Arts and Sciences, Irving K. Barber School of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Erland, Lauren Alexandra Elizabeth
Master of Science - MSc
University of British Columbia
This study aimed to improve essential oil composition by modifying terpene production in Lavandula x intermedia cv Grosso via mutagenesis to more closely resemble the oil of the commercially valuable essential oil from L. angustifolia or the medicinally active essential oil from L. latifolia. Additionally this study aimed to identify genes that control essential oil production in lavenders, and to determine the effect of essential oil composition on biological activity, specifically insecticidal and insect repellent properties. This study resulted in an improved method for the efficient regeneration of Grosso lavender, and applied this method to generate ten unique mutants. The transcriptomes of some mutants were sequenced, and thirty seven differentially expressed transcripts were identified as being involved in the biosynthesis and production of essential oil terpenes. The transcript expression results were confirmed by real-time quantitative polymerase chain reaction analysis. The lavender essential oil showing greatest biological activity against an invasive pest, spotted wing drosophila, was identified as Lavandula latifolia cv Medikus and the active constituents were identified through fumigation and spray toxicity assays as the monoterpenes 1,8-cineole and linalool. These oils showed strong fumigation and contact toxicity. In all, this thesis presents the generation, screening and analysis of unique L. x intermedia essential oil mutants, which represent both potential new commercial cultivars and model organisms for the investigation of the regulation and biosynthesis of essential oil terpenes.
ENHANCEMENT OF SPECIALIZED METABOLISM, REGENERATION EFFICIENCY AND BIOLOGICAL ACTIVITY IN LAVANDIN (LAVANDULA X INTERMEDIA CV ‘GROSSO’) by Lauren Alexandra Elizabeth Erland B.Sc., University of British Columbia, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) March 2015 ©Lauren Alexandra Elizabeth Erland, 2015 ii Abstract This study aimed to improve essential oil composition by modifying terpene production in Lavandula x intermedia cv Grosso via mutagenesis to more closely resemble the oil of the commercially valuable essential oil from L. angustifolia or the medicinally active essential oil from L. latifolia. Additionally this study aimed to identify genes that control essential oil production in lavenders, and to determine the effect of essential oil composition on biological activity, specifically insecticidal and insect repellent properties. This study resulted in an improved method for the efficient regeneration of Grosso lavender, and applied this method to generate ten unique mutants. The transcriptomes of some mutants were sequenced, and thirty seven differentially expressed transcripts were identified as being involved in the biosynthesis and production of essential oil terpenes. The transcript expression results were confirmed by real-­‐time quantitative polymerase chain reaction analysis. The lavender essential oil showing greatest biological activity against an invasive pest, spotted wing drosophila, was identified as Lavandula latifolia cv Medikus and the active constituents were identified through fumigation and spray toxicity assays as the monoterpenes 1,8-­‐cineole and linalool. These oils showed strong fumigation and contact toxicity. In all, this thesis presents the generation, screening and analysis of unique L. x intermedia essential oil mutants, which represent both potential new commercial cultivars and model organisms for the investigation of the regulation and biosynthesis of essential oil terpenes. iii Preface A version of a portions of Chapter 1 has been accepted for publication as the following book chapter, while a version of a separate portion has been submitted for publication in the below review: Erland LAE, Mahmoud SS. 2013. Lavender (Lavandula angustifolia). In: Essential oils in food flavour, preservation and safety. Elsevier. Invited review – accepted A version of Chapter 2 has been published in the following manuscript: Erland, L.A.E., Mahmoud, S.S., 2014. An efficient method for regeneration of lavandin (Lavandula x intermedia cv. 'Grosso'). In vitro cellular and developmental biology -­ plant. 50(5) 646-­‐654. Chapter 3 is based on work I conducted in Dr. Soheil Mahmoud’s lab at UBC Okanagan. NMR analysis was performed independently by Dr. Paul Shipley at the University of British Columbia, Okanagan and by Dr. Andrew Lewis at Simon Fraser University. I completed all other experimental work and data analysis. Chapter 4 is based on work I conducted in Dr. Soheil Mahmoud’s lab at UBC Okanagan. Preparation of cDNA libraries for Illumina© sequencing (performed by Dr. Andrey Golubov), and Illumina© sequencing, quality control, trimming, transcriptome assembly, differential expression analysis, hierarchial clustering and single nucleotide variant analysis for the transcriptome assembled by alignment to the expressed sequence tag libraries (performed by Dr. Yaroslav Illnysky) were done at PlantBiosis (Lethbridge, iv Albetra, Canada) through a paid service. I performed all other experimental work and data analysis. Chapter 5 is based on work I conducted in Dr. Soheil Mahmoud’s lab at UBC Okanagan. Initial mixed fly cultures were provided by Dr. Susanna Acheampong at the Canadian Food Inspection Agency. I completed all experimental work and data analysis. v Acknowledgments This thesis would not have been possible without the support and assistance of many mentors, colleagues and advisors. I would first and most sincerely like to thank my supervisor Dr. Soheil Mahmoud for his continued support, guidance and most of all optimism for the success of this project. I would also like to thank my past and present committee members Dr. Deanna Gibson, Dr. Paul Shipley and Dr. Cedric Saucier for their feedback, insights and direction. I must acknowledge my collaborators across the country without whose expertise, and hardwork this project could never have succeded: Dr. Andrey Golubov and Dr. Yaroslav Illnystkyy at the University of Lethbridge who performed and assisted in analysis of Illumina sequencing, Dr. Sanjoy Ghosh for use of his equipment, Ms. Amy Botta for her limitless experience in design and analysis of qPCR experiments, Dr. Paul Shipley for NMR analysis, Dr. Andrew Lewis and Mr. Colin Zhang for additional NMR analysis at Simon Fraser University, Dr. Mark Rheault for his assistance in rearing and testing of spotted wing drosophila cultures, Dr. Susanna Acheampong for supplying the source cultures for the spotted wing drosophila colony and lastly but certainly not least Dr. Susan Murch for her advice in the treatment of cultures and sharing of supplies. I would also like to thank my fellow lab members, research assistants and volunteers who provided support, assistance and advice: Dr. Zerihun Demissie, Mr. Lukman Sarker, Ms. Mariana Galata, Mr. Ayelign Mengesha, Ms. Brooke Finney, Ms. Ashley Lemke and Ms. Katie Del Buono. I am also grateful to my past mentors who are the reason I was inspired to pursue a graduate degree, in particular Dr. Tom Lowery, Ms. Naomi DeLury and Dr. Howard Thistlewood. Funding for this research was provided through grants to Dr. Soheil Mahmoud and Lauren Erland by Natural Sciences and Engineering Research Council of Canada, and to Dr. Soheil Mahmoud by Canada Foundation for Innovation, University of vi British Columbia, industrial collaborators, and by Agriculture and Agri-­‐Food Canada and the BC Ministry of Agriculture through programs delivered by the Investment Agriculture Foundation of BC. vii Table of Contents Abstract ...................................................................................................................................................ii Preface ................................................................................................................................................... iii Acknowledgments................................................................................................................................ v Table of Contents................................................................................................................................vii List of Tables .......................................................................................................................................... x List of Figures .................................................................................................................................... xiii Table of Symbols and Abbreviations ........................................................................................ xxii Chapter 1. Introduction......................................................................................................................1 1.1. Lavender ..................................................................................................................................................1 1.2. Essential Oils.........................................................................................................................................3 1.3. Biosynthesis of essential oils ..........................................................................................................6 1.3.1. Regulation of essential oil biosynthesis.............................................................................................10 1.4. Plant Tissue Culture ........................................................................................................................ 14 1.5. In vitro mutagenesis of plants and determination of gene targets ................................. 19 1.6. Biological activity screening ........................................................................................................ 21 1.7. Research Objectives and Hypotheses........................................................................................ 22 Chapter 2. Development of a protocol for regeneration of L. x intermedia cv Grosso 24 2.1. Abstract ............................................................................................................................................... 24 2.2. Materials and Methods................................................................................................................... 24 2.3. Results.................................................................................................................................................. 26 2.3.1. Organogenesis from leaf callus ..............................................................................................................26 2.3.2. Addition of phenolic inhibitors, adsorbents and reducing agents .........................................28 2.3.3. Effect of light and media conditions on rooting .............................................................................30 viii 2.3.4. Effect of polyamines ...................................................................................................................................33 2.4. Discussion........................................................................................................................................... 36 Chapter 3. Modification of Essential Oil Composition in L. x intermedia cv Grosso.... 40 3.1. Abstract ............................................................................................................................................... 