms, in vitro assays showed that co-purified recombinant proteins of LiGPPS.LSU and LiGPPS.SSU1 were active as heteromeric GPPS, and catalyzed the condensation of IPP and DMAPP to form GPP. However, neither individual subunits nor heteromeric LiGPPS derived from LiGPPS.LSU and LiGPPS.SSU2 produced detectable GPP from these substrates under the same conditions. LiGPPS.SSU1 modified the chain-length specificity of L. x intermedia geranylgeranyl diphosphate synthase (LiGGPPS) in E. coli and endogenous GGPPS in N. 135 benthamiana, leading to an increased GPPS activity. Previously reported GPPS.SSU1 from different plant species have shown a similar chain-length specific modification of GGPPS into GPPS in vitro (Burke and Croteau 2002; Wang and Dixon 2009; Rai et al. 2013). However, such responses were not consistent in different plants, depending on the source plants from where this gene was isolated. This has been shown previously in N. tabacum using a snapdragon GPPS.SSU1 that functioned as a 'modifier' of GGPPS to GPPS activity (Orlova et al. 2009). Different responses were also observed in N. tabacum and N. benthamiana using a peppermint GPPS.SSU1 that functioned as a 'modifier' and an 'accelerator' of GGPPS activities (Yin et al. 2016). Expression of LiGPPS.SSU1 in N. benthamiana resulted in an increase in GPPS activity at the expense of GGPP-derived metabolites, as had aslo been observed with snapdragon GPPS.SSU1. Additionally, the transcript levels of LiGPPS.SSU1 along with LiGPPS.LSU paralleled the levels of total monoterpenes, with more transcripts of these genes in flowers where dominant monoterpenes are produced. Therefore, the identification of GPPS subunits, particularly a large subunit and a small subunit-1 appear to influence GPP biosynthesis in Lavandula, and that could eventually control the amount of supplied GPP for the formation of monoterpenes in these plant EO-producing tissues. The cloned LiGPPS subunits would also find utility in metabolic engineering of monoterpene biosynthesis in microbial systems or plants. The cDNAs encoding GPPS subunits that we discovered function as heteromeric GPPS in Lavandula. In vitro and in vivo experiments confirmed the regulatory roles of LiGPPS small subunits in chain-length specificity of GGPPS and eventually in the formation of GPP. However, because N. benthamiana produces a very low amount of terpenoids, it was difficult to extract detectable terpenoids from transgenic plants expressing LiGPPS.SSU, leading to an inability to assess LiGPPS.SSU roles directly in monoterpene accumulation in plants. Another challenge of this section was determining the roles of LiGPPS.SSU2. Despite sequence similarity across GPPS.SSU2 genes from different plant species, several attempts failed to show its connection 136 to the biosynthesis of GPP through in vitro and heterologous expressions. Hence, a reverse genetic approach would be needed to study the regulation of GPP formation by LiGPPS subunits. Although expression of GPPS subunits paralleled the total monoterpenes in Lavandula tissues, regulatory genes (i.e., transcription factors) that are specific to GPPS subunits or TPSs may also have a significant impact on precursor / monoterpene biosynthesis, as observed in spearmint (Wang et al. 2016; Reddy et al. 2017). Given that, identification of transcription factors specific to GPPS subunits would be useful to further investigate the regulation of GPPS subunit expression to produce GPP, and its derived products. 5.3 Biosynthesis of 3-carene and S-linalool The last aim of this thesis was to identify and characterize cDNAs encoding monoterpene synthases that have potential roles in plant defense and essential oil quality. A portion of this objective was met by identifying and functionally characterizing a cDNA corresponding to a methyl jasmonate-responsive 3-carene synthase (Li3CARS) in vitro (Chapter 4). The recombinant Li3CARS catalyzed the conversion of GPP into 3-carene as the major product, and NPP into multiple products with a small amount of 3-carene, suggesting that monoterpene metabolism may be controlled in part through the availability of precursors. Li3CARS was expressed in leaf and floral tissues, with strong expression in leaves. In fact, 3-carene concentration increased in coniferous plants when the plants were attacked by important pests (e.g., white pine weevil and bark beetle) or treated with the signaling compound methyl jasmonate (MeJA) (Miller et al. 2005; Boone et al. 2011; Ott et al. 2011; Byun-McKay et al. 2012). In Lavandula, 3-carene appears to be a minor monoterpene in flowers, but is highly accumulated in leaf tissues. Since most pests affect the vegetative parts of the plants, the leaf-specificity of 3-carene accumulation may be a sign of its defensive role in Lavandula. When the foliage of L. x intermedia was treated with MeJA, the transcriptional expression of Li3CARS and 3-carene accumulation were up-regulated. Thus, the results suggest that the induction of 137 Li3CARS expression and accumulation of 3-carene by MeJA could have a role in induced defense responses in lavenders. In this section, we described the discovery of a cDNA encoding Li3CARS and characterized its catalytic functions in the synthesis of 3-carene (as a major product from GPP) and other multiple products (from NPP) in vitro. However, whether Li3CARS is involved mainly in 3-carene biosynthesis or production of other monoterpenes using an alternative precursor (e.g., NPP) in vivo remains an open question due to lack of information about NPP as a precursor of monoterpenes in Lavandula. Another limitation of this study may be the use of simulating inducer MeJA rather than directly using pests (insects or pathogens). Although the response of Li3CARS and 3-carene to MeJA showed their potential role in plant defense, their defensive response against specific pests is unknown in Lavandula. Therefore, future studies would be needed, particularly targeting functions of Li3CARS on the biosynthesis of monoterpenes directly in Lavandula to identify Li3CARS-specific monoterpenes, providing insights on the presence/ absence of an alternative precursor (NPP) in these plants. The molecular mechanisms of how MeJA results in the transient increase in levels of Li3CARS transcripts and 3-carene concentration should be targeted to lavender foliage. Furthermore, the defensive role of Li3CARS along with 3-carene should be assessed in Lavandula foliar tissues after exposing them to potential insects or pathogens. Another