40 3.2. Methods ............................................................................................................................................... 40 3.2.1. Induction of point mutations in L. x intermedia cv Grosso ........................................................40 3.2.2. Screening of essential oil for modified composition ....................................................................41 3.2.3. Isolation of α-­‐cadinol .................................................................................................................................46 3.2.4. Confirmation of essential oil screening method.............................................................................47 3.3. Results.................................................................................................................................................. 47 3.3.1. Production of lavender essential oil for essential oil mutants.................................................47 3.3.2. Identified novel leaf EO mutants...........................................................................................................48 3.3.3. Mutants with novel growth patterns ..................................................................................................54 3.3.4. Lavender leaf essential oil composition at different leaf ages .................................................54 3.3.5. Purification of unknown from wild-­‐type leaf essential oil ........................................................56 3.3.6. Confirmation of accuracy of relative percent composition calculations .............................57 3.4. Discussion........................................................................................................................................... 58 Chapter 4. Identification of mutation targets in novel essential oil mutants ............... 63 4.1. Abstract ............................................................................................................................................... 63 4.2. Methods ............................................................................................................................................... 63 4.2.1. Identification of gene targets through next-­‐generation transcriptome sequencing ......63 4.2.2. Differential Expression Analysis ...........................................................................................................66 4.3. Results.................................................................................................................................................. 73 4.3.1. Transcriptome assembly ..........................................................................................................................73 4.3.2. Annotation and functional classification ...........................................................................................74 4.3.3. Differential expression analysis ............................................................................................................75 ix 4.4. Discussion........................................................................................................................................... 92 Chapter 5. Screening of lavender and mutant essential oil for modified biological activity ................................................................................................................................................103 5.1. Abstract .............................................................................................................................................103 5.2. Methods .............................................................................................................................................103 5.2.4. Rearing .......................................................................................................................................................... 103 5.2.5. Optimization of a method for testing fumigation toxicity and fumigation toxicity testing 104 5.2.6. Contact toxicity .......................................................................................................................................... 105 5.2.7. Oviposition deterrent activity ............................................................................................................. 107 5.3. Results................................................................................................................................................108 5.4. Discussion.........................................................................................................................................114 Chapter 6. Conclusion ....................................................................................................................117 References .........................................................................................................................................121 Appendices ........................................................................................................................................133 Appendix A – Structure and main biological activities of the major essential oil constituents of lavender (subsection Lavandula) floral essential oil. .....................................133 Appendix B – Interactions plots for polyamine treatments ........................................................136 Appendix C – Standard curves for absolute quantification of essential oil samples...........139 Appendix D – Primer Efficiencies .........................................................................................................146 Appendix E – Selection of qPCR reference genes.............................................................................147 Appendix F – Plots of qPCR data............................................................................................................149 x List of Tables Table 1.1 The major essential oil constituents of L. latifolia, L. x intermedia and L. angustifolia..............................................................................................................................................1 Table 1.2. Examples of some transcription factor families identified in plants and their functions. ..............................................................................................................................................12 Table 1.3. Examples of some common plant growth regulators .......................................................18 Table 2.1. Average vigor of lavandin cultures after exposure to various treatments, where 1 indicates a dead explant and 4 a healthy mature explant with no browning.z ............................................................................................................................................27 Table 3.1. Quantification ions for terpene constituents included in analysis of leaf and bud essential oils...............................................................................................................................44 Table 3.2. Quantification ions for terpene consituents included in analysis of floral essential oils. .......................................................................................................................................44 Table 3.3. Summary of explants, shoots and mature plants surviving the culture process after treatment with EMS.............................................................................................48 Table 3.4. Mutants identified by oil screening -­‐ time for regeneration from initial explant to soil, proportions of major leaf oil constituents and oil yield, where yield is the averaged across all ages of leaf. ............................................................51 Table 3.5. Mutants flowering in spring 2014 identified by oil screening -­‐ time for regeneration from initial explant to soil and proportions of major bud oil constituents. ........................................................................................................................................52 Table 3.6. Mutants flowering in Spring 2014 identified by oil screening -­‐ time for regeneration from initial explant to soil, proportions of major 30% flower oil constituents and oil yield. .......................................................................................................53 xi Table 3.7. Comparison of composition as determined by relative percent composition and absolute quantification from standard curves ............................................................57 Table 4.1. qPCR primer sequences, amplicon length and melting temperature used in qPCR experiments. ...........................................................................................................................70 Table 4.2. Contig measurements for de novo assembly.........................................................................73 Table 4.3. Summary statistics for de novo assembly...............................................................................73 Table 4.4. Summary statistics for read mapping to L. angustifolia EST library (Lane et al. 2010) and de novo assembled library ................................................................................73 Table 4.5. Genes identified as showing differential expression during expression analysis in CLC, normalized expression values are given as reads per kilobase of target per million mapped reads (RPKM). Samples for which there is no RNA-­‐Seq data but expression was still studied by qPCR are noted by “qPCR” and results that are supported by significant qPCR results are given in bold ................................................................................................................................83 Table 4.6. Expression levels of genes identified as differentially expressed candidates in wild-­‐type plants expressed as mean fold change as determined by qPCR analysis. Different letters indicate statistical significance by ANOVA with alpha = 0.05. Results that significantly support results obtained from transcriptome profiling are bolded...........................................................................................86 Table 4.7. Expression levels of genes identified as differentially expressed candidates in leaves of mutant plants expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by xii * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. ..............................................................................88 Table 4.8. Expression levels of genes identified as differentially expressed candidates in tissues/plants for which there is no RNA-­‐Seq data available expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. ...................................................................................................................................................90 Table 5.1. Oils used for testing, source tissue and company. ........................................................... 105 Table 2. Relative percent composition lavender essential oils tested (ISO) (Woronuk et al. 2010) ........................................................................................................................................ 110 Table 5.3. Fumigation toxicity of lavender essential oils against 4-­‐7 day old D. suzukii adults, half lethal concentrations (LC50) and confidence intervals (CI) calculated by probit analysis (alpha = 0.05) ...................................................................... 