portion of this objective was met by identifying and functionally characterizing a cDNA corresponding to a scent-determining S-linalool synthase (LiS-LINS) in vitro (Chapter 4). LiS-LINS is a TPS-g subfamily member (mTPSs without RR(x)8W motif) identified from Lavandula. The recombinant LiS-LINS catalyzed the conversion of GPP (and also NPP) into S-linalool. LiS-LINS was highly expressed in flowers, but had much less expression than those corresponding to R-linalool synthase (LiR-LINS), paralleling the concentration of linalool isomers in L. x intermedia oils. The enantiomeric distribution of R-linalool varies from 87 - 97 % and S-138 linalool varies from 3 - 13 % in L. x intermedia oils (Renaud et al. 2001; Özek et al. 2010). These data indicate that production of S-linalool is at least partially controlled by the expression of LiS-LINS. The cloned LiS-LINS would be useful in studies aimed at producing recombinant S-LINS in microbial systems, and in improving essential oil composition and scent quality in lavenders and other plants through metabolic engineering. The work described in this section revealed a TPS-g subfamily monoterpene synthase from Lavandula, in which the recombinant protein of this TPS catalyzes the conversion of precursors to S-linalool in vitro. LiS-LINS transcripts also corresponded to the concentration of S-linalool in lavender oils. Compared with LiR-LINS and R-linalool, the low expression of LiS-LINS and S-linalool concentration could be due to poor transcriptional expression of the LiS-LINS gene, or to the instability of the LiS-LINS mRNA. However, the molecular mechanisms for such low expression of LiS-LINS, which can control S-linalool contents, have not yet been determined in Lavandula. Investigating the mechanism of how S-LINS controls S-linalool biosynthesis in Lavandula should target S-LINS-specific regulatory elements, as transcription factors were identified to be involved in regulation of TPSs in orange fruit and tomato (Spyropoulou et al. 2014; Li et al. 2017). 5.4 Closing summary Overall, experiments reported in this thesis generated information in three areas. Firstly, they identified SSRs from L. angustifolia and L. x intermedia ESTs, a few of which were validated in the donor species and in closely related species. Secondly, using genomic resources, they mediated the cloning and investigating the function of L. x intermedia GPPS, the enzyme that produces precursor for regular monoterpenes, in plants. Thirdly, they led to the identification, cloning and functional characterization of two monoterpene synthases responsible for formation of 3-carene and S-linalool from L. x intermedia. 3-Carene synthase is a leaf-specific and methyl jasmonate-responsive gene, and its enzyme has a preference for GPP 139 rather than NPP in producing 3-carene. The floral-based S-linalool synthase gene encodes a protein that accepts GPP/NPP as substrates to produce S-linalool. The EST-SSR markers, and the genes encoding short-chain trans-isoprenyl diphosphate synthases and monoterpene synthases identified in this thesis would be useful for improving Lavandula EO yield and quality through marker assisted breeding, and plant biotechnology. For example, the cloned trans-IDS and terpene synthase genes could be used to enhance monoterpene production through overexpression of these genes under the control of strong and tissue-specific promoters in plants. Further, these genes could be useful for the mass production of monoterpenes in the heterologous hosts (e.g., bacterial, yeast, and other plants, such as tobacco) through metabolic engineering. 140 Bibliography Adal AM, Demissie ZA, Mahmoud SS (2015) Identification, validation and cross-species transferability of novel Lavandula EST-SSRs. 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Proc Natl Acad Sci 201705689. doi: 10.1073/pnas.1705689114 169 Appendices Appendix A: Reagents and buffers used in EST-SSR validation 1. 0.5 M EDTA (pH 8.0) Add 93.05 g of Na2EDTA in 400 ml of RO water Stir thoroughly and adjust pH 8.0 with pellet NaOH (the solution becomes transparent when the pH reaches to 8) Bring volume to 0.5 L with RO water Autoclave and store at RT 2. 50x TAE buffer, 0.5 L 121 g Tris base (MW=121.1 g/mol) 28.5 ml glacial acetic acid 50 ml 0.5 M EDTA (pH 8.0) a. Dissolve 121 g Tris in 300 ml sterile reverse osmosis (RO) water with gently stirring b. Add EDTA and glacial acetic acid c. Bring the final volume to 0.5 L with sterile RO water d. Labeled properly and autoclave at 121 °C for 30 min e. Store at RT 3. 1x TAE buffer, 4 L a. Measure 80 ml 50x TAE b. Bring the final volume to 4 L with RO water c. Store at RT 4. 5x TBE buffer, 1 L, pH 8.3 54 g Tris base (MW=121.1 g/mol) 27.5 g boric acid (MW=61.83 g/mol) 20 ml of 0.5 M EDTA (pH 8.0) 170 a. Dissolve Tris base in 600 ml RO water with gentle stirring b. Add boric acid c. Add EDTA solution d. Adjust pH to 8.3 and bring up the final volume to 1 L e. Autoclave at 121 °C for 30 min and store at RT 5. 0.5x TBE working solution, 2 L a. Measure 200 ml 5x TBE b. Bring the final volume to 2 L with RO water c. Store at RT 6. 10 % Ammonium per sulfate (APS) (v/w) Dissolve 1 g APS (MW= 228.2 g/mol) in 10 ml sterile RO water Store in 4 °C (prepared fresh after 30 days) 7. 1M Tris (pH 8.0), 0.5 L a. Dissolve 60.55 g Tris base in 400 ml b. Add 20 ml concentrated HCl c. Bring the final volume to 0.5 L with RO water Autoclave at 121 °C for 30 min and store at RT 8. 10 mM Tris (pH 8.0) buffer, 100 ml Take 2 ml 1 M Tris (pH 8.0) Bring the final volume to 100 ml with sterilized RO water Store at 4 °C 9. 1 % agarose gel Add 0.5 g of Agarose into 250 ml Erlenmeyer flask containing 50 ml 1x TAE buffer Boil in microwave with frequent monitoring until it gets clear Cool down to ~50 °C 171 Add 1 µl SYBR safe and mix gently Pour into the gel cast to solidify 10. 6 % polyacrylamide gel (PAGE), 1.5 mm gel (10 ml) Add sequentially: a. 5.73 RO water b. 1.67 ml Acrylamide 40 % solution [Acrylamide and Bis-Acrylamide (19:1)] c. 2.4 ml 5x TBE buffer d. 200 µl 10 % APS e. 10 µl TEMED Mix thoroughly and pour into gel cast using pasture pipette (avoid bubbles!) Wait till completely polymerized 172 Appendix B: List of Lavandula EST-SSR motifs classified under Category I (≥18 bp in length). Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO L. angustifolia cv lady flower EST-SSRs La_flower_EST-2 758 (GCT)7 21 96-117 CDS AT4G29860.1 :(EMBRYO DEFECTIVE 2757) La_flower_EST-3 819 (TCG)8 24 440-464 CDS AT1G31190.1 : inositol monophosphatase family La_flower_EST-4 819 (TCGTCA)4 24 466-490 CDS AT1G31190.1 La_flower_EST-5 826 (TC)13 26 37-63 5' UTR AT3G27810.1 : MYB DOMAIN PROTEIN La_flower_EST-6 595 (AT)24 48 462-510 3' UTR . La_flower_EST-8 419 (CTT)6 18 200-218 CDS . La_flower_EST-9 729 (TA)23 46 107-153 5' UTR B9SF29_RICCO : putative TF La_flower_EST-10 747 (AAT)7 21 189-210 CDS . La_flower_EST-11 839 (CT)12 24 118-142 5' UTR . La_flower_EST-12 839 (AAG)6 18 293-311 CDS . La_flower_EST-13 827 (GCA)6 18 232-250 CDS . La_flower_EST-14 890 (GAG)6 18 234-252 CDS Q2V9B8_SOLTU : RNA-binding protein AKIP1-like La_flower_EST-16 790 (GGC)6 18 189-207 5' UTR . La_flower_EST-17 770 (CT)10 20 195-215 CDS Q43115_9ROSA : Ribulose bisphosphate carboxylase small chain La_flower_EST-18 761 (TC)11 22 670-692 3' UTR . La_flower_EST-19 904 (TC)16 32 49-81 5' UTR Q700A6_CICAR : non-specific lipid transporter protein La_flower_EST-20 853 (CAA)6 18 791-809 CDS B9RZT1_RICCO : WRKY TF La_flower_EST-21 848 (TTC)12 36 35-71 5'UTR C6FFS5_CAPAN : Homeodomain leucine-zipper1 173 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_flower_EST-23 803 (AG)11 22 226-248 CDS B9SKU3_RICCO : root phototropism protein La_flower_EST-24 661 (GTG)8 24 438-462 CDS . La_flower_EST-25 1048 (AG)18 36 329-365 5' UTR A9PFP2_POPTR: Glutathione peroxidase La_flower_EST-26 749 (TC)10 20 129-149 5' UTR . La_flower_EST-27 737 (TTATTT)6 24 606-630 3' UTR AT5G50850.1: pyruvate dehydrogenase La_flower_EST-29 703 (CAGCC)4 20 81-101 5' UTR B0BLJ6_LOTJA La_flower_EST-30 811 (AGAAAA)4 24 158-182 CDS . La_flower_EST-31 789 (TA)14 28 625-653 3' UTR RAC13_GOSHI :Rac-like GTP-binding protein RAC13 La_flower_EST-32 593 (GGA)6 18 379-397 CDS . La_flower_EST-33 723 (GA)25 50 61-111 5' UTR . La_flower_EST-34 776 (GCT)7 21 343-364 CDS . La_flower_EST-35 911 (AG)14 28 178-106 CDS B1Q3F6_SOLLC : Succinic semialdehyde reductase La_flower_EST-36 739 (AGA)7 21 310-331 CDS . La_flower_EST-37 464 (TC)14 28 353-381 3' UTR . La_flower_EST-38 389 (GA)11 22 223-245 CDS . La_flower_EST-39 670 (GAA)7 21 143-164 CDS . La_flower_EST-40 652 (AG)16 32 32-64 5' UTR . La_flower_EST-41 667 (CT)14 28 544-572 3' UTR . La_flower_EST-42 1529 (AAGCCA)4 24 413-437 CDS . La_flower_EST-44 564 (GA)11 22 363-384 3' UTR . La_flower_EST-45 589 (AT)14 28 415-443 3' UTR . La_flower_EST-46 1064 (TCT)8 24 38-62 5' UTR A9XU50_GOSHI : Phosphoethanolamine N-methyltransferase 174 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_flower_EST-47 944 (AG)13 26 44-70 5' UTR D2CFI9_TOBAC :Leucine-rich repeat protein La_flower_EST-48 860 (GAT)8 24 22-46 5' UTR . La_flower_EST-50 792 (CA)15 30 22-52 5' UTR La_flower_EST-51 991 (CT)14 28 165-193 5' UTR GAPN_NICPL :NADP-dependent glyceraldehyde-3-phosphate dehydrogenase La_flower_EST-52 751 (ACC)8 24 98-122 CDS . La_flower_EST-53 1236 (GA)14 28 22-50 5' UTR . La_flower_EST-54 836 (AT)25 50 713-763 3' UTR . La_flower_EST-55 819 (CTCCT)5 25 26-51 CDS . La_flower_EST-56 1228 (GGC)8 24 841-865 CDS . La_flower_EST-57 1573 (GGTGCA)4 24 1306-1330 CDS . La_flower_EST-58 1287 (AG)16 32 585-617 5' UTR . La_flower_EST-59 842 (GATTT)6 30 497-527 5' UTR . La_flower_EST-60 714 (CA)10 20 28-48 CDS . La_flower_EST-61 843 (AT)10 20 382-402 5' UTR . La_flower_EST-62 946 (CAC)8 24 451-475 CDS . La_flower_EST-63 809 (CAT)7 21 244-265 CDS . La_flower_EST-64 728 (CGCCT)4 20 211-231 5' UTR . La_flower_EST-65 772 (GGC)8 24 86-110 CDS . La_flower_EST-66 771 (TC)12 24 682-706 3' UTR . La_flower_EST-67 837 (CTCCAC)4 24 359-383 CDS . La_flower_EST-68 752 (TA)18 36 79-115 CDS . La_flower_EST-69 689 (AG)10 20 553-573 3'UTR . La_flower_EST-70 712 (CT)17 34 118-152 5'UTR . La_flower_EST-71 763 (TC)13 26 245-271 CDS . La_flower_EST-72 478 (CCACAT)5 30 100-130 CDS . 175 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_flower_EST-73 896 (TTC)7 21 46-67 CDS . La_flower_EST-74 743 (GA)10 20 664-684 CDS . La_flower_EST-75 727 (CT)10 20 31-51 5' UTR . La_flower_EST-76 1055 (AG)12 24 84-108 5' UTR . La_flower_EST-77 758 (TC)12 24 511-535 3' UTR . La_flower_EST-79 733 (GA)21 42 35-77 5' UTR . La_flower_EST-80 861 (GAG)7 21 520-541 CDS . La_flower_EST-81 1473 (TTTA)5 20 280-300 CDS . La_flower_EST-82 613 (CT)21 42 36-78 5' UTR . La_flower_EST-84 820 (ATAG)5 20 95-115 5' UTR . La_flower_EST-85 868 (TCATCC)5 30 267-297 CDS . La_flower_EST-86 799 (CCACAT)5 30 172-202 CDS . La_flower_EST-87 852 (GA)14 28 26-54 5'UTR . La_flower_EST-88 404 TA)32 64 116-180 CDS . La_flower_EST-89 745 (AG)11 22 62-84 5' UTR . La_flower_EST-90 655 (TA)22 44 376-418 3' UTR . La_flower_EST-93 786 (CT)13 26 78-104 5' UTR . La_flower_EST-94 739 (AG)11 22 247-269 CDS . La_flower_EST-95 714 (CT)16 32 508-540 3' UTR . La_flower_EST-96 1305 (TC)11 22 1198-1220 3' UTR . La_flower_EST-98 830 (TC)10 20 118-138 5' UTR . La_flower_EST-99 876 (CATCTC)4 24 46-70 5' UTR . La_flower_EST-101 811 (GAT)7 21 541-562 CDS SCS_SALOF : 1,8-cineole synthase La_flower_EST-102 567 (TC)10 20 461-481 3' UTR . La_flower_EST-103 849 (AGGAGA)4 24 725-749 3' UTR . La_flower_EST-104 837 (GCT)9 27 32-59 5' UTR . La_flower_EST-106 813 (CT)14 28 97-125 5' UTR . 176 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_flower_EST-107 726 (TA)10 20 50-70 CDS . La_flower_EST-108 971 (TC)11 22 25-47 5' UTR . La_flower_EST-109 1162 (TG)10 20 304-324 5' UTR . La_flower_EST-111 419 (CCTCT)4 20 355-375 CDS . La_flower_EST-112 647 (TC)18 36 494-530 CDS . La_flower_EST-113 942 (AG)23 46 196-242 5' UTR . La_flower_EST-114 674 (GA)12 24 588-612 CDS . La_flower_EST-115 633 (AG)10 20 27-47 5' UTR . La_flower_EST-116 739 (TCCCTC)5 30 20-50 5' UTR . La_flower_EST-117 782 (TC)12 24 118-142 5' UTR . La_flower_EST-118 616 (TA)14 28 483-511 3' UTR Q6E0V0_NICGL : Non-specific lipid-transfer protein La_flower_EST-119 2142 (AG)10 20 814-834 5' UTR . La_flower_EST-120 763 (CCATCA)10 60 634-694 CDS . La_flower_EST-121 723 (CCA)8 24 347-371 CDS . La_flower_EST-122 653 (TC)20 40 88-128 5' UTR . La_flower_EST-124 442 (GGT)8 24 159-183 CDS . La_flower_EST-125 779 (GAGGGA)4 24 437-461 CDS . La_flower_EST-127 631 (GAA)7 21 204-225 CDS . La_flower_EST-128 438 (CAC)7 21 133-154 CDS . La_flower_EST-130 1026 (GGGAAT)4 24 309-333 CDS . La_flower_EST-131 765 (AG)22 44 232-276 5' UTR . La_flower_EST-133 1442 (CCTCTC)4 24 238-262 5' UTR A9PIY0_POPJC :Peptidyl-prolyl cis-trans isomerase La_flower_EST-135 631 (GGTTT)4 20 158-178 CDS . La_flower_EST-136 826 (GA)17 34 109-143 5' UTR . La_flower_EST-138 632 (TC)13 26 208-234 CDS . La_flower_EST-139 795 (TC)10 20 35-65 CDS . 