112 Table 5.4. Contact toxicity of lavender essential oils against 4-­‐7 day old D. suzukii adults, half lethal concentrations (LC50) and confidence intervals (CI) calculated by probit analysis (alpha = 0.05) ...................................................................... 112 Table A1. Structure and biological activity of the major floral essential oil constituents of lavender (subsection Lavandula), LAT = Lavandula latifolia, INT = Lavandula x intermedia, ANG = Lavandula angustifolia. ............................................... 133 Table D1. Mean primer efficiency across all tissue types as calculated by linreg analysis ............................................................................................................................................... 146 xiii List of Figures Figure 1.1 Scanning electron microscope photographs of (from left to right) the front and back view of a glandular trichome. (Erland and Mahmoud, 2013a). .......................................4 Figure 1.2. Overview of the terpenoid biosynthetic pathway, where: AA-­‐CoA – acetoacetyl-­‐CoA; CDP-­‐ ME – 4-­‐(cytidine 5’-­‐diphospho)-­‐2-­‐C-­‐methyl-­‐D-­‐erythritol; CDP-­‐ME2PP – 2-­‐phospho-­‐4-­‐(cytidine 5’ di-­‐phospho)-­‐2-­‐C-­‐methyl-­‐D-­‐erythritol; DXP – 1-­‐deoxy-­‐D-­‐xylulose-­‐5-­‐phosphate; FPP – farnesyl disphosphate; GAP – glyceraldehyde-­‐3-­‐phosphate; GPP – geranyl diphosphate; GGPP – geranylgeranyl diphosphate; HMBPP 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐disphosphate; HMG-­‐CoA – 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐CoA; LPP – lavandulyl diphosphate; ME-­‐2,4-­‐cPP – 2-­‐C-­‐methyl-­‐D-­‐erythritol-­‐2,4-­‐cyclodiphosphate; MEP – 2-­‐C-­‐methyl-­‐D-­‐erythritol-­‐4-­‐phosphate; MVA – mevalonate; MVAP – mevanolate-­‐5-­‐phosphate; MVAPP – mevalonate-­‐5-­‐diphosphate.......................................................................................................................................................8 Figure 1.3. Examples of some specific terpene synthase pathways leading to production of common constituents of lavender essential oil. .............................................................................10 Figure 1.4. Transcription factor families putatively identified by a BLAST search of a L. x intermedia cv Grosso floral glandular trichome EST library, given as total number of contigs identified within each family as a proportion of all transcription factors putatively identified. Lighter coloured bars represent families that have been implicated in the regulation of terpene biosynthesis. ......................................................13 Figure 1.5. Indirect organogenesis in L. x intermedia cv Grosso: callus induction (top left), shoot initiation (bottom left), root induction (centre) and acclimatization (right) (Erland and Mahmoud, 2013b) .............................................................................................................16 xiv Figure 1.6. Indirect somatic embryogenesis in Coriandrum sativum: callus induction (far left), somatic embryo production (centre left), embryo development (centre right), acclimatization (far right). .........................................................................................................16 Figure 2.1. Regeneration of lavandin from leaf cuttings where: (a) callus induction, (b) shoot initiation, (c) shoot multiplication........................................................................................................27 Figure 2.3. Contingency analysis of explant vigor after exposure to various treatments, n=12 for all treatments, colour blocks represent proportional distribution of explants by health. .........................................................................................................................................................30 Figure 2.4. Boxplot for rooting efficiency observed after four weeks on light, media type and media strength treatments where solid bars represent the mean, boxes encompass the first and third quartiles and whiskers extend to range of data with experiments performed in triplicate and n = 4 for all treatments. Letters are assigned based using Tukey’s HSD (α = 0.05) with treatments are significantly different assigned different letters.......................................................................................................31 Figure 2.5. Explants after four weeks on light, media type and media strength treatments where treatments are as follows: (a) red light, half MS (b) red light, full MS (c) white light, half MS (d) white light, full MS (e) red light, half WPM (f) red light, full WPM (g) white light, half WPM (h) white light, full WPM..................................................32 Figure 2.6. Boxplot of rooting efficiency observed after four weeks on 10, 100 and 1000 µM polyamine treatments (Spd, Spm or Put on either MS or WPM) where solid bars represent the mean, boxes encompass the first and third quartiles and whiskers extend to range of data with experiments performed in triplicate and n = 6 for all treatments. Letters are assigned based using Tukey’s HSD (a = 0.05), with xv treatments that are significantly different assigned different letters. From top to bottom treatment plots are: Spd, Put, Spm.......................................................................................34 Figure 2.7. Explants after four weeks on polyamine treatments (Spd, Spm or Put on either MS or WPM) where treatments are from left to right as follows: a-­‐c 10, 100 and 1000 µM Spm on MS; d-­‐f 10, 100 and 1000 µM Spm on WPM; g-­‐i 10, 100 and 1000 µM Spd on MS; j-­‐l 10, 100 and 1000 µM Spd on WPM; m-­‐o 10, 100 and 1000 mM Put on MS; p-­‐r 10, 100 and 1000 µM Put on WPM...................................................35 Figure 3.1. Stages of lavender flowering (a) bud, (b) antethesis, (c) 10% flower and (d) 30% flower. ...............................................................................................................................................................43 Figure 3.2. Sample chromatograms with (top three panes) and without (bottom three panes) labels of leaf (top), bud (middle) and flower (bottom) chromatograms from wild-­‐type Grosso leaf samples extracted by sonication...................................................45 Figure 3.3. Stages of lavender leaves: (a) young, (b) medium and (c) old leaf. .......................................46 Figure 3.4. Overview of identified mutants by phenotype................................................................................49 Figure 3.5. EG 108 (a) showing light coloured flower and trailing flower morphology and wild-­‐type (b) showing typical upright flower colour and morphology. ..............................54 Figure 3.6. Average percent composition of major essential oil constituents across young, medium and old leaves from wild-­‐type and mutant Grosso plants, as calculated by equation 1, error bars represent standard error.....................................................................55 Figure 3.7. Average total essential oil yield from young, medium and old leaves from mutant and wild-­‐type Grosso plants as calculated using equation 2, error bars represent standard error. ..............................................................................................................................................56 Figure 3.8. Fraction 10 (10:90 pentane:chloroform) containing primarily α-­‐cadinol. ........................57 Figure 4.1 Overview of RNA-­‐Seq Analysis Pipeline (Wolf 2013)..................................................................64 xvi Figure 4.2. Percentage distribution of gene sequences after GO mapping for GO cellular components, biological processes and molecular function at GO level 2 and top metabolic pathways with sequences representing enzymes from the KEGG database. ..........................................................................................................................................................75 Figure 4.3. qPCR Fold-­‐changes normalized to wild-­‐type expression levels for differentially expressed the gene candidates 3-­‐hydroxy-­‐3-­‐methylglutaryl CoA reductase (HMGR), 3-­‐hydroxy-­‐3-­‐methylglutaryl CoA synthase (HMGS), unknown terpene synthase (UTPS) 1-­‐4 and cadinol synthase (CADS), selected from alignment to the EST database, in the leaves of mutants e123-­‐3, e47-­‐5, e47-­‐3 and 28, as well as wild-­‐type Grosso leaf. Values are compared to wild-­‐type using Student’s t-­‐test where significance values are as follows * alpha = 0.05, ** alpha = 0.01, *** alpha = 0.001 and ns = no significant difference. .......................................................................................77 Figure 4.4. Heatmap of comparisons between differentially expressed genes in mutants as compared to wild-­‐type expressed as fold change. Comparisons were performed using the Wilcoxon test and FDR corrected z values plotted. Red indicates under represented groups, while blue indicates over represented groups.....................................78 Figure 5.1. Experimental set-­‐up for optimization and testing fumigation toxicity of essential oils against adult D. suzukii. .................................................................................................................. 105 Figure 5.2. Exposure chambers used for contact toxicity testing of lavender essential oils against adult D. suzukii. .......................................................................................................................... 106 Figure 5.3. Experimental set-­‐up for testing oviposition deterrent activity of lavender essential oils. ............................................................................................................................................... 108 xvii Figure 5.4. Percent survival of SWD under control conditions (nothing added), with Drosophila medium added (control with food) and with damp filter paper added (control with humidity). ........................................................................................................................ 109 Figure 5.5. SWD larvae (a), oviposition marks (b) and egg (c) viewed under 50x magnification. ............................................................................................................................................. 113 Figure 5.6. Oviposition deterrent activity of lavender essential oils against 4-­‐7 day old D. suzukii adults after 48 hours. ............................................................................................................... 114 Figure C1. Standard curve for alpha-­‐bisabolol standard. .............................................................................. 139 Figure C2. Standard curve for borneol standard............................................................................................... 140 Figure C3. Standard curve for α-­‐cadinol standard. ......................................................................................... 141 Figure C4. Standard curve for camphor standard. ........................................................................................... 142 Figure C5. Standard curve for carene standard. ................................................................................................ 143 Figure C6. Standard curve for caryophyllene standard. ................................................................................ 144 Figure C7. Standard curve for 1,8-­‐cineole standard ........................................................................................ 145 Figure E1. Expression levels of candidate reference genes: ubiquitin, actin, 18S rRNA and 26S rRNA across all tissue samples including both mutant and wild-­‐type calculated by global normalization. Actin and 18S rRNA were selected as reference genes for further analysis due to lower Cq values and decreased variability across samples..................................................................................................................... 147 Figure E2. Amplification plots of candidate reference genes: (a) 18s rRNA, (b) 26S rRNA, (c) actin, (d) ubiquitin.................................................................................................................................... 148 Figure F1. Differential expression analysis by qPCR of wild-­‐type plants against terpene synthase genes, significant differences between groups was determined by ANOVA analysis and multiple comparisons model with significant differences xviii indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. MTPS – monoterpene synthase; TPS – terpene synthase. ......................................................................................................................... 149 Figure F2. Differential expression analysis by qPCR of terpene synthase genes in wild-­‐type plants. Significant differences between groups were determined by ANOVA analysis and multiple comparisons model with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. TPS – terpene synthase. ................................... 150 Figure F3. Differential expression analysis by qPCR of terpene synthase genes in wild-­‐type plants. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. BDH – borneol dehydrogenase; CINS – cineole synthase; CADS – cadinol synthase; TPS – terpene synthase. ............................... 151 Figure F4. Differential expression analysis of by qPCR tested against genes involved in isoprenoid precursor biosynthesis in wild-­‐type plants. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. HMGR – 3-­‐hydroxy-­‐3-­‐methylglutaryl coA reductase; MVAPP – mevalonate diphosphate................................................................................................................................................. 152 Figure F5. Differential expression analysis by qPCR tested against transcription factor genes involved in regulation of isoprenoid biosynthesis in wild-­‐type plants. Significant difference between groups was determined by ANOVA analysis and xix multiple comparisons model with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. ................................................................................................................. 153 Figure F6. Differential expression analysis by qPCR of mutant leaf samples against terpene synthase genes, significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample. MTPS – monoterpene synthase .......................................................................................................... 154 Figure F7. Differential expression analysis by qPCR of mutant leaf samples against terpene synthase genes, significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by * (p < 0.05)/** (p < 0.01). Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample. BDH – borneol dehydrogenase; CADS – cadinol synthase. ................................... 155 Figure F8. Differential expression analysis by qPCR of mutant leaf samples against genes involved in isoprenoid precursor biosynthesis. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by * (p < 0.05)/** (p < 0.01). Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the xx average of the wild-­‐type sample. HMGR – 3-­‐hydroxy-­‐3-­‐methylglutaryl coA reductase; MVAPP – mevalonate diphosphate............................................................................. 156 Figure F9. Differential expression analysis by qPCR of mutant leaf samples against transcription factor genes involved in regulation of isoprenoid biosynthesis. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by * (p < 0.05)/** (p < 0.01). Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample. ............................ 157 Figure F10. Differential expression analysis by qPCR of mutant flower samples against terpene synthase genes, significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso flower samples with significant differences indicated by different letters. Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample. MTPS – monoterpene synthase ......................................................................................... 158 Figure F11. Differential expression analysis by qPCR of mutant flower samples against genes involved in isoprenoid precursor biosynthesis. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by * (p < 0.05)/** (p < 0.01). Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample. HMGR – 3-­‐hydroxy-­‐3-­‐methylglutaryl coA reductase; MVAPP – mevalonate diphosphate. ........................................................................... 159 xxi Figure F12. Differential expression analysis by qPCR of mutant flower samples against transcription factor genes involved in regulation of isoprenoid biosynthesis. Significant difference between groups was determined by ANOVA analysis and multiple comparisons model compared to wild-­‐type Grosso leaf samples with significant differences indicated by * (p < 0.05)/** (p < 0.01). Mean fold change values are plotted with error bars representing standard error of the mean. All mutants are normalized against the average of the wild-­‐type sample.............................. 160 xxii Table of Symbols and Abbreviations AA – L-­‐ascorbic acid AACT -­‐ acetoacetly-­‐CoA thiolase AC – activated charcoal AIP – 2 aminoindane-­‐2-­‐phosphonic acid ANOVA – analysis of variance BDH – borneol dehydrogenase BLAST – basic local alignment search tool CADS – cadinol synthase cDNA – copy deoxyribonucleic acid CDP-­‐ME -­‐ 4-­‐(cytidine 5’-­‐diphospho)-­‐2-­‐C-­‐methyl-­‐D-­‐erythritol CI – confidence interval CINS – cineole synthase CMK -­‐ 4-­‐(cytidine 5’-­‐diphospho)-­‐2-­‐C-­‐methyl-­‐D-­‐erythritol kinase DMAPP – dimethylallyl diphosphate DNA – deoxyribonucleic acid DNAse – deoxyribonuclease DXP – deoxy-­‐D-­‐xylulose-­‐5-­‐phosphate DXR – deoxy-­‐D-­‐xylulose-­‐5-­‐phosphate reductase DXS – deoxy-­‐D-­‐xylulose-­‐5-­‐phosphate synthase EMS – ethylmethane sulfonate EST – expressed sequence tag F – fraction FDR – Benjamin-­‐Hochberg false discovery rate xxiii GA3 – gibberellic acid GABA -­‐ gamma aminobutyric acid GAP – glyceraldehyde-­‐3-­‐phosphate GC – MS – gas chromatography mass spectrometry GO – gene ontology GPP – geranyl diphosphate GPPS – geranyl diphosphate synthase GrL – Lavandula x intermedia cv Grosso leaf GrF – Lavandula x intermedia cv Grosso flower HDR – 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐disphosphate reductase HDS -­‐ 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐diphosphate synthase HMBPP -­‐ 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐diphosphate HMG-­‐CoA -­‐ 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐CoA HMGR -­‐ 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐CoA reductase HMGS -­‐ 3-­‐hydroxy-­‐3-­‐methylglutaryl Co-­‐A synthase HSD – Tukey’s honestly significant difference IAA – indole-­‐3-­‐acetic acid IBA – indolebutyric acid IPP – isopentenyl diphosphate IPPI – isopentenyl disphosphate isomerase KEGG – Kyoto encyclopedia of genes and genomes LaL – Lavandula angustifolia cv Lady leaf LaF – Lavandula angustifolia cv Lady flower LC50 – half lethal concentration xxiv LPP – lavadulyl diphosphate LPPS – lavandulyl diphosphate synthase MCS -­‐ 2-­‐C-­‐methyl-­‐D-­‐erythritol 2,4-­‐cyclodiphosphate synthase MCT -­‐ 2-­‐C-­‐methyl-­‐D-­‐erythritol-­‐4-­‐phosphate cytidyltransferase ME-­‐2,4cPP -­‐ 2-­‐C-­‐methyl-­‐D-­‐erythritol 2,4-­‐cyclodiphosphate MEP -­‐ 2-­‐C-­‐methyl-­‐D-­‐erythritol-­‐4-­‐phosphate MIQE – minimum information for publication of quantitative real-­‐time polymerase chain reaction experiments MK – mevalonate kinase MS – Murashige and Skoog MTPS – monoterpene synthase MVA – mevalonate MVAP – mevalonate 5-­‐phosphate MVAPP – mevalonate 5-­‐disphosphate NAA – naphthylacetic acid NMDA -­‐ N-­‐methyl-­‐D-­‐aspartate NMR – nuclear magnetic resonance spectroscopy NR – non-­‐redundant ns – no significant difference PCR – polymerase chain reaction PMK – phosphomevalonate kinase Put – putrescine PVP -­‐ polyvinylpyrrolidone RNA – ribonucleic acid xxv RNAse – ribonuclease RPKM – reads per kilobase of transcript per million reads mapped qPCR – real-­‐time quantitative polymerase chain reaction SNV – single nucleotide variant Spd – spermidine Spm – spermine SWD – spotted wing drosophila TDZ – thiadiazuron TPS – terpene synthase UTPS – unknown monoterpene synthase WPM – Llyod and McCown woody plant mediu 1 Chapter 1. Introduction 1.1. Lavender Lavenders are a diverse group of essential oil producing perennial shrubs in the genus Lavandula, in the Lamiaceae family. Comprised of more than 40 species and more than 100 cultivars and hybrid varieties, only three are grown commercially for their essential oil: English lavender (Lavandula angustifolia Mill.), spike lavender (L. latifolia) and their hybrid lavandin (L. x intermedia) (Upson and Andrews 2004a). English lavender is considered to have the highest quality oil in the fragrance industry with very little camphor and high levels of linalool and linalyl acetate. Spike lavenders have significantly higher amounts of camphor, 1,8-­‐cineole and borneol, which, although contributing a less pleasing odour to their oil, have significant bioactivity and are therefore of interest to the alternative medicine industry (Harborne and Williams 2004). Lavandin exhibits traits of both parents with intermediate levels of the major constituents of English and spike lavender (Table 1). Of the commercially grown lavenders Grosso lavandin (L. x intermedia) shows characteristic hybrid vigor and is the most widely grown for its essential oil, producing up to ten times more oil than English lavender (Upson and Andrews 2004b). Table 1.1 The major essential oil constituents of L. latifolia, L. x intermedia and L. angustifolia. L. latifolia L. x intermedia L. angustifolia 1,8 -­‐ Cineole 25-­‐36% 4-­‐10% -­‐ Camphor 5.3-­‐15.3% 6-­‐12% -­‐ Borneol 0.8-­‐4.9% 1.5-­‐3.7% -­‐ Linalool 26-­‐44% 20-­‐35% 10-­‐50% Linalyl acetate 0-­‐1.5% 19-­‐38% 12-­‐54% Lavandulyl acetate 0.2-­‐1.5% 0.5-­‐3% 0.1-­‐14% Although they are now grown worldwide, all lavenders originate from the Mediterranean region where they have been used for their pleasant scent in strewing herbs, 2 aromatherapy, traditional medicine and washing since Greek and Roman times. The genus name itself originates from its original use in washing water with Lavandula being derived from the Latin “lavare” meaning to wash. In more recent history lavender essential oil has been used in the flavour and fragrance industries as well as in traditional and alternative medicine (Castle and Lis-­‐Balchin 2004). Lavender essential oil has been reported to have insect repellent, insecticidal, anti-­‐inflammatory, antioxidant, pro-­‐apoptotic, anti-­‐tumor, anti-­‐depressant, anti-­‐spasmolytic, anxiolytic, antifungal, antibacterial, antiparasitic, carminative, sedative, immunomodulatory and analgesic properties (Cavanagh and Wilkinson 2002; Moon et al. 2006; Woronuk et al. 2010; Baker et al. 2012). This diverse activity has made it an appealing candidate for both medicinal and agricultural applications. The neurological activity, particularly the effect of lavender on the limbic system, has been reported to be due to its interaction with serotonergic, N-­‐methyl-­‐D-­‐aspartate (NMDA) and gamma aminobutyric acid (GABA) A receptors. In particular linalool and linalyl acetate have been found to be able to enter the blood stream through ingestion across the skin during applications such as massage and also via inhalation and have an effect on these receptors (Lis-­‐Balchin 2004; Landelle et al. 2008; Chioca et al. 2013). Lavandula essential oils and particularly camphor has also been shown to block sodium and calcium channels leading to anti-­‐muscarinic effects and inhibition of acetylcholinesterase has also been reported in both insect and mammalian models (El-­‐Sheikh et al. 2005; Acikalin et al. 2012). Immunomodulatory and anti-­‐inflammatory properties have been reported to be due to the activity of 1,8-­‐cineole and borneol through their interaction with intestinal microbiota and suppression of tumor necrosis factor alpha (Abe et al. 2003; Baker et al. 2012). Many of the mono-­‐ and sesquiterpenes making up lavender essential oils have been tested for 3 bioactivity and in many cases the activity of the individual constituents does not match that of the overall activity of the essential oil indicating that bioactivity is a result of synergistic activity of all the constituents including diverse minor components found as less than 1% of overall oil composition. The mode of action of lavender essential oil as an antimicrobial agent has been related to its solvent like qualities which leads to disruption of cell membranes as well as hyphal degradation and cytoplasmic coagulation in fungi among other effects (Soylu et al. 2010). Appendix 1 details reported bioactivities of the major constituents of L. angustifolia, L. latifolia and L. intermedia. 