177 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_flower_EST-141 799 (TGACGA)4 24 241-265 CDS . La_flower_EST-142 573 (TCAGGT)4 24 291-115 CDS . La_flower_EST-143 797 (TC)13 26 26-52 5' UTR . La_flower_EST-144 809 (CTC)7 21 365-386 CDS . La_flower_EST-145 763 (TA)16 32 260-302 CDS . L. angustifolia cv. Lady leaves EST-SSRs La_leaf_EST-1 817 (AAG)6 18 386-404 CDS . La_leaf_EST-3 830 (TTG)7 21 286-307 CDS . La_leaf_EST-4 821 (GAG)6 18 225-243 CDS . La_leaf_EST-6 746 (TGC)7 21 568-589 CDS . La_leaf_EST-7 788 (AG)15 30 75-105 CDS . La_leaf_EST-8 788 (TA)12 24 712-736 3'UTR . La_leaf_EST-9 777 (AAT)11 33 161-194 5'UTR . La_leaf_EST-10 1303 (AG)16 32 774-806 CDS . La_leaf_EST-11 847 (TTTTA)4 20 188-208 CDS . La_leaf_EST-12 749 (TC)19 38 42-80 5'UTR . La_leaf_EST-13 737 (AAATGC)4 24 139-163 CDS . La_leaf_EST-14 737 (TGCAAG)4 24 159-183 CDS . La_leaf_EST-15 762 (TATTT)4 20 123-143 5'UTR . La_leaf_EST-17 786 (TCT)6 18 460-478 CDS . La_leaf_EST-18 692 (TTC)7 21 46-67 CDS . La_leaf_EST-19 935 (CT)15 30 33-63 5'UTR . La_leaf_EST-20 735 (AG)20 40 61-100 5'UTR . La_leaf_EST-21 808 (AT)16 32 31-63 5'UTR . La_leaf_EST-22 808 (CAG)7 21 194-215 5'UTR . La_leaf_EST-23 450 (TG)10 20 296-316 3'UTR . 178 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO La_leaf_EST-24 813 (AGA)6 18 415-433 CDS . La_leaf_EST-25 600 (GA)10 20 491-511 CDS . La_leaf_EST-26 679 (CCG)8 24 56-80 CDS . La_leaf_EST-27 795 (ACC)6 18 623-641 CDS . La_leaf_EST-29 1264 (CCA)6 18 164-282 CDS . La_leaf_EST-30 752 (TC)14 28 287-315 5'UTR . La_leaf_EST-31 289 (GGC)6 18 171-189 3'UTR . La_leaf_EST-32 766 (AG)16 32 34-66 5'UTR . La_leaf_EST-34 500 (CTC)8 24 154-178 CDS . La_leaf_EST-35 1415 (TC)16 32 1295-1327 3'UTR . La_leaf_EST-36 839 (CT)13 26 31-57 5'UTR . La_leaf_EST-37 822 (TGGAGG)9 54 744-798 CDS . La_leaf_EST-38 763 (AG)11 22 210-232 5'UTR . La_leaf_EST-39 824 (TC)10 20 23-43 5'UTR . La_leaf_EST-40 780 (AG)14 28 25-53 5'UTR . La_leaf_EST-41 697 (GA)15 30 489-519 CDS . La_leaf_EST-42 1264 (AGA)6 18 523-541 CDS . L. x intermedia cv. Grosso oil glands EST-SSRs Li_gland_EST-2 804 (TC)16 32 217-249 CDS AT5G43270.1: squamosa promoter binding protein-like Li_gland_EST-3 694 (GGC)6 18 126-144 5' UTR Q8L8H3_9ROSI: Iron-stress related protein Li_gland_EST-4 849 (GCAGTT)5 30 54-73 CDS AT5G07990.1: Cytochrome P450 superfamily protein Li_gland_EST-5 773 (AG)18 36 51-87 5' UTR C5X955_SORBI : Phosphatidate cytidylyltransferase 179 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO Li_gland_EST-6 391 (TC)22 44 43-87 . AT3G47750.1 : ABCA4 | ATP binding cassette subfamily A4 Li_gland_EST-7 794 (AAAAT)4 20 497-517 CDS AT4G38590.2 : BGAL14 | beta-galactosidase Li_gland_EST-9 567 (CGC)6 18 426-444 CDS AT5G65700.2: Leucine-rich receptor-like protein kinase Li_gland_EST-10 1018 (AACCC)4 20 72-92 5' UTR AT3G10980.1: PLAC8 family protein Li_gland_EST-11 663 (TTC)6 18 499-517 3' UTR AT4G09720.3: RAB GTPase homolog G3A Li_gland_EST-12 550 (AGGTCC)4 24 105-129 CDS AT1G50920.1 : Nucleolar GTP-binding protein Li_gland_EST-13 1009 (AG)18 36 30-66 5' UTR AT1G69120.1: AP1 | K-box region and MADS-box TF Li_gland_EST-15 816 (CCTTT)4 20 178-198 CDS . Li_gland_EST-16 507 (GCG)7 21 201-222 CDS B9SLM9_RICCO: Arginyl-tRNA--protein transferase Li_gland_EST-17 1184 (CGC)7 21 63-84 CDS TPIC_SPIOL : Triosephosphate isomerase Li_gland_EST-18 766 (GGA)6 18 457-475 CDS B9SKK4_RICCO: ELMO domain-containing protein Li_gland_EST-19 692 (AT)10 20 441-461 CDS . Li_gland_EST-20 693 (TCTGA)4 20 439-459 CDS . Li_gland_EST-21 723 (TGA)7 27 516-543 CDS Q9ATD9_GOSHI : BNLGHi233 Li_gland_EST-22 867 (CT)11 22 188-110 CDS . Li_gland_EST-24 789 (TC)11 22 55-77 5' UTR Q8LPD2_TOBAC: Protein kinase Ck2 regulatory subunit Li_gland_EST-25 845 (AG)10 20 760-780 CDS . Li_gland_EST-27 609 (GA)10 20 397-417 CDS . 180 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO Li_gland_EST-30 985 (TC)11 22 47-69 5' UTR B9RKT6_RICCO: Aldehyde dehydrogenase Li_gland_EST-32 707 (CT)10 20 32-52 5' UTR . Li_gland_EST-33 1310 (ATG)6 18 914-932 CDS Q9SNY7_TOBAC: Sucrose-6-phosphate synthase A Li_gland_EST-34 166 (CT)10 20 119-139 CDS . Li_gland_EST-35 645 (GGT)7 21 474-495 CDS . Li_gland_EST-36 1406 (TCACCT)4 24 826-850 CDS B9T4W3_RICCO: Zinc ion binding protein Li_gland_EST-37 694 (CCG)6 18 194-112 CDS . Li_gland_EST-38 437 (TC)11 22 71-93 CDS . Li_gland_EST-39 437 (TC)14 28 239-267 CDS . Li_gland_EST-40 711 (ATCTCT)4 24 364-388 CDS . Li_gland_EST-41 1404 (GCT)6 18 848-866 CDS . Li_gland_EST-42 1017 (ATT)7 21 455-476 CDS . Li_gland_EST-44 638 (AT)15 32 512-544 3' UTR . Li_gland_EST-45 818 (GGC)6 18 239-257 CDS Li_gland_EST-46 736 (CTG)6 18 555-573 3' UTR . Li_gland_EST-50 723 (CCT)8 24 244-268 CDS . Li_gland_EST-51 240 (GTG)7 21 155-176 CDS . Li_gland_EST-52 845 (AT)10 20 699-719 3' UTR Q45W76_ARAHY: Ubiquitin-conjugating enzyme 2 Li_gland_EST-53 769 (GA)10 20 56-76 5' UTR . Li_gland_EST-54 832 (TC)22 44 195-139 CDS AT5G52920.1: plastidic pyruvate kinase beta subunit Li_gland_EST-55 855 (TC)13 26 74-100 CDS . Li_gland_EST-56 693 (CT)10 20 492-512 CDS . Li_gland_EST-57 765 (CAT)6 18 352-370 CDS . 181 Seq /gene ID validated SSR name Seq length (bp) Motif SSR Length SSR position SSR location GO Li_gland_EST-60 400 (CTCCT)5 25 33-58 CDS . Li_gland_EST-61 715 (AG)10 20 23-43 CDS Q6LB28_SOLLC: Histone H3 Li_gland_EST-62 604 (CCG)6 18 51-69 5' UTR . Li_gland_EST-63 465 (GAG)7 21 178-200 CDS . Li_gland_EST-64 709 (GCTCCA)4 24 94-118 CDS C5YW39_SORBI: 40S ribosomal protein S12 Li_gland_EST-65 962 (TC)10 20 58-78 CDS B9RZT4_RICCO: Galactokinase, mevalonate Li_gland_EST-66 798 (CT)11 22 58-80 5' UTR . Li_gland_EST-67 872 (GGTGAG)4 24 281- 305 CDS . Li_gland_EST-68 808 (AG)10 20 174-194 5' UTR B9RK53_RICCO: Serine/ threonine-protein kinase 182 Appendix C: Estimated effective multiplex ratio, mean heterozygosity and marker index for validated makers Validation of 31 EST-SSR markers in L. angustifolia and L. x intermedia species as shown by other marker efficiency indices (E = effective multiplex ratio; H.av = mean heterozygosity; MI = marker index). The highest values of these indices indicate good markers. EST-SSRs L. angustifolia (n= 8) L. x intermedia (n=7) Na Pa E H.av MI Na Pa E H.av MI LAF1 6 6 3.78 0.009 0.033 6 6 1.86 0.010 0.019 LAF2 4 4 1.67 0.014 0.023 2 2 1.29 0.033 0.042 LAF3 - - - - - - - - - - LAF4 4 4 1.11 0.011 0.012 3 2 1.14 0.022 0.026 LAF5 4 4 2.11 0.014 0.029 4 4 2.86 0.015 0.042 LAF6 3 0 3.00 0.000 0.000 5 1 4.43 0.006 0.026 LAF7 - - - - - - - - - - LAF8 2 2 3.22 0.009 0.028 1 1 0.57 0.070 0.040 LAF9 4 4 1.78 0.014 0.024 3 3 1.29 0.023 0.030 LAF10 2 2 1.00 0.028 0.028 2 2 1.00 0.036 0.036 LAF11 4 4 