1.2. Essential Oils Essential oils are volatile oils comprised primarily of isoprenoids, in particular the mono-­‐ and sesquiterpenes, and in plants are involved in allelopathy, plant defense and pollinator attraction. The isoprenoids are a diverse class of compounds, which encompasses not only the C10 monoterpenes and C15 sesquiterpenes but also hemi-­‐ (C5), di-­‐ (C20), tri-­‐ (C30), and poly-­‐ (>C30) terpenes. These constituents may be produced in any number of specialized structures in plants, but are generally secreted and stored in specialized cavities and structures as they are highly insoluble in water and have solvent like properties making them difficult and sometimes toxic to store within a plant cell. These specialized structures include resin ducts and glandular trichomes. In lavender, essential oils are produced in glandular trichomes present on stem, leaf and particularly flower surfaces and in general they are composed of three main part: a secretory cell, a stalk and a storage cavity (1, 2 and 3 respectively in Figure 1)(Huang et al. 2008). Essential oils are commercially valuable due to both their pleasant scent and their bioactivity. ! ^!!J/O&'(!A4A!56%))/)O!(+(61';)!>/6';:6;V(!V2;1;O'%V2:!;<!N<';>!+(<1!1;!'/O21P!12(!<';)1!%)-!"%6S!9/(X!;<!%!O+%)-&+%'!1'/62;>(4!N.'+%)-!%)-!E%2>;&-7!?@A[%P4!!!B2(!/)-&:1'/%+!%VV+/6%1/;):!%)-!"/;+;O/6%+!%61/9/1/(:!;<!(::()1/%+!;/+:!%)-!12(/'!6;):1/1&()1:!6%)!"(!V'(-/61(-!"#!12(/'!)%1&'%+!<&)61/;):!/)!V+%)1:4!J;'!(,%>V+(!>%)#!>;);1('V()(:!%'(!(+/6/1(-!/)!'(:V;):(!1;!V%12;O()!%11%6S7!:&62!%: %+V2%YV/)()(!NE('6/('!(1!%+4!?@@m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eP4!B2(!6;):/:1()6#!;<!12(:(!6;>V;&)-:!%6';::!+/<(!<;'>:!!/:!%!V;X('<&+!1;;+!<;'!V+%)1:!%++;X/)O!12(>!1;!6;>>&)/6%1(!X/12!12(/'!:&'';&)-/)O:4!$(%<!(::()1/%+!;/+:!%'(!%+:;!2/O2!/)!:(:W&/1('V()(:7!X2/62!%'(!X(++!S);X)!1;!2%9(!/):(61/6/-%+!V';V('1/(:!%)-!%'(!V';-&6(-!"#!>%)#!/):(61:!%:!-(<():(!6;>V;&)-:!N$sV(0!(1!%+4!?@A[P4!K)(!:&62!(,%>V+(!/:!(>/::/;)!;<!12(!<%')(:()(!"#!12(!V(%!%V2/-!A58-+>31*6>3$(6*1&.!C%''/:!N*V2/-/-%(P!X2/62!/:!V';-&6(-!%:!%!S%/';>;)(!/)!'(:V;):(!1;!%11%6S!"#!V'(-%1;':!:&62!%:!+%6(X/)O:!%)-!+%-#"/'-!"((1+(:!Nt;%62/>!(1!%+4!?@A[P4!D)!12/:!6%V%6/1#!<%')(:()(!<&)61/;)!/)!%V2/-!-(<():(!"#!%11'%61/)O!V'(-%1;':!;'! 5 parasitoids of these aphid predators. In plants farnesene is also a minor but common constituent of plant essential oils. Although these constituents often have multiple biological activities, their in planta activity provides important clues on potential new applications. In addition to their medicinal applications essential oils are used extensively in the food and fragrance industries. Monoterpenes such as 1,8-­‐cineole and menthol are well-­‐recognized active ingredients in many cough and cold products as they possess soothing and cough suppressant activities. Additionally if you look at many scented products it is likely that one or more of the listed ingredients will be a mono-­‐ or sesquiterpene, with one of the most common being linalool. The most commonly associated industrial application with essential oils is its use in the flavour and fragrance industries, which makes up the majority of the demand for lavender essential oils, however, it is becoming increasingly important in other industries as well. Essential oils have found applications in the food industry as inhibitors of both fungal and bacterial food spoilage microorganisms both in the field and in post-­‐harvest applications as well as in meat and seafood (Zambonelli et al. 1996; Moon et al. 2007; Lodhia et al. 2009; Imelouane et al. 2009; Gómez-­‐Estaca et al. 2010; Soylu et al. 2010; Djenane et al. 2012). Essential oils, such as lavender essential oils have also been found to have preservative properties due to their antioxidant potential, acting as stabilizers to preserve the quality of food during storage, heating or cooking (Da Porto et al. 2009; Rodrigues et al. 2012) . In one study it was found that addition of lavender essential oils to soybean oil during microwaving preserved the quality of the oil by stabilizing the lipid fraction, preventing its oxidation (Rodrigues et al. 2012). The use of essential oils as natural insect repellents and insecticides in one of the most promising industrial applications of lavender essential oils with products using lavender essential oil 6 already having been developed (van Tol et al. 2007). EOs have been found to possess both repellency and toxic activity towards a variety of insects that are pests in field, and during food storage and are generally active due to their solvent-­‐like activity, damaging the insect cuticle on contact as well as disrupting insect metabolism (El-­‐Sheikh et al. 2005; Rozman et al. 2007). Some well-­‐known already registered essential oils based insecticides include pyrethrum and neem oil. 1.3. Biosynthesis of essential oils All isoprenoids are built from five carbon isoprene units, isopentenyl diphosphate (IPP) and dimethylallyl disphosphate (DMAPP), which are produced by two distinct but interactive pathways in plants, the cytosolic mevalonate (MVA) pathway and the plastidial 1-­‐deoxy-­‐D-­‐xylulose-­‐5-­‐phosphate (DXP) pathway. The MVA pathway leads primarily to sesqui-­‐ and triterpenes and the DXP pathway gives rise to the mono-­‐, di-­‐ and polyterpenes, though both pathways have been found to contribute to some members of all classes (Eisenreich et al. 1998; Hemmerlin et al. 2003; Vranova et al. 2012)(Figure 2). The DXP pathway starts with the addition of glyceraldehyde-­‐3-­‐phosphate (GAP) and pyruvate, which are derived from glycolytic reactions, to form DXP by DXP synthase (DXS), the enzyme catalyzing the first committed step in the DXP pathway. DXP reductase (DXR) then reduces DXP to form 2-­‐C-­‐methyl-­‐D-­‐erythritol-­‐4-­‐phosphate (MEP), which is then phosphorylated and a cytidine added by MEP cytidyltransferase (MCT) to form 4-­‐(cytidine 5’-­‐diphospho)-­‐2-­‐C-­‐methyl-­‐D-­‐erythritol (CDP-­‐ME). Another phosphate is then added to CDP-­‐ME by CDP-­‐ME kinase (CMK) and is then cyclized by 2-­‐C-­‐methyl-­‐D-­‐erythritol 2,4-­‐cyclodiphosphate synthase (MCS) form 2-­‐C-­‐methyl-­‐D-­‐erythritol 2,4-­‐cyclodiphosphate (ME-­‐2,4cPP). The cyclic group is then oxidized by 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐diphosphate synthase (HDS) to form 1-­‐hydroxy-­‐2-­‐methyl-­‐2-­‐butenyl 4-­‐diphosphate (HMBPP), which can 7 then be reduced to form IPP and DMAPP (Eisenreich et al. 1998; Vranova et al. 2012; Xiang et al. 2012). In the MVA pathway three acetyl Co-­‐A molecules derived from the citrate cycle are added by acetoacetly-­‐CoA thiolase (AACT) and 3-­‐hydroxy-­‐3-­‐methylglutaryl Co-­‐A synthase (HMGS) to form 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐CoA (HMG-­‐CoA) via acetoacetyl-­‐CoA. HMG-­‐CoA is then reduced by HMG-­‐CoA reductase (HMGR) in the first committed and the rate limiting step in this pathway to form MVA. MVA is then phosphorylated twice, first by mevalonate kinase (MK) to form MVA 5-­‐phosphate (MVAP) then by phosphomevalonate kinase (PMK) to form MVA 5-­‐diphosphate (MVAPP), which is then decarboxylated to form IPP. In the MVA pathway IPP can then be isomerized by isopentenyl diphosphate isomerase ! Z!!!J/O&'(!A4?4!K9('9/(X!;<!12(!1('V();/-!"/;:#)12(1/6!V%12X%#7!X2('(\!**Y=;*!]!%6(1;%6(1#+Y=;*o!=FHY!E.!]!^YN6#1/-/)(!TpY-/V2;:V2;PY?Y=Y>(12#+YFY('#12'/1;+o!=FHYE.?HH!]!?YV2;:V2;Y^YN6#1/-/)(!Tp!-/YV2;:V2;PY?Y=Y>(12#+YFY('#12'/1;+o!FqH!]!AY-(;,#YFY,#+&+;:(YTYV2;:V2%1(o!JHH!]!<%')(:#+!-/:V2;:V2%1(o!M*H!]!O+#6('%+-(2#-(Y[YV2;:V2%1(o!MHH!]!O('%)#+!-/V2;:V2%1(o!MMHH!]!O('%)#+O('%)#+!-/V2;:V2%1(o!CE3HH!AY2#-';,#Y?Y>(12#+Y?Y"&1()#+!^Y-/:V2;:V2%1(o!CEMY=;*!]![Y2#-';,#Y[Y>(12#+O+&1%'#+Y=;*o!!$HH!]!+%9%)-&+#+!-/V2;:V2%1(o!E.Y?7^Y6HH!]!?Y=Y>(12#+YFY('#12'/1;+Y?7^Y6#6+;-/V2;:V2%1(o!E.H!]!?Y=Y>(12#+YFY('#12'/1;+Y^YV2;:V2%1(o!EQ*!]!>(9%+;)%1(o!EQ*H!]!>(9%);+%1(YTYV2;:V2%1(o!EQ*HH!]!>(9%+;)%1(YTY-/V2;:V2%1(4!!NDHHDP!1;!<;'>!FE*HH!N=';1(%&!%)-!$;;>/:!Ame?o!E%2>;&-!%)-!=';1(%&!?@@?P4!B;!<;'>!12(!6%'";)!62%/)!"%6S";)(!DHH!%)-!FE*HH!%'(!6;)-():(-!"#!V'()#+1'%):<('%:(:7!%)-!-(V()-/)O!;)!12(!1('V()(!1;!"(!:#)12(:/0(-7!-/<<('()1!V'()#+1'%):<('%:(:!%'(!&:(-!NJ/O&'(![P!N3&'S(!?@@Ao!Q'%);9%!(1!%+4!?@A?P4!D)!12(!6%:(!;<!>;);1('V()(!"/;:#)12(:/:!/)!+%9()-('7! 9 two prenyltransferases are involved, geranyl diphosphate synthase (GPPS) which synthesizes geranyl diphosphate (GPP), the precursor for regular monoterpenes or lavandulyl diphosphate synthase (LPPS), which synthesizes lavandulyl diphosphate (LPP), the precursor for irregular monoterpenes (Figure 4) (Burke 2001; Demissie et al. 2013). Regular or irregular monoterpenes are defined by the manner in which the isoprene units are joined; in the former they are joined in a head to tail manner whereas irregular monoterpenes involve joining in different configurations (Demissie et al. 2013). Once LPP or GPP has been synthesized they are then modified to produce unique monoterpene end-­‐products by individual monoterpene synthases (TPS) (Figure 3). These chemical modifications include reduction, oxidation and cyclization reactions among others and the diversity of the activity, function and substrate specificity of terpene synthases is the mechanism behind the vast biodiversity of terpenes (Mahmoud and Croteau 2002). ! A@!!J/O&'(!A4[4!.,%>V+(:!;<!:;>(!:V(6/</6!1('V()(!:#)12%:(!V%12X%#:!+(%-/)O!1;!V';-&61/;)!;<!6;>>;)!6;):1/1&()1:!;<!+%9()-('!(::()1/%+!;/+4!!ITSTIT 3ANKL?=DEF(EB(A<<AF=D?L(EDL(;DE<YF=HA<D<(5;>(!1('V()(:!V+%#!9('#!:V(6/%+/0(-!';+(:!/)!%!V+%)1:!+/<(7!%)-!%:!:&62!12(/'!"/;:#)12(:/:!%)-!%66&>&+%1/;)!/:!);1!'(W&/'(-!%1!%++!1/>(:7!"&1!'%12('!;)+#!/)!'(:V;):(!1;!:V(6/</6!"/;1/67!%"/;1/6!%)-!-(9(+;V>()1%+!1'/OO(':!X/12/)!12(!V+%)1!NL&%O+/%!(1!%+4!?@A?o!Q'%);9%!(1!%+4!?@A?o!H%1'%!(1!%+4!?@A[P4!B2/:!+(%-:!1;!%!'(W&/'(>()1!<;'!:1'/61!'(O&+%1/;)!;<!1('V()(!V';-&61/;)!1;!V'(9()1!X%:1/)O!9%+&%"+(!'(:;&'6(:4!D)!%--/1/;)!1;!12(!';+(!6;>V%'1>()1%+/0%1/;)!V+%#:!/)!>;-&+%1/)O!6%'";)!<+&,!12';&O2!12(!/:;V'();/-:!V%12X%#:7!12('(!%'(!1X;!>%/)!V;/)1:!%1!X2/62!1('V()(!V';-&61/;)!6%)!"(!'(O&+%1(-\!1'%):6'/V1/;)%+!'(O&+%1/;)!%)-!-/'(61!/)2/"/1/;)!;<!()0#>(:!X/12/)!%!"/;:#)12(1/6!V%12X%#!NQ'%);9%!(1!%+4!?@A?P4! 11 Transcriptional regulation occurs due to the action of transcription factors, deoxy-­‐ribonucleic acid (DNA) binding proteins or protein complexes which interact with the promoter region of particular genes to activate or deactivate gene expression (Patra et al. 2013). Transcription factors have diverse roles, and participate in the regulation of virtually every process occurring within a plant. Although the amount of information known about transcriptional regulation of some secondary metabolites, such as the phenolics and the alkaloids is increasing steadily, little is still known about the transcription factors regulating terpene biosynthesis (Patra et al. 2013). Of the 84 gene families of transcription factors identified in plants to date, only three have been found to play an active role in regulation of terpene biosynthesis: WRKY, bHLH and AP2-­‐EREBP (Pérez-­‐Rodríguez et al. 2010; Hong et al. 2012; Yang et al. 2012; Patra et al. 2013). A BLAST (basic local alignment search tool) search of an expressed sequence tag (EST) library created from L. x intermedia cv Grosso putatively identified 20 families of transcription factors in lavender which included 76 individual contigs, Figure 1.4 summarizes these results. 12 Table 1.2. Examples of some transcription factor families identified in plants and their functions. Gene Family Processes regulated in plants Reference AP2-­‐EREBP Flower development, cell proliferation, secondary metabolism, absisic acid and ethylene response modulator, abiotic and biotic stress responses, terpenoid biosynthesis (Riechmann and Ratcliffe 2000; Dietz et al. 2010; Patra et al. 2013) ARF Auxin primary response, development, floral meristem patterning (Guilfoyle et al. 1998) AUX/IAA Repression auxin response (Tiwari et al. 2004; Pérez-­‐Rodríguez et al. 2010)} bHLH Anthocyanin biosynthesis, sesquiterpene biosynthesis, phytochrome signaling, globulin expression, fruit dehiscence, carpel and epidermal development (Buck and Atchley 2003; Hong et al. 2012; Yang et al. 2012) bZIP Pathogen defense, light and stress signaling, seed maturation, flower development, seed-­‐storage gene expression, leaf development, absisic acid response, gibberellin biosynthesis (Riechmann and Ratcliffe 2000; Jakoby et al. 2002) C2C2-­‐Dof Seed germination, endosperm-­‐specific expression, carbon metabolism, plant growth and development (Riechmann and Ratcliffe 2000; Yanagisawa 2004) C2C2-­‐GATA Light and nitrogen dependent control (Reyes et al. 2004) GRAS Gibberellin signal transduction, root radial patterning, axillary meristem formation, phytochrome A signal transduction, gametogenesis (Tian et al. 2004; Pérez-­‐Rodríguez et al. 2010) MADS Flower development, floral meristem and organ identity (Ng and Yanofsky 2001) MYB and MYB related Plant development, defense response, anthocyanin biosynthesis, secondary metabolism, cell morphogenesis, meristem formation, floral and seed development, cell cycle control, light and hormone signaling pathways, abiotic and biotic stress response (Riechmann and Ratcliffe 2000; Yanhui et al. 2006) NAC Embryonic, floral and vegetative development, pattern formation, defense and abiotic stress response (Riechmann and Ratcliffe 2000; Olsen et al. 2005) SBP Floral timing, fetility, plant defense, development, shoot maturation (Unte et al. 2003; Birkenbihl et al. 2005; Stone et al. 2005; Schwarz et al. 2008) ! A[!M()(!J%>/+#! H';6(::(:!'(O&+%1(-!/)!V+%)1:! I(<('()6(!B=H! M';X12!%)-!-(9(+;V>()1!!N=&"%:!(1!%+4!AmmmP!nIcR! B('V();/-!"/;:#)12(:/:7!V+%)1!-(<():(7!1'/62;>(!-(9(+;V>()17!:()(:6()6(7!"/;1/6!%)-!%"/;1/6!:1'(::!'(:V;):(!N.&+O(>!?@@@o!Hu'(0YI;-'vO&(0!(1!%+4!?@A@o!H%1'%!(1!%+4!?@A[Pw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a;'!';+(!/)!12(!%++;6%1/;)!;<!6%'";)!<+;X!N51('>('!(1!%+4!AmmAo!I;-'/O&(0Y=;)6(V6/;)!?@@`P4!B2(!</':1!>%/)!V;/)1!<;'!'(O&+%1/;)!/:!/)!12(!:#)12(:/:!;<!DHH!%)-!FE*HH!9/%!12(!EQ*!%)-!FqH!V%12X%#:4!B2/:!-(1('>/)(:!12(! 14 amount of carbon that is being diverted from other processes towards terpene biosynthesis, and as such has been a major point of focus for efforts in metabolic engineering (Mahmoud and Croteau 2002; Botella-­‐Pavía et al. 2004; Vranova et al. 2012; Kampranis and Makris 2012). In the MVA pathway HMGR catalyzes the rate limiting step in the pathway and is the main regulatory enzyme. This enzyme is subject to regulation by phosphorylation as well as hormone and sterol mediated feedback inhibition (stermer et al. 1994; Newman and Chappell 1999; Goldstein et al. 2006; Burg and Espenshade 2011). Other enzymes in the MVA pathway have been found to play more minor but still important roles in regulation of the MVA pathway, for example AACT, which catalyzes the first committed step in the pathway is under feedback inhibition in response to abiotic stresses (Soto et al. 2011). In contrast the DXP pathway has been found to be regulated at multiple points: the main regulatory enzyme is DXS which catalyzes the rate limiting reaction and is subject to feedback inhibition as well as transcriptional regulation, the last enzyme in the pathway HDR which synthesizes IPP and DMAPP is also involved in modulating carbon flux, though the mechanisms are more poorly understood (Botella-­‐Pavía et al. 2004; Rodriguez-­‐Concepcion 2006; Vranova et al. 2012; Xiang et al. 2012). 1.4. Plant Tissue Culture Plant tissue culture and particularly regeneration is based on the concept of totipotency, the potential for individual plant cells to give rise to a new plant, and plant tissue culture is the process through with this occurs in vitro (Krikorian 2004). Plant tissue culture, broadly, can be grouped into three classes, indirect morphogenesis, direct morphogenesis and cuttings. The latter of these techniques is not commonly used in biotechnology but is very common in commercial nurseries. Cuttings are generally taken at meristems or nodes where the plant is already actively dividing and are then placed on media or in soil to 15 promote root formation and continued plant growth, this allows for direct clonal propagation of a specific plant (Cassells 2004). Although there are many variations in cultures techniques depending on the aims of a project, generally it can be simplified to three steps (Figure 1.5, 1.6). In indirect morphogenesis the first step is callus induction. Mature tissue such as leaf, stem or floral tissue is excised from a parent plant, often injured, and then placed in liquid, solid or semi-­‐solid medium containing plant growth regulators which induce essentially a cancerous state in which the plant cells at the injury sites start to divide rapidly without differentiation. This callus can then be induced to go through organogenesis or in some conditions to produce somatic embryos (Cassells 2004). In the case of direct morphogenesis callus production is not observed and instead plant cells at the injury site directly differentiate and undergo organogenesis or embryogenesis. Organogenesis is the production of specific plant organs such as roots or shoots, whereas somatic embryogenesis is the process by which the callus cells are stimulated to form a plant embryo which is essentially identical to a zygotic embryo produced through sexual reproduction but is produced from somatic cells (Arnold 2008; Raghavan 2004). Organogenesis is generally easier to attain and is therefore often the preferred technique when tissue culture is applied in biotechnology (Cassells 2004; George et al. 2008). ! A`!!J/O&'(!A4T4!D)-/'(61!;'O%);O()(:/:!/)!!/()(*$+,-.,%*"!69!M';::;\!6%++&:!/)-&61/;)!N1;V!+(<1P7!:2;;1!/)/1/%1/;)!N";11;>!+(<1P7!';;1!/)-&61/;)!N6()1'(P!%)-!%66+/>%1/0%1/;)!N'/O21P!N.'+%)-!%)-!E%2>;&-7!?@A["P!!J/O&'(!A4`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o!E%62%S;9%!(1!%+!?@@Zo!9%)!51%-()!(1!%+!?@@Zo!E;:2S;9!(1!%+!?@@ZP4!*1!%!V+%)1p:!6(++&+%'!+(9(+!()-;O();&:!%&,/):!%'(!/)9;+9(-!/)!6(++!-/9/:/;)!%)-!(+;)O%1/;)!%)-!%'(!%+:;!/)9;+9(-!/)!12(!<;'>%1/;)!%)-!>%/)1()%)6(!;<!>('/:1(>:7!V;+%'/1#7!%V/6%+!-;>/)%)6(!%)-!>(-/%1/;)!;<!1';V/:>:4!D)!6(++!6&+1&'(! 17 techniques, auxins are generally used to induce rooting. Chemically, auxins are characterized by the presence of either an indole or an aromatic ring in their structure. In culture they are useful in inducing callus formation and organogenesis, with their most common application being rooting induction (Machakova et al 2008). A good example is the use of the auxin indolebutyric acid (IBA) in commercially available rooting powder rooting powders are commonly used in both home and commercial situations to induce rooting of cuttings. When used in combination with cytokinins the second class of plant growth regulator auxins lead to callus formation and bud and growth initiation (Murch and Saxena 2008). Cytokinins, like auxins, have diverse effects with the ability to modulate plant growth and behavior in conjunction with or contrary to other plant growth regulators that are present and with differing effects depending on species and tissue type. Generally, however, cytokinins stimulate protein synthesis and are involved in cell cycle control in whole plants and are particularly involved in chloroplast maturation and senescence. In culture, cytokinins are able to overcome apical dominance allowing for shoot multiplication by releasing lateral buds from dormancy (van Staden et al. 2008). The first cytokinin to be discovered was the synthetic cytokinin kinetin, discovered to be a product of the breakdown of herring sperm that was used in cell culture (Amasino 2005). Most cytokinins are purines with the class comprising all chemicals that showed activity similar to that of kinetin, essentially the ability to stimulate cell division. Since then many more cytokinins, including naturally produced examples have been discovered, however, in general the natural cytokinins, such as zeatin, are significantly more expensive as compared with synthetic alternatives, with the synthetic cytokinin benzylaminopurine being one of the most common (van Staden et al. 2008). ! AZ!B%"+(!A4[4!.,%>V+(:!;<!:;>(!6;>>;)!V+%)1!O';X12!'(O&+%1;':!G%>(! 51'&61&'(! =+%::! B#V(!`Y3()0#+%>/);V&'/)(!N3*P!=#1;S/)/)! 5#)12(1/6!G`Y<&'<&'#+%>/);V&'/)(!NS/)(1/)P!!=#1;S/)/)! 5#)12(1/6!b(%1/)!!=#1;S/)/)! G%1&'%+!B2/%-/%0&';)!NBFbP!!=#1;S/)/)l!%&,/)!/)-&6('!5#)12(1/6!D)-;+("&1#'/6!%6/-!ND3*P!!*&,/)! G%1&'%+l!5#)12(1/6j!jV';-&6(-!/)!9('#!+;X!+(9(+:!)%1&'%++#!D)-;+(Y[Y%6(1/6!%6/-!ND**P!!*&,/)! G%1&'%+!G%V212#+%6(1/6!%6/-!NG**P!!*&,/)! 5#)12(1/6!M/""('(++/6!%6/-!NM*[P!!M/""('(++/)! G%1&'%+!*":/:/6!%6/-!!*":/6/6!%6/-! G%1&'%+!.12#+()(! .12#+()(!!G%1&'%+! 19 The applications for plant tissue culture are multifaceted ranging from plant conservation to biotechnology and bioreactors. Regardless of the application, however, an optimal protocol for regeneration of the species of interest is first required. This is frequently a time consuming and often complex process which is effected not only by the growth factors present in the media but also media type and composition, i.e. micro and macronutrient content, light conditions, amount and type of gelling agent, type of tissue used and the presence or absence of many different additives. The choice of all these conditions can be governed by protocols in similar species and regeneration type desired and must be modified through experimental analysis to determine optimum conditions (George and Davies 2008; George and de Klerk 2008; Thorpe et al 2008). 