2.22 0.014 0.030 4 4 1.57 0.017 0.027 LAF12 - - - - - - - - - - LAF13 7 4 4.11 0.008 0.032 7 7 4.00 0.010 0.040 LAF14 - - - - - - - - - - LAF15 5 4 2.67 0.011 0.029 5 5 3.14 0.013 0.042 LAF16 6 6 2.00 0.011 0.021 1 1 0.71 0.058 0.042 LAF18 3 3 3.22 0.009 0.028 4 4 2.86 0.015 0.042 LAF19 5 5 2.78 0.011 0.030 5 5 2.00 0.014 0.027 LAF20 5 5 2.44 0.011 0.027 4 4 2.14 0.018 0.038 LAF21 4 3 2.22 0.014 0.030 2 1 1.86 0.009 0.018 LAL2 7 7 2.89 0.008 0.022 7 6 3.14 0.010 0.032 LAL3 - - - - - - - - - - LAL4 10 8 5.11 0.006 0.031 9 5 6.29 0.007 0.042 Subtotal 85 75 74 63 Mean 4.7 4.2 2.63 0.011 0.026 4.1 3.5 2.34 0.021 0.034 LINT1 - - - - - - - LINT4 6 5 4.22 0.008 0.033 7 5 4.14 0.010 0.041 LINT5 1 1 0.11 0.022 0.002 2 2 1.43 0.029 0.042 LINT6 2 2 0.78 0.038 0.030 3 2 1.71 0.023 0.040 LINT10 3 2 0.67 0.049 0.033 3 2 1.43 0.029 0.042 LINT11 - - - - - - - - - - LINT12 2 2 1.44 0.022 0.032 2 2 1.14 0.035 0.040 LINT14 4 2 3.00 0.010 0.031 7 6 4.14 0.010 0.041 Subtotal 18 14 24 19 Mean 3 2.3 1.70 0.025 0.027 4 3.2 2.33 0.023 0.041 Total 103 89 98 82 Mean 4.3 3.7 2.40 0.015 0.026 4.1 3.4 2.34 0.022 0.036 Max 10 8 5.11 0.049 0.033 9 7 6.29 0.070 0.042 183 EST-SSRs L. angustifolia (n= 8) L. x intermedia (n=7) Na Pa E H.av MI Na Pa E H.av MI Min 1 0 0 0 0 1 1 0.57 0.006 0.018 n= number of cultivars; Na= number of bands (alleles); Pa= polymorphic bands; E = effective multiplex ratio; H.av = mean heterozygosity; MI = marker index; "-" represents no results from markers failed to amplify detectable products. 184 Appendix D: List of GenBank accessions for protein sequences used in phylogenetics Table D.1 Short-chain trans-IDSs Genes Accession No. Source Species Short-chain trans-IDS AGH33891.1 Lavandula x intermedia AAS82859.1 Antirrhinum majus GPPS.SSU-1 AAF08792.1 Mentha x piperita BAF98300.1 Hevea brasiliensis AAS82870.1 Clark ia breweri AGL91646.1 Catharanthus roseus ACQ90681.1 Humulus lupulus CAO38946.1 Vitis vinifera AEZ55678.1 Salvia miltiorrhiza MN064856 Lavandula x intermedia EAY87007.1 Oryza sativa Indica GPPS.SSU-2 BAF98303.1 Hevea brasiliensis CAO64763.1 Vitis vinifera NP_001312125.1 Nicotiana tabacum AEZ55680.1 Salvia miltiorrhiza NP_195558.1 Arabidopsis thaliana JAT53303.1 Anthurium amnicola NP_001310395.1 Solanum pennellii MN064857 Lavandula x intermedia AAS82860.1 Antirrhinum majus GPPS.LSU AF182828_1 Mentha x piperita ACQ90682.1 Humulus lupulus AGL91645.1 Catharanthus roseus GGPPS MN064858 Lavandula x intermedia P34802.2 Arabidopsis thaliana MN064859 Lavandula x intermedia AGQ04160.1 Lavandula angustifolia ADT70780.1 Salvia miltiorrhiza AAR27053.1 Ginkgo biloba FPPS AAL82595.1 Musa acuminata AAD17204.1 Artemisia annua AAQ14871.1 Zea mays ACF21779.1 Gentiana lutea ALT07952.1 Leucosceptrum canum AAY87903.1 Panax ginseng AAQ56011.1 Hevea brasiliensis CAA57893.1 Parthenium argentatum AFW98441.1 Chrysanthemum lavandulifolium Short-chain cis-IDS CPPS ADO17798.1 Tanacetum cinerariifolium zFPPS B8XA40.1 Solanum habrochaites NPPS ACO56895.1 Solanum lycopersicum LPPS AGH33890.1 Lavandula x intermedia Note: GenBank accession numbers with boldfaced show the genes from the current study. 185 Table D.2 Selected plant TPSs and Li3CARS. Accession No. Source Species Gene/ protein name Accession No. Source Species Gene/ protein name AAB70907.1 Abies grandis (-)-4S-limonene synthase AAF65545.1 Perilla citriodora limonene synthase AAF61453.1 Abies grandis beta-phellandrene synthase AAF76186.1 Perilla frutescens mercene synthase AAB70707.1 Abies grandis (-)-camphene synthase ACN42009.1 Perilla setoyensis linalool synthase AAB71085.1 Abies grandis pinene synthase AAK39127.2 Picea abies beta-phellandrene synthase-like AAC24192.1 Abies grandis alpha-Bisabolene Synthase AAS47689.1 Picea abies alpha-Bisabolene Synthase BAC92722.1 Chamaecyparis obtusa limonene/borneol synthase AAO73863.1 Picea abies (+)-3-carene synthase AAM53945.1 Citrus limon (-)-beta-pinene synthase AAP72020.1 Picea sitchensis pinene synthase BAD91046.1 Citrus unshiu (E)-beta-ocimene synthase AAO61225.1 Pinus taeda (-)-alpha-pinene synthase AAU05952.1 Cucumis sativus beta-Caryophyllene Synthase AAO61228.1 Pinus taeda (+)-alpha-pinene synthase AAC12784.1 Gossypium hirsutum delta-Cadinene Synthase AAO61227.1 Pinus taeda alpha-terpineol synthase ADQ73631.1 Lavandula angustifolia beta-phellandrene synthase AAX07267.1 Pseudotsuga menziesii (-)-alpha-pinene synthase ABB73045.1 Lavandula angustifolia linalool synthase AAX07264.1 Pseudotsuga menziesii terpinolene synthase ABB73044.1 Lavandula angustifolia limonene synthase AAX07265.1 Pseudotsuga menziesii beta-Farnesene Synthase ABB73046.1 Lavandula angustifolia trans-alpha-Bergamotene Synthase AAX07266.1 Pseudotsuga menziesii gamma-Bisabolene Synthase AGL98420.1 Lavandula angustifolia germacrene-D synthase ABP01684.1 Rosmarinus officinalis pinene synthase AGL98418.1 Lavandula angustifolia cadinol synthase ABI20515.1 Rosmarinus officinalis cinenol synthase AGN72803.1 Lavandula stoechas germacrene A Synthase ABH07677.1 Salvia fruticosa cineole synthase AGN72805.1 Lavandula viridis alpha pinene AAC26016.1 Salvia officinalis 1,8-cineole synthase AFL03421.1 Lavandula x intermedia 1,8-cineole synthase AAC26017.1 Salvia officinalis (+)-bornyl diphosphate synthase AGU13712.1 Lavandula x intermedia 9-epi-caryophyllene synthase AAC26018.1 Salvia officinalis (+)-sabinene synthase KX024762 Lavandula x intermedia 3-carene synthase ABH07678.1 Salvia pomifera sabinene synthase Q8H2B4.1 Mentha aquatica R-linalool synthase AAM89254.1 Salvia stenophylla 3-carene synthase AAD50304.1 Mentha longifolia limonene synthase AEM05858.1 Solanum lycopersicum Germacrene Synthase 186 Accession No. Source Species Gene/ protein name Accession No. Source Species Gene/ protein name AAV63791.1 Ocimum basilicum beta-myrcene synthase ADR74206.1 Vitis vinifera (E)-beta-ocimene/myrcene synthase AAV63790.1 Ocimum basilicum fenchol synthase AAS79352.1 Vitis vinifera (-)-a-terpineol synthase AAV63792.1 Ocimum basilicum terpinolene synthase ADR66821.1 Vitis vinifera Germacrene A Synthase E2E2P0.1 Origanum vulgare Gamma-terpinene synthase Table D.3 S-linalool synthases from selected plants, and LiS-LINS and TPSs from Lavandula. Accession No. Source Species Gene/ protein name Accession No. Source Species Gene/ protein name ADQ73631.1 Lavandula angustifolia beta-phellandrene synthase XP_019083713.1 Camelina sativa (S)-linalool synthase ABB73045.1 Lavandula angustifolia (R)-linalool synthase XP_009113120.2 Brassica rapa (S)-linalool synthase ABB73044.1 Lavandula angustifolia limonene