1.5. In vitro mutagenesis of plants and determination of gene targets Induction of mutations in plants has allowed for not only the evolution of plant species in nature, but has become an important laboratory technique. There are diverse techniques by which this can be accomplished but they can be broadly grouped into two categories: those induced by physical agents and those induced by chemical agents. Physical treatments include x-­‐rays, gamma radiation and ultraviolet radiation, while chemical agents include alkylating, intercalating and base analogues (Mba et al. 2010). These techniques lead to changes in appearance, traits or characteristics of the organism of interest due to DNA modification. These DNA modifications may be minor such as the point mutations induced by the alkylating agent ethyl methane sulfonate (EMS) or may result in changes to entire chromosomes (Mba et al. 2010). In plants mutagenesis is most often induced at the single cell stage, generally in the form of callus, though embryos and whole plants have also been used. The application of a chemical or physical mutagen to the callus stage has the advantage of being able to reliably produce entire plants containing a 20 particular mutation, however, these techniques do require broad scale screening to determing those individuals producing the desired traits as they are non-­‐specific in their activity. Tissue culture techniques allow for these single callus cells to be grown into shoots and then whole organisms that can be moved to the field, subcultured and multiplied (Mba et al. 2010). Once individuals with desired mutations have been identified the mutated gene can be identified using various molecular techniques. Primarily sequencing, both of the genome and transcriptome, have been used to compare mutated individuals to wild-­‐type individuals and in particular, next-­‐generation sequencing techniques such as Illumina© sequencing, which provide large numbers of reads, thereby increasing accuracy of sequencing, are being used (Blumenstiel et al. 2009; Uchida et al. 2011; Uchida et al. 2014). Other techniques such as polymerase chain reaction (PCR), gene cloning and target sequencing can also be used on a smaller scale to target and examine specific genes instead of the whole genome or transcriptome (Lis et al. 2008). To identify gene targets for EMS mutation in plants expressing a unique and desirable essential oil composition next-­‐generation sequencing techniques will be used. Illumina© is a next-­‐generation sequencing technique that can be used to sequence either the genome or transcriptome of a plant and will be used to perform both on wild-­‐type and mutant plants, as has been previously reported (Uchida et al. 2014). Illumina© sequencing is a fluorescence based sequencing-­‐by-­‐synthesis technology that is more cost effective than other sequencing technologies, allows for high throughput analysis and produces large numbers of 20-­‐150 bp reads for every sequence. This greater read number allows for more accurate identification of single base pair mutations. 21 1.6. Biological activity screening Drosophila suzukii or spotted wing drosophila (SWD) is a vinegar fly species native to Japan. It has recently become an invasive pest in North America. Reported as far back as the 1930’s in Japan, first reports of SWD in North America came from berry growers in California in 2008 (Hauser 2011). Spotted wing drosophila quickly moved up the west coast of North America and the first reports of SWD in British Columbia came from a cherry orchard near Kelowna in 2009 (Acheampong and Thistlewood 2011; Lee et al. 2011b; Thistlewood et al. 2012). Since this first report, SWD has become a major invasive pest in British Columbia and the Pacific Northwest (Washington and Oregon) and has spread as far as Europe, having been able to colonize diverse stone fruits and berry crops in the regions including: blueberry, raspberry, strawberry, cherry and blackberry (Lee et al. 2011b; Lee et al. 2011a). Unlike related Drosophila species that oviposite only in decaying or damaged fruit, SWD posses a unique serrated ovipositor that is capable of piercing the skin of unripe and ripening fruit (Atallah et al. 2014). Due to the speed and efficiency with which the pest has spread integrated pest management programs continue to develop and evolve as both growers, food inspectors and scientists try to develop effective pest management protocols (Dreves 2011; Lee et al. 2011b). Current control methods for SWD management rely on frequent application of traditional pesticides, SWD is able to go through numerous life cycles in a single growing season due to its relatively short life cycle. Fruit processors often have zero tolerance policies for infested fruit (Beers and Van Steenwyk 2011; Bruck et al. 2011). Unfortunately frequent application of insecticides are raising alarm due to their damaging effects on beneficial insects, such as pollinators and predacious insects as well as contamination of local ecosystems and water sources (Lin et al. 2011; Tan et al. 2014). As a result of this, 22 there is a growing interest in less ecologically damaging control methods and a strong push to develop new, organic and ecologically sustainable methods, which are still effective in controlling this economically devastating pest (Cleveland et al. 2002; Ioriatti et al. 2011; Lee et al. 2011b). Essential oils are produced by many plant species and serve many important commercial purposes including use in cooking, medicine and agriculture. Plants produce these important compounds in nature for many purposes including allelopathy, pollinator attraction and plant defense (Mahmoud and Croteau 2002). From an agricultural perspective their functions in insect attraction and plant defense are of greatest interest. Some essential oil constituents, such as linalool, are also insect pheromones, while many others both alone and as whole oils such as have shown strong activity as repellents, feeding and oviposition deterrents and insecticides in diverse genera of insects (Mant et al. 2005; Sfara et al. 2009; Regnault-­‐Roger et al. 2012). One such commercial example is the impregnation of grafting strips with lavender essential oil for the repellency of the red bud borer, which show 95% efficacy (van Tol et al. 2007). 1.7. Research Objectives and Hypotheses Essential oils and their individual components are important plant natural products exploited by humans for many diverse purposes and industries including cosmetics and perfumery, medicine, insect control and biofuels among others (Woronuk et al. 2010; Reinsvold et al. 2011; Regnault-­‐Roger et al. 2012). Though the composition of lavender essential oils is well defined the mechanisms by which they are produced and through which this process is regulated is as yet to be fully defined. In particular there is a lack of literature on transcriptional and post-­‐transcriptional regulatory mechisms in essential oil producing plants. The production of essential oil mutants will provide a model system in 23 which to study the contribution of differential biosynthetic and regulatory mechanisms in producing specific phenotypes. Additionally these plants may represent new and valuable commercial cultivars which provide higher yield per acre while also producing an oil of improved quality. The last objective of this hypothesis was to examine the effect of different essential oils on an invasive pest Drosophila suzukii for which the effect of essential oils and in particular lavender essential oils has yet to be reported. This thesis will address these concerns via four hypotheses: (1) regeneration efficiency of L. x intermedia cv Grosso can be improved through adjustment of culture conditions(2) cultures treated with the chemical mutagen ethyl methanesulfonate will yield genetically distinct mutants with an altered essential oil profile due to mutations of structural and/or regulatory genes (3) EMS-­‐induced mutations in regenerated plants can be identified using molecular techniques such as sequencing and qPCR and, (4) essential oils with different compositions will have varying activities against the invasive pest Drosophila suzukii. 24 Chapter 2. Development of a protocol for regeneration of L. x intermedia cv Grosso 2.1. Abstract An improved protocol for the regeneration of lavandin (Lavandula x intermedia cv Grosso) is reported. Thiadazuron (9 µM), a plant growth modulating phenylurea, was used to induce callus formation and shoot initiation from cultured leaf explants. Newly emerged shoots were maintained on media containing 0.05 µM NAA to allow maturation, and then transferred to media containing 2.9 µM IAA to allow root formation. The phenolic control agents polyvinylpyrrolidone (PVP), ascorbic acid (AA), 2-­‐aminoindane-­‐2-­‐phosphonic acid (AIP) and activated charcoal (AC) were tested for their ability to prevent shoot browning and death in culture. All agents except PVP were found to be effective, with ascorbic acid being most consistent in promoting development of healthy mature shoots. The effect of light type (red light vs. white light) and culture medium composition (full and half strength Murashige and Skoog (MS) medium, and Llyod and McCown’s woody plant medium (WPM)) on rooting efficiency was also evaluated. Cultures on half strength WPM in white light were found to have the highest rooting efficiency. Additionally application of the polyamines putrescine (Put), spermine (Spm) and spermidine (Spd) were tested for their effect on rooting. While rooting efficiency was not improved with any of the polyamine treatments, Spm and Spd were found to have an inhibitory effect at concentrations greater than 10 µM. 2.2. Materials and Methods Mature Lavandula x intermedia var Grosso plants were obtained from Okanagan Lavender Herb Farm, Kelowna, BC, Canada. Leaf or node cuttings were removed and surface 25 sterilized by immersion in 20% sodium hypochlorite with 0.1% Triton-­‐X100 (Fisher Scientific, Canada) for 20 minutes with stirring. Explants were then washed three times with sterile water for 5 minutes each. To propagate shoots for shoot survival trials a previously reported method, used for propagating L. angustifolia, was used with minor modifications. Briefly leaf explants were lacerated along the edges then plated abaxial side down on MS media including vitamins (Murashige and Skoog 1962) (PhytoTechnology Laboratories, USA) containing 3% (w/v) sucrose (Fisher Scientific, Canada), 2.35 % (w/v) gellan gum (PhytoTechnology Laboraties, USA) and 9 µM TDZ (PhytoTechnology Laboratories, USA), pH was adjusted to 5.8 prior to autoclaving. Cultures were kept in the dark at 25oC for approximately three weeks until shoot production was observed (Falk et al. 2009). Shoots were then excised from the callus and moved to a 16-­‐hour photoperiod and allowed to multiply. Newly emerged shoots were sub-­‐cultured every 14 days for a month to establish sufficient number of shoots. These shoots (n=12 for each treatment group) were then placed one per culture box on MS medium (as described above) containing 0.05 µM NAA and one of each of the following treatments: 10 mg/L AA (Acros Organics, Canada), 250 g/mL PVP (Sigma-­‐Aldrich, Canada), 0.21 mg/L AIP (sv Chembiotech Inc. Edmonton, Canada), 0.5 g/L AC (EMD Millipore, USA), 1 g/L AC or no treatment. AA and PVP were filter-­‐sterilized (0.22 µm, EMD Millipore, USA) and added after autoclaving, while AIP and AC were added prior to autoclaving. Cultures were kept at 25oC, 16-­‐hour photoperiod for 4 weeks and browning and leaf morphology changes recorded. At the end of four weeks, shoot vigor was recorded on a scale of zero to four with categories defined as follows: 0 – brown and dead; 1 – brown, immature; 2 – brown, mature; 3 – green, immature; 4 – green, mature. 