synthase XP_013607704.1 Brassica oleracea (S)-linalool synthase ABB73046.1 Lavandula angustifolia trans-alpha-bergamotene Synthase XP_010533841 Tarenaya hassleriana (S)-linalool synthase AGL98420.1 Lavandula angustifolia germacrene-D synthase XP_006648251. Oryza brachyantha (S)-linalool synthase AGL98418.1 Lavandula angustifolia cadinol synthase XP_015623808.1 Oryza sativa (S)-linalool synthase AJW68082.1 Lavandula angustifolia bornyl diphosphate synthase Q84UV0.2 Arabidopsis thaliana (S)-linalool synthase AGN72803.1 Lavandula stoechas germacrene A Synthase ADD81295.1 Actinidia polygama (S)-linalool synthase AGN72805.1 Lavandula viridis alpha pinene ADD81294.1 Actinidia arguta (S)-linalool synthase AFL03421.1 Lavandula x intermedia 1,8-cineole synthase P0CV95.1 Fragaria x ananassa (S)-linalool /(E)-nerolidol synthase 2 AGU13712.1 Lavandula x intermedia 9-epi-caryophyllene synthase P0CV94.1 Fragaria x ananassa (E)-nerolidol synthase 1 KX024762 Lavandula x intermedia 3-carene synthase NP_001280833.1 Malus domestica (E)-nerolidol synthase 1-like MG870571 Lavandula x intermedia (S)-linalool synthase NM_001281061.1 Vitis vinifera (S)-linalool/(E)-nerolidol 187 Accession No. Source Species Gene/ protein name Accession No. Source Species Gene/ protein name EF433761 Antirrhinum majus (S)-linalool synthase EF433762 Antirrhinum majus (E)-nerolidol synthase 188 Appendix E: Maps of plasmids used in this thesis. Figure E.1 A map of p326::sGFP vector. (A gift from Dr I. Hwang, Pohang University of Science and Technology, Korea). 189 Figure E.2 A map of pET32a(+) vector (Novagen). 190 Figure E.3 A map of pET41b(+) vector (Novagen). 191 Figure E.4 A schematic representation of pGA (pGA482) vector carrying GUS. Using a pair of restriction sites (EcorI/ KpnI), GUS gene was removed and replaced by each of LiGPPS subunit. CaMV 35S- Cauliflower mosaic virus promoter; Kan R- a gene encoding Neomycin phosphotransferase (Kanamycin resistance protein); NOS- a nopaline synthase terminator; upstream MCS - EcorI and NcoI; downstream MCS- NcoI, BamHI and KpnI; T-DNA LB - left border; T-DNA RB - right border; Tet R- a gene encoding tetracycline resistant protein; Ori V- replication origin, and Ori T- origin of transfer site (An 1986). 192 Figure E.5 A map of pGEM-T vector (Promega). 193 Appendix F: Bacteria strains used in this thesis. Organism Strains Antibiotic resistant Purpose E. coli Jm109 - Cloning DH5a - Rosetta (DE3)pLysS Chloramphenicol Expression BL21 (DE3) - A. tumefaciens GV3101 Rifampicin Plant transformation 194 Appendix G: Media recipes used for N. benthamiana transformation and regeneration a) Co-cultivation medium:  2.215 g L-1 (half-strength) Murashige and Skoog salts with vitamins (PhytoTechnology laboratories)  0.4 % Gellan gum (PhytoTechnology laboratories)  3.0 % (w/v) sucrose (Fisher scientific)  0.1 mg L-1 indole butyric acid (Sigma Aldrich),  0.8 mg L-1 6-benzylaminopurine (Sigma Aldrich),  adjusted pH 5.7 and autoclaved  100 µM Acetosyringone (PhytoTechnology laboratories) (added after autoclaving) b) Regeneration (Shooting) medium:  4.43 g L-1 Murashige and Skoog salts with vitamins  0.4 % Gellan gum  3.0 % (w/v) sucrose  0.1 mg L-1 indole butyric acid  0.8 mg L-1 6-benzylaminopurine  adjusted pH to 5.7 and autoclaved  200 mg L-1 Timentin (ticarcillin and clavulanate) (Gold Biotechnology, USA),  250 mg L-1 cefotaxime (Gold Biotechnology, USA)  100 mg L-1 Kanamycin (Bio Basic Inc.) c) Rooting medium:  4.43 g L-1 Murashige and Skoog salts with vitamins,  0.8 % Gellan gum, 195  3.0 % (w/v) sucrose,  0.5 mg L-1 indole butyric acid  adjusted pH to 5.7 and autoclaved  200 mg L-1 Timentin (ticarcillin and clavulanate)  250 mg L-1 cefotaxime  200 mg L-1 Kanamycin Note: all antibiotics used in regeneration and rooting media were added after autoclaving 196 Appendix H: Multiple sequence alignment of short-chain trans-IDSs LiGPPS.SSU1 1 MALNLSHL-PI-------KTRSFFC--NSSA-----------QPV--SAAQNASYWSSID AmGPPS.SSU1 1 MAHGLTHF-NTKSGLFPSITKSKTT--RPST-----------RPVILAMTRTQTYRATIE CrGPPS.SSU1 1 MAKAISSFINGGRTTFLSISRSDLSPTTPPH-----------RPNSVI-CMKSNSWANIE MpGPPS.SSU1 1 MAINLSHI-NSKT-CFPLKTRSDLS--RSSSARCMPTAAAAAFPTIATAAQSQPYWAAIE CbGPPS.SSU1 1 MLLVPLKP-NLR-------HPSFFR--RKSA-----------SPTA-TMATLSTYWDSIN HlGPPS.SSU1 1 MSRTHENH----------------------H-----------VPTS----TSIVVSASIT LiGPPS.SSU1 38 ADINTYLKKAIPIRSPETVFEPMHHLTFAAPTTTASALCVAACELFGGNRTQALPAASAI AmGPPS.SSU1 47 SDIESYLKKAIPIRAPESVFEPMHHLTFAAPRTSASALCVAACELVGGDRSDAMAAAAAV CrGPPS.SSU1 49 SDIQTHLKKSIPIRAPEDVFEPMHYLTFAAPRTTAPALCIAACEVVGGDGDQAMAAAAAI MpGPPS.SSU1 57 ADIERYLKKSITIRPPETVFGPMHHLTFAAPATAASTLCLAACELVGGDRSQAMAAAAAI CbGPPS.SSU1 39 DKIDGHLKAAVPVRKPLSVFEPLHHLTLAAPRSSAPALCIAACELVGGSSSQAMDAAAAL HlGPPS.SSU1 24 ADIEAHLKQSITLKPPLSVHEPMYNLVFSAPPNSAPSLCVAACELVGGHRSKAIAAASAL LiGPPS.SSU1 98 HLIHAAAYTHDHLPLTDRPRPESKPDIQHKFNPNIELLTGDGIAPFGFELLANSVDT--- AmGPPS.SSU1 107 HLMHVAAYTHENLPLTDGPMS--KSEIQHKFDPNIELLTGDGIIPFGLELMARSMDPTR- CrGPPS.SSU1 109 HLVHAAAYAHENLPLTDRRRP--KPPIQHKFNSNIELLTGDGIVPYGFELLAKSMDS--- MpGPPS.SSU1 117 HLVHAAAYVHEHLPLTDGSRPVSKPAIQHKYGPNVELLTGDGIVPFGFELLAGSVDPART CbGPPS.SSU1 99 HLIHASLSVHKRLYRG---------GSGSGFGSNIELLTGDGMLPLAFELLANSEGAGDE HlGPPS.SSU1 84 RLLHAANFTHEHLPLTDSPSP--SPVIHNSYDPSIQLLMPDAILPLGFELLAQSYNPAQ- LiGPPS.SSU1 155 THPDRILRVVIEISRASGSQGLVDGLYREKQIV----DQ-------RSRFEFIEYVSKKK AmGPPS.SSU1 164 NNPDRILRAIIELTRVMGSEGIVEGQYHELGL-----NQ-------LNDLELIEYVCKKK CrGPPS.SSU1 164 NNSDRILRVIIEITQAAGSKGIIDGQFRELDVI----DS-------EINMGLIEYVCKKK MpGPPS.SSU1 177 DDPDRILRVIIEISRAGGPEGMISGLHREEEIV----DG-------NTSLDFIEYVCKKK CbGPPS.SSU1 150 SRSRRILRVIVEISKAAGSEGLLKGEYNELQLG----RLAGFTGGDWRDAFWMDHVCGKK HlGPPS.SSU1 141 NNSDRVLRVIVEFARAFGSKGILDGQYRQRVVSISNGDE-------VDNAERVDCSGREK LiGPPS.SSU1 204 YGEMHACGAACGVILAGGDEEQIQKLRNFGLYAGTVRGMIEMKSGLEKKNIAQIEKIIIK AmGPPS.SSU1 212 EGTLHACGAACGAILGGCDEDKIEKLRRFGLYVGTVQGLLG---KN----RSGFEGRIKE CrGPPS.SSU1 213 EGELNACGAACGAILGGGSEEEIGKLRKFGLYAGMIQGLVHGVGKN----REEIQELVRK MpGPPS.SSU1 226 YGEMHACGAACGAILGGAAEEEIQKLRNFGLYQGTLRGMMEMKNSH----QLIDENIIGK CbGPPS.SSU1 206 EGGLHACAAACGGILGGGTEEEIEKLRKLGYLVGMIRGIVSVKGGAAGE-DEEWMKVVEE HlGPPS.SSU1 194 EGKMHACAAACGAILGDANEEETEKLRTFGLYVGMIQGYSIKFMRE-REEEKEAERTIKE LiGPPS.SSU1 264 FQQLAMKEMEGFQGKNAELISSLVT-EPILCP----A AmGPPS.SSU1 265 LKELAVKELESFGGEKIELIRGVFELEHSLAG----V CrGPPS.SSU1 269 LRYLAMEELKSLKNRKIDTISSLLE-T-DLCS----V MpGPPS.SSU1 282 LKELALEELGGFHGKNAELMSSLVA-EPSLYA----A CbGPPS.SSU1 265 MAETALKLLTGFSGELKSKITNICQ-YEDLFPLIKSY HlGPPS.SSU1 253 LRNLALKELEHFHGRKLEPISSFIY-CL--------- Figure H.1 Selected GPPS.SSU1 from angiosperms. Comparison of the predicted amino acids of GPPS.SSU1 from L. x intermedia to those of other functionally characterized plant GPPS.SSU1 subfamily members. All contain the two conserved 'CxxxC' motifs, essential for physical interaction between different subunits, and lack the two conserved aspartate-rich motifs that function in catalysis and substrate binding. LiGPPS.SSU1 shares the highest amino acids identity with M. piperita MpGPPS.SSU1 (67.35 %) and the lowest with C. breweri CbGPPS.SSU1 (43.2 %) and H. lupulus HlGPPS.SSU1 (44 %). * * CxxxC CxxxC * * 197 LiGPPS.SSU2 1 MVFAPVITST-PHVCFPK--------VNRSALRRTIR----CSAVSMTPGFDLKTYWTSL OsGRP 1 MALSSFSMSL-PFAKLPS--------TSKSTRFLPIRASSAAAAAAASPSFDLRLYWTSL AtGPPS.SSU2 1 MLFSGSAIPLSSFCSLPEKPHTLPMKLSPAAIRSS------SSSAPGSLNFDLRTYWTTL LiGPPS.SSU2 48 IKEIDQKLDEAIPVKYPQQIYEAMRYSVLA-----KGAKRSPPVMCVAACELFGGNRLAA OsGRP 52 IADVEAELDAAMPIRTPERIHSAMRYAVLPGAGNEGTAKRAPPVLCVAACELLGAPREAA AtGPPS.SSU2 55 ITEINQKLDEAIPVKHPAGIYEAMRYSVLA-----QGAKRAPPVMCVAACELFGGDRLAA LiGPPS.SSU2 103 FPTACALEMVHAASLIHDDLPCMDDDPSRRGLPSNHTIFGEDMAILAGDALFPLGFRHIV OsGRP 112 LPAAVALEMLHAASLVHDDLPCFDAAPTRRGRPSTHAAYGTDMAVLAGDALFPLAYTHVI AtGPPS.SSU2 110 FPTACALEMVHAASLIHDDLPCMDDDPVRRGKPSNHTVYGSGMAILAGDALFPLAFQHIV LiGPPS.SSU2 163 SHTP-TDLVPETRLLRVIAEIARAVGSTGMAAGQFLDLEGG----PNAVDFVQEKKFGEM OsGRP 172 AHTPSPDPVPHAVLLRVLGELARAVGSTGMAAGQFLDLAGATALGEAEVMKVLTKKFGEM AtGPPS.SSU2 170 SHTP-PDLVPRATILRLITEIARTVGSTGMAAGQYVDLEGG----PFPLSFVQEKKFGAM LiGPPS.SSU2 218 GQCSAVCGGLLAGASDDEIEHLRKYGRAVGILYQVVDDIRDAKSEPEEKDEGKRKGKSYV OsGRP 232 AECSAACGAMLGGAGPDEEAALRRYGRTIGVLYQLVDDIRSASGNGK-----MRSNASVL AtGPPS.SSU2 225 GECSAVCGGLLGGATEDELQSLRRYGRAVGMLYQVVDDITEDKKK------------SYD LiGPPS.SSU2 278 SVYGVEK-AMEVAEDLRSQAKKELDRL-DKYG--DKVLPLYSFVDYAADR---------- OsGRP 287 RALGMDR-ALGIVEELKAQAKMEADRFGDKYG--ERVLPLYSFVDYAVERGFELQDAATT AtGPPS.SSU2 273 --GGAEKGMMEMAEELKEKAKKELQVFDNKYGGGDTLVPLYTFVDYAAHRHFLLPL---- LiGPPS.SSU2 - OsGRP 344 P AtGPPS.SSU2 - Figure H.2 Sequence alignment of LiGPPS.SSU2 with other plant GPPS.SSU2 proteins. The GPPS.SSU2-like protein, OsGRP, from O. sativa is involved in accelerating GGPPS activity and then enhancing chlorophyll concentration (Zhou et al. 2017). Arabidopsis AtGPPS.SSU2 slightly modifies the activity of GGPPS to GPPS to enhance the formation of GPP (Chen et al. 2015). L. x intermedia LiGPPS.SSU2 shares ~70 % amino acid identity with AtGPPS.SSU2, and 55 % with OsGRP. Unlike the latter proteins, LiGPPS.SSU2 impacts neither chlorophyll synthesis nor GPP formation. 198 LiGGPPS 1 MRSMNL---GVQNLSILKQ------VQA---------------YPSKSLVGFRFE-PV-- LiGPPS.LSU 1 MSILIA---KSAPSCPLQR------RDVW--RSRS---SFF--HPPTIKLPLS------- HlGPPS.LSU 1 MSSVNL---TWVQTCSMFN------QAG---RSRSSTFNLLHYHPLK-KVPFSFQTPK-- SmGPPS.LSU 1 MSLLVNPLAT---T-CVKD-----VHGGR--RSRS---GL-------------------- MpGPPS.LSU 1 MSALVNPVAKWPQTIGVKD-----VHGGRRRRSRS---TLFQSHPLRTEMPFSLYFSS-- AtGGPP1 1 MASVTL--GSWIVVHHHNHHHPSSILTKS--RSRS---C-----PITLTKPISFRSKR-- AmGPPS.LSU 1 MSLVNP-ITTWSTTTTSKS--PKNVQTTT--RSRS----II--LPHK----ISLFPSNPK CrGPPS.LSU 1 MSFVNS-ITTWVPAQSIYC--LENGRS-S--SMRS---NLC--HPLKNQLPISFFLSGTI LiGGPPS 34 --FL-KSRKPICVSAVLTGEEAR-------------ISTQAFNFNAYVVEKANQVNKALD LiGPPS.LSU 38 ---R-NQNTNFSVSALLIKEDN----------------PTSFDFNKYMLEKANSVNGALD HlGPPS.LSU 46 --QRRPTSSFSSISAVLTEQEAVTE---------DEEQKSTFNFKSYMVQKANSVNQALD SmGPPS.LSU 27 ---L-STSVKTRISAVYTKEDKN-------------PTWAAFDFKRYMVEKADSVNKALE MpGPPS.LSU 51 --PL-KAPATFSVSAVYTKEGSEIR-----DKDPAPSTSPAFDFDGYMLRKAKSVNKALE AtGGPP1 47 --TV-SSSSSIVSSSVVTKEDNLR-----------QSEPSSFDFMSYIITKAELVNKALD AmGPPS.LSU 46 SK-S-KTHLRFSISSILTKNPQESSQKT------SKDPTFTLDFKTYMLEKASSVNKALE CrGPPS.LSU 50 RKPI-FSCSRLSISAIITKEQTQEESESKSKKEVAFSSTSSFDFKAYMIGKANSVNKALE LiGGPPS 78 EAVAVKNPPMIHEAMRYSLLAGGKRVRPMLCIAACEVVGGHQEAAIPAACAVEMIHTMSL LiGPPS.LSU 78 EAVALKEPLGIHESMRYSLLAGGKRVRPMLCIAACEVVGGHESAAIPAACAVEMIHTMSL HlGPPS.LSU 95 AAVLLREPQMIHEAMRYSLLAGGKRVRPVLCLSACELVGGDESVAMPAACAVEMIHTMSL SmGPPS.LSU 70 AVVQMKEPLKIHESMRYSLLAGGKRVRPMLCIAACELVGGEESTAMPAACAVEMIHTMSL MpGPPS.LSU 103 AAVQMKEPLKIHESMRYSLLAGGKRVRPMLCIAACELVGGDESTAMPAACAVEMIHTMSL AtGGPP1 93 SAVPLREPLKIHEAMRYSLLAGGKRVRPVLCIAACELVGGEESTAMPAACAVEMIHTMSL AmGPPS.LSU 98 QAVLLKEPLKIHESMRYSLLAGGKRVRPMLCIAACELVGGLESTAMPSACAVEMIHTMSL CrGPPS.LSU 109 DAVLVREPLKIHESMRYSLLAGGKRVRPMLCIAACELFGGTESVAMPSACAVEMIHTMSL LiGGPPS 138 IHDDLPCMDNDDLRRGKPTNHKVFGEDVAVLAGDALLAFAFEFIATATVG--VAPE--R- LiGPPS.LSU 138 MHDDLPCMDNDDLRRGKPTNHKVFGEDVAVFAGDALLSFAFEFVAAATVG--VAHGEDRD HlGPPS.LSU 155 IHDDLPCMDNDDLRRGKPTNHKVFGEDVAVLAGDALLAYAFEHVAVSTVG--VPAA--R- SmGPPS.LSU 130 MHDDLPCMDNDDLRRGKPTNHKVFGEDVAVLAGDALLSLAFEHVAVATRG--SAPE--R- MpGPPS.LSU 163 MHDDLPCMDNDDLRRGKPTNHMAFGESVAVLAGDALLSFAFEHVAAATKG--APPE--R- AtGGPP1 153 IHDDLPCMDNDDLRRGKPTNHKVFGEDVAVLAGDALLSFAFEHLASATSSDVVSPV--R- AmGPPS.LSU 158 IHDDLPCMDNDDLRRGKPTNHKIYGEDVAVLAGDALLAFSFEHVAKSTKG--VSSD--R- CrGPPS.LSU 169 MHDDLPCMDNDDLRRGKPTNHKVFGEDVAVLAGDALLAFAFEHIATATKG--VSSE--R- LiGGPPS 193 ILATVVELAKSIGTEGLVAGQVVDLDCTGN--SDVGLKTLEFIHIHKTAALLEASVVLGA LiGPPS.LSU 196 GCGGVGEVDRVGGAGG-RAGGGPLLGGDGQ---MWGSTTLEFIHLHKTAALLEGSVVLGA HlGPPS.LSU 210 IIRAIGELAKSIGSEGLVAGQVVDIDSEGL--ANVGLEQLEFIHLHKTAALLEASVVLGA SmGPPS.LSU 185 ILRALGQLAKSIGAEGLVAGQVVDICSEGM--AEVGLDHLEFIHLHKTAALLQGSVVMGA MpGPPS.LSU 218 IVRVLGELAVSIGSEGLVAGQVVDVCSEGM--AEVGLDHLEFIHHHKTAALLQGSVVLGA AtGGPP1 210 VVRAVGELAKAIGTEGLVAGQVVDISSEGLDLNDVGLEHLEFIHLHKTAALLEASAVLGA AmGPPS.LSU 213 IVRVIGELAKCIGSEGLVAGQVVDISSEGM--TEVGLEHLEFIHVHKTAALLEASVVLGA CrGPPS.LSU 224 IVRVVGELAKCIGSEGLVAGQVVDVCSEGI--ADVGLEHLEFIHIHKTAALLEGSVVLGA LiGGPPS 251 VLGGGSDEQVEKLRIFARKIGLLFQVVDDILDVTKSSEELGKTAGKDLAVDKTTYPKLLG LiGPPS.LSU 252 ILGGGSDEQVAKLRIFARWIGLLFQVVDDIFDVTKSSEELGKTAGKDLAVNKTTYPKLLG HlGPPS.LSU 268 ILGGGTDEQVEELRSFARCIGLLFQAVDDILDVTKSSQELGKTAGKDLVADKATYPRLMG SmGPPS.LSU 243 ILGGAKEEEVERLRKFAKCIGLMFQVVDDILDVTKSSHELGKTAGKDLVADKTTYPKLLG MpGPPS.LSU 276 ILGGGKEEEVAKLRKFANCIGLLFQVVDDILDVTKSSKELGKTAGKDLVADKTTYPKLIG AtGGPP1 270 IVGGGSDDEIERLRKFARCIGLLFQVVDDILDVTKSSKELGKTAGKDLIADKLTYPKIMG AmGPPS.LSU 271 IVGGADDEDVEKLRKFARCIGLLFQVVDDILDVTKSSQELGKTAGKDLVADKTTYPKLLG CrGPPS.LSU 282 IVGGANDEQISKLRKFARCIGLLFQVVDDILDVTKSSQELGKTAGKDLVADKVTYPKLLG CxxxC DDxxxxD DDxxD * * * * ** * 199 LiGGPPS 311 LDKAREFAEKLNEEAKQQLLDFDPVKAAPLVALADYIAHRQN LiGPPS.LSU 312 LEKAVEFAEKLNEEAKQQLLDFDPIKVAPLVALADYIAHRQN HlGPPS.LSU 328 IEKSREFAEKLNREAQEHLGGFDPQKAAPLIALANYIAYRQN SmGPPS.LSU 303 VQKSKEFADDLNREAQEQLLHFDSHKAAPLIAIANYIAYRNN MpGPPS.LSU 336 VEKSKEFADRLNREAQEQLLHFHPHRAAPLIALANYIAYRDN AtGGPP1 330 LEKSREFAEKLNREARDQLLGFDSDKVAPLLALANYIAYRQN AmGPPS.LSU 331 IEKSREFAEKLNREAQEQLEGFDSVKAAPLIALANYIAYRDN CrGPPS.LSU 342 IDKSREFAEKLNREAQEQLAEFDPEKAAPLIALANYIAYRDN Figure H.3 Sequence alignment of selected plant GPPS.LSU and GGPPS proteins. Alignment of amino acid sequences of LiGPPS.LSU and LiGGPPS was made with functionally characterized GPPS.LSUs and GGPPSs from other selected plants. Of the given proteins, HlGPPS.LSU from H. lupulus and CrGPPS.LSU from C. roseus are bifunctional as GPPS and GGPPS. Despite the presence of two aspartate-rich motifs in the sequence, M. piperita MpGPPS.LSU is catalytically inactive by itself. L. x intermedia LiGPPS.LSU shares ~72 % amino acid identity with LiGGPPS in the same species, and from 62 -65 % identity with the other proteins. LiGPPS.LSU functions as a GPPS when it physically interacts with LiGPPS.SSU1. 200 LiFPPS 1 MA-NMNGAASDLRAKFLEVYSVLKSELLNDTAFEWTDGSRQWVERMLDYNVPGGKLNRGL AtFPPS 1 M--------ADLKSTFLDVYSVLKSDLLQDPSFEFTHESRQWLERMLDYNVRGGKLNRGL ZmFPPS 1 MAAGGNGAGGDTRAAFARIYKTLKEELLTDPAFEFTEESRQWIDRMVDYNVLGGKCNRGL SmFPPS 1 MA-NLNGESADLRATFLGVYSVLKSELLNDPAFEWTDGSRQWVERMLDYNVPGGKLNRGL LiFPPS 60 SVIDSYKLLKEGKDLSENEVFLASALGWCVEWLQAYFLVLDDIMDNSHTRRGQPCWFRVP AtFPPS 53 SVVDSYKLLKQGQDLTEKETFLSCALGWCIEWLQAYFLVLDDIMDNSVTRRGQPCWFRKP ZmFPPS 61 SVVDSYKLLKGADALGEEETFLACTLGWCIEWLQAFFLVLDDIMDDSHTRRGQPCWFRVP SmFPPS 60 SVIDSYKLLKGGKDLTDDEVFLASALGWCVEWLQAYFLVLDDIMDNSHTRRGQPCWFRVP LiFPPS 120 KVGMIAVNDGIVLRNHIPRILKNHFREKPYYVDLLDLFNEVEFQTASGQMIDLITTIEGE AtFPPS 113 KVGMIAINDGILLRNHIHRILKKHFREMPYYVDLVDLFNEVEFQTACGQMIDLITTFDGE ZmFPPS 121 QVGLIAANDGIILRNHISRILRRHFKGKPYYADLLDLFNEVEFKTASGQLLDLITTHEGE SmFPPS 120 KVGMIAINDGIILRNHIPRILKKHFRTKPYYVDLLDLFNEVEFQTASGQMIDLITTIEGE LiFPPS 180 KDLSKYSLPLHRRIVQYKTAYYSFYLPVACALLMAGENLEKHTLVKDVLIDMGIYFQVQD AtFPPS 173 KDLSKYSLQIHRRIVEYKTAYYSFYLPVACALLMAGENLENHTDVKTVLVDMGIYFQVQD ZmFPPS 181 KDLTKYNITVHGRIVQYKTAYYSFYLPVACALLLSGENLDNYGDVENILVEMGTYFQVQD SmFPPS 180 KDLSKYSLPLHRRIVQYKTAYYSFYLPVACALLMAGEDLEKHPTVKDVLINMGIYFQVQD LiFPPS 240 DYLDCFGEPEKIGKIGTDIEDFKCSWLVVKALELCNEEQKKTLFDHYGKEDKDDVSKIKA AtFPPS 233 DYLDCFADPETLGKIGTDIEDFKCSWLVVKALERCSEEQTKILYENYGKAEPSNVAKVKA ZmFPPS 241 DYLDCYGDPEFIGKIGTDIEDYKCSWLVVQALERADESQKRILFENYGKKDPACVAKVKN SmFPPS 240 DYLDCFGEPEKIGKIGTDIEDFKCSWLVVKALELCNEEQKKTLFEHYGKEDPADVAKIKV LiFPPS 300 LYDEINLQGVFAEFESKSYEKITGSIEAHPSKSVQAVLKSFLGKIYKRQK AtFPPS 293 LYKELDLEGAFMEYEKESYEKLTKLIEAHQSKAIQAVLKSFLAKIYKRQK ZmFPPS 301 LYKELDLEAVFQEYENESYKKLIADIEAQPSIAVQKVLKSFLHKIYKRQK SmFPPS 300 LYNEINLQGVFAEFESKSYEKLNSSIEAHPSKSVQAVLKSFLGKIYKRQK Figure H.4 Multiple sequence alignment of selected plant FPPSs. LiFPPS has the two aspartate-rich conserved motifs, but lacks any of cystein-rich motif(s). It exhibits 69 %, 78 % and 92 % amino acid sequence similarity to FPPS from Z. mays (ZmFPPS), A. thaliana (AtFPPS) and S. miltiorrhiza (SmFPPS), respectively. DxxD D DDxxD ** * * * * 201 Appendix I: Responses of N. benthamiana when over-expressing LiGPPS.SSU1 or LiGPPS.SSU2. Figure I.1 Boxplots depicting the phenotypes LiGPPS.SSU1 expression in N. benthamiana. The transgenic lines expressing LiGPPS.SSU1 had a decrease in plant height, leaf area and number of leaves compared to the corresponding control plants. * P < 0.01, n= 3 individual plants. Control – N. benthamiana expressing GUS. 202 Table I.1 GGPP-derived plant pigments from N. benthamiana expressing LiGPPS.SSU2. Gene Lines Chl a (µg ml-1 plant extract) Chl b (µg ml-1 plant extract) Chl a/b Tot. Car. (µg ml-1 plant extract) Chl:Car ratio Control 5.86 ± 0.37 1.80 ± 0.06 3.55 ± 025 1.18 ± 0.06 6.59 ± 0.15 LiGPPS.SSU2 SSU2-1 5.83 ± 0.28 1.51 ± 0.07 3.86 ± 0.17 1.12 ± 0.06 6.54 ± 0.12 SSU2-2 5.86 ± 0.18 1.46 ± 0.05 4.01 ± 0.11 1.15 ± 0.06 6.35 ± 0.09 SSU2-3 5.57 ± 0.12 1.57 ± 0.05 3.56 ± 0.08 1.12 ± 0.04 6.37 ± 0.06 SSU2-4 5.82 ± 0.17 1.57 ± 0.07 3.71 ± 0.12 1.20 ± 0.06 6.17 ± 0.09 ns ns ns ns ns Note: GGPP-derived plant pigments were compared between leaves of GUS transformed control line and transgenic lines expressing LiGPPS.SSU2. The pigments appeared to be similar between the control and LiGPPS.SSU2 transgenic lines. Chl – chlorophyll, Tot. Car – total carotenoids. Data are means ± standard error (n= 3), ns - non-significant at p< 0.05, Student's t-test Table I.2 LiGPPS.SSU2 transgenic plant phenotypes. Gene Lines Leaf number Plant height (cm) Leaf area (cm2) Flowering time (days) Control 21.67 ± 0.33 23.40 ± 0.31 45.47 ± 2.22 45 LiGPPS.SSU2 SSU2-1 20.33 ± 1.45 22.00 ± 0.58 41.52 ± 1.66 46 SSU2-2 21.67 ± 1.20 23.93 ± 1.03 43.86 ± 0.82 46 SSU2-3 18.67 ± 0.67 21.27 ± 0.54 48.45 ± 1.83 45 SSU2-4 19.00 ± 0.58 19.17 ± 0.33 42.53 ± 2.20 46 ns ns ns ns All LiGPPS.SSU2 transformed lines displayed similar growth phenotypes to the control plants. Data are means ± standard error (n = 3), ns - non-significant at p< 0.05, Student's t-test. All plant growth parameters were collected at 8 weeks after seeding. 203 Appendix J: Kinetic analysis of Li3CARS using NPP with respect to different products Michaelis-Menten saturation plot of Li3CARS for (a) α-thujene, (b) limonene and (c) terpinolene, and the Lineweaver–Burk (double-reciprocal) plot of Li3CARS for (d) α-thujene, (e) limonene and (f) terpinolene. (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). a) d) b) e) c) f) 204 Appendix K: Monoterpene profile of young leaf tissues of L. x intermedia (cv. Grosso). Products RT (min) % Total (peak area) Products RT (min) % Total (peak area) α-Thujene 5.895 0.378 ± 0.008 1,8-Cineole 7.721 28.281 ± 0.707 α-Pinene 6.042 4.594 ± 0.118 Terpinolene 8.955 1.147 ± 0.039 Camphene 6.300 1.687 ± 0.045 Camphor 10.987 23.821 ± 0.99 β-Pinene 6.765 4.138 ± 0.176 Borneol 11.635 12.809 ± 1.138 3-Carene 7.257 8.977 ± 0.354 α-Terpineol 12.207 2.421 ± 0.046 Limonene 7.598 4.624 ± 0.1 *Others ... 7.121 ± 0.46 a) Gas chromatogram of monoterpenes and b) their relative abundance in essential oil of young leaves. Mean peak areas and standard errors are calculated from seven independent samples. Unidentified monoterpenes are labeled from 1-7. *Others indicates the sum of products labeled as 1-7 from panel 'a'. The percentage values were determined relative to an internal standard (Thymol) injected together with the samples. (Reprinted by permission from Springer Nature: Plant Molecular Biology, 93: 641-657, Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia by Ayelign M. Adal, Lukman S. Sarker, Ashley Lemke, Soheil S. Mahmoud, © 2017). a) b)
Thesis/Dissertation
eng
Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Development of molecular markers and cloning of genes involved in the biosynthesis of monoterpenes in Lavandula
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