26 For rooting trials node cuttings were placed on either MS media or WPM (Lloyd and McCown 1980)(PhytoTechnology Laboratories, USA) at full or half strength with 3% (w/v) sucrose, 2.5% (w/v) gellan gum and 2.9 µM IAA (Sigma-­‐Aldrich, Canada) with pH adjusted to 5.8. Nodes cultured on each of the four media types were then kept on a 16-­‐hour photoperiod under either white fluorescent or red light emitting diode (LED) light. Cultures were maintained for 4 weeks and root formation was recorded. For each treatment group n = 4, experiments were performed in triplicate. The effect of the polyamines Put (PhytoTechnology Laboratories, USA), Spm (Sigma-­‐Aldrich, Canada) and Spd (PhytoTechnology Laboratories, USA) on rooting efficiency was examined on plants that were grown on half-­‐strength MS or half-­‐strength WPM media in white light as described above. Polyamines were filter sterilized and added to the medium after autoclaving to obtain final concentrations of 10, 100 or 1000 µM. Cultures were maintained for 4 weeks and root formation was recorded. For each treatment n = 6, experiments were performed in triplicate. Statistical analysis was carried out by analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) multiple comparisons model using R with α = 0.05. Contingency analysis was performed on shooting data using JMP v. 4.0. In all cases a full factorial design was used, with treatments randomly assigned to cultures. 2.3. Results 2.3.1. Organogenesis from leaf callus A procedure previously developed for the regeneration of L. angustifolia plants (Falk et al. 2009) was used to regenerate Grosso plants from leaf segments. Noticeable callus was observed at injury sites on leaf tissue within two weeks of culture start date, and after three weeks calli readily produced viable shoots Approximately 5-­‐10 shoots were ! ?e!V';-&6(-!V('!6%++&:7!X2/62!6;&+-!12()!"(!(,6/:(-!%)-!:&"Y6&+1&'(-!;)!E5!>(-/%!6;)1%/)/)O!m!xE!BFb!NJ/O&'(!?4AP4!52;;1:!X('(!1'%):<(''(-!1;!>(-/%!6;)1%/)/)O!G**!1;!%++;X!<&'12('!-(9(+;V>()17!%)-!12()!1;!>(-/%!6;)1%/)/)O!D**!1;!V';>;1(!';;1/)O7!2;X(9('7!>;:1!:2;;1:!-/-!);1!:&'9/9(!12/:!V';6(::!-&(!1;!"';X)/)O!%)-!);)(!;<!12;:(!12%1!:&'9/9(-!V';-&6(-!9/%"+(!';;1:!NB%"+(!?4AP4!!J/O&'(!?4A4!I(O()('%1/;)!;<!+%9%)-/)!<';>!+(%<!6&11/)O:!X2('(\!N%P!6%++&:!/)-&61/;)7!N"P!:2;;1!/)/1/%1/;)7!N6P!:2;;1!>&+1/V+/6%1/;)4!!B%"+(!?4A4!*9('%O(!9/O;'!;<!+%9%)-/)!6&+1&'(:!%<1('!(,V;:&'(!1;!9%'/;&:!1'(%1>()1:7!X2('(!A!/)-/6%1(:!%!-(%-!(,V+%)1!%)-!^!%!2(%+12#!>%1&'(!(,V+%)1!X/12!);!"';X)/)O40!B'(%1>()1! *9('%O(!Q/O;'0! 51%)-%'-!F(9/%1/;)! mTf!=;)</-()6(!D)1('9%+!G;)(! @4^?"! @4TA! N@4@m7!@4e^P!@4T!Ol$!*=! ?4TZ%! A4[Z! NA4eA7![4^`P!A!Ol$!*=! ?4T@%! A4e[! NA4^@7![4`@P!@4?A!>Ol$!*DH! ?4`e%! A4^m! NA4e?7![4`?P!A@!>Ol$!**! [4`e%! @4ZZ! N[4A@7!^4?[P!?T@!>Ol$!HQH! @4T@"! @4T?! N@4Ae7!@4Z[P!0B&S(#p:!C5F!N%!i!@4@T7!)!i!A?P!X%:!&:(-!1;!-(1('>/)(!:/O)/</6%)1!-/<<('()6(:!X/12!1'(%1>()1:!"(/)O!:/O)/</6%)1+#!-/<<('()1!%::/O)(-!-/<<('()1!+(11(':7!%)-!12;:(!12%1!%'(!);1!:/O)/</6%)1+#!-/<<('()1!%::/O)(-!12(!:%>(!+(11('4!!! 28 2.3.2. Addition of phenolic inhibitors, adsorbents and reducing agents Phenolic compounds secreted by cultured plants are a well-­‐known cause of culture browning so in an attempt to reduce this phenomenon, the phenolic control agents AA, AC, AIP and PVP were added to the culture media. On a ranking scale where a dead explant was assigned a score of zero and a healthy mature shoot scored a four (Figure 2.2), the control group ranked at 0.42; treatment with PVP was not significantly better. AA, AC and AIP treatments all showed a significant improvement in shoot vigor, with AA being the highest at 3.67. All additives except PVP showed a significant improvement in shoot vigor as compared to the control, although AA was the most effective agent with very little variability in results (Table 2.1). Both the control and PVP treatment also showed very low variability, however, AC and AIP treatments were less consistent with large variability (Table 6). Only 42% of cultures in the control survived, and those that did were of poor quality. In comparison 83% of all cultures started on AA treatment were of good quality and reached maturity with 100% surviving the culture process. In comparison, AC (0.5 g/L and 1 g/L) and AIP treatments had 92, 75 and 92 % survival, respectively, with 42, 50 and 50% being of good quality and reaching maturity, respectively. Only 50% of shoots treated with PVP survived, with none reaching maturity (Figure 2.3). ! ?m!!J/O&'(!?4?4!.,%>V+(:!;<!:2;;1:!6+%::/</(-!%:!N%P!2(%+12#7!>%1&'(!:2;;17!2(%+12!i!^!N"P!>%1&'(!:2;;1!X/12!"';X)/)O7!2(%+12!i!?!N6P!2(%+12#7!/>>%1&'(!:2;;17!2(%+12!i[!N-P!/>>%1&'(!:2;;1!X/12!"';X)/)O7!2(%+12!i!A4!!!!!! [@!!J/O&'(!?4[4!=;)1/)O()6#!%)%+#:/:!;<!(,V+%)1!9/O;'!%<1('!(,V;:&'(!1;!9%'/;&:!1'(%1>()1:7!)iA?!<;'!%++!1'(%1>()1:7!6;+;&'!"+;6S:!'(V'(:()1!V';V;'1/;)%+!-/:1'/"&1/;)!;<!(,V+%)1:!"#!2(%+124!!RTSTST !BBA@=(EB(LDNH=(?FJ(PAJD?(@EFJD=DEF<(EF(>EE=DFN(!B;!-(1('>/)(!12(!(<<(61:!;<!+/O21!W&%+/1#!%)-!>(-/&>!:1'()O127!);-%+!6&11/)O:!<';>!M';::;!V+%)1:!X('(!O';X)!;)!<&++!;'!2%+<!:1'()O12!E5!;'!nHE!>(-/%!&)-('!X2/1(!<+&;'(:6()1!;'!'(-!$.F!+/O214!!D)1('%61/;)!V+;1:!X('(!6;):1'&61(-!<;'!%++!1'(%1>()1!6;>"/)%1/;):!"(<;'(!*GKQ*!%)%+#:/:!X%:!V('<;'>(-!N-%1%!);1!:2;X)P!1;!-(1('>/)(!/<!:/O)/</6%)1!/)1('%61/;):!X('(!;66&''/)O4!*+12;&O2!);!/)1('%61/;)!X%:!:(()!X/12!>(-/%!1#V(7!%!:/O)/</6%)1!/)1('%61/;)!X%:!:(()!"(1X(()!+/O21!1#V(!%)-!>(-/%!:1'()O127!X/12!'(-!+/O21!2%9/)O!%!:/O)/</6%)1!(<<(61!;)+#!;)!6&+1&'(:!;)!<&++!:1'()O12!>(-/&>!NV!i!@4@AP4!E(-/%!1#V(!2%-!12(!:1';)O(:1!(<<(61!;)!';;1/)O!(<</6/()6#!X/12!:/O)/</6%)1+#!>;'(!:2;;1:!O';X)!;)!nHE!:2;X/)O!';;1/)O!NV!k!@4@@AP7!+/O21!W&%+/1#!2%-!%!>/)/>%+!(<<(61!NV!i!@4@AP4!*!>&+1/V+(!6;>V%'/:;):!>;-(+!X%:!V('<;'>(-!;)!%++!1'(%1>()1!6;>"/)%1/;):!%)-!<;&)-!12(!(<<(61:!;<!+/O21!1;!"(!-/>/)/:2(-7!X/12!1'(%1>()1!O';&V:!"(1X(()!'(-!%)-!X2/1(!+/O21!;<1()!);1!:2;X/)O!%)#!:/O)/</6%)1! 31 difference (Figure 2.4). Although cultures under red light on full or half strength WPM had the best rooting efficiency, these treatments were not significantly better than cultures (at either strength) on WPM or half strength MS under white light. Red light treatments under WPM did induce a higher rooting efficiency than white light treatments, however, cultures under red light showed some etiolation, and increased callus production, while explants under white light showed no etiolation and generally had less callus production and more robust roots (Figure 2.5). Figure 2.4. Boxplot for rooting efficiency observed after four weeks on light, media type and media strength treatments where solid bars represent the mean, boxes encompass the first and third quartiles and whiskers extend to range of data with experiments performed in triplicate and n = 4 for all treatments. Letters are assigned based using Tukey’s HSD (α = 0.05) with treatments are significantly different assigned different letters. ! [?!!J/O&'(!?4T4!.,V+%)1:!%<1('!<;&'!X((S:!;)!+/O217!>(-/%!1#V(!%)-!>(-/%!:1'()O12!1'(%1>()1:!X2('(!1'(%1>()1:!%'(!%:!<;++;X:\!N%P!'(-!+/O217!2%+<!E5!N"P!'(-!+/O217!<&++!E5!N6P!X2/1(!+/O217!2%+<!E5!N-P!X2/1(!+/O217!<&++!E5!N(P!'(-!+/O217!2%+<!nHE!N<P!'(-!+/O217!<&++!nHE!NOP!X2/1(!+/O217!2%+<!nHE!N2P!X2/1(!+/O217!<&++!nHE!!!!!!!!!!!!!!!!!!!!! 33 2.3.4. Effect of polyamines Putrescine treatment did not have a significant effect on rooting efficiency at any level (p = 0.35) while spermine (p <0.001) and spermidine (P < 0.001) treatments showed significant inhibition of rooting. No significant interactions were found between media type and polyamine treatment, with the exception of spermine at the 10 μM level (Appendix II). Both spermine and spermidine showed significant rooting inhibition on both WPM and MS at concentrations greater than 10 μM with 50 % inhibition at 100 μM on MS for both Spd and Spm, and 38 % inhibition on WPM for Spd and 36 % for Spm. Complete inhibition occurred for Spm at 1 mM on both media types while the effect of Spd was slightly weaker, with 83% inhibition occurring on both WPM and MS media. Multiple comparison’s analysis found a significant difference in rooting efficiency at the 10 μM level for spermine treatment on WPM, and a significant difference at the 1 mM level for spermidine treatments, and spermine on MS media (Figure 2.6). Furthermore, explants on higher polyamine levels showed significant browning and tissue death while explants at lower concentrations were of higher quality (Figure 2.7). 34 Figure 2.6. Boxplot of rooting efficiency observed after four weeks on 10, 100 and 1000 µM polyamine treatments (Spd, Spm or Put on either MS or WPM) where solid bars represent the mean, boxes encompass the first and third quartiles and whiskers extend to range of data with experiments performed in triplicate and n = 6 for all treatments. Letters are assigned based using Tukey’s HSD (a = 0.05), with treatments that are significantly different assigned different letters. From top to bottom treatment plots are: Spd, Put, Spm. 35 Figure 2.7. Explants after four weeks on polyamine treatments (Spd, Spm or Put on either MS or WPM) where treatments are from left to right as follows: a-­‐c 10, 100 and 1000 µM Spm on MS; d-­‐f 10, 100 and 1000 µM Spm on WPM; g-­‐i 10, 100 and 1000 µM Spd on MS; j-­‐l 10, 100 and 1000 µM Spd on WPM; m-­‐o 10, 100 and 1000 mM Put on MS; p-­‐r 10, 100 and 1000 µM Put on WPM. 36 2.4. Discussion A method for the regeneration of L. angustifolia plants from leaf tissue was recently reported (Falk et al. 2009). Although callus production and shoot initiation was highly successful from Grosso leaf tissue (with up to 15 shoots per explant), shoot maturation and root induction were problematic as most shoots did not survive due to browning and none of those that did produced roots. After shoot initiation and elongation, maturation must occur before shoots are able to produce viable roots. A major obstacle in this process, browning, can occur while shoots are still in an immature state. Though in nature phenolic compounds such as trans-­‐cinnamic acid act as allelochemicals improving plant survival by reducing competition, in culture the build up of phenolic compounds in media during the regeneration process may lead to tissue browning, which in many cases is lethal to the explant (Laukkanen et al. 1999; Tang and Newton 2004; Jones and Saxena 2013). Several classes of additives were used in this study to reduce browning and improve regeneration efficiency of Grosso plants. Polyvinylpyyrolidone and AC adsorb free phenolics in culture medium to decrease the phenolic compounds that are in contact with the explant (Weatherhead et al. 1978; Weatherhead et al. 1979; Pan and Staden 1998). Ascorbic acid accomplishes the same by acting as a reducing agent and has previously been reported to have been used in the culture of Lavandula pedunculata (Zuzarte et al. 2010). 2-­‐Aminoindane-­‐2-­‐phosphonic acid (AIP) is a relatively novel reversible competitive inhibitor of the first regulatory step in the phenylpropanoid pathway, PAL. By inhibiting this step the overall carbon flow into the pathway is reduced and the quantity of phenolics produced is limited. Though this reduces the risk of browning due to phenolic build-­‐up in species which require phenylpropanoids for structural components such as the ferulic acid in the cell wall of Zea mays (L.) it can have a negative effect on growth (Jones et al.). To date the use of AIP 37 in culture has had varying effebeen reported only in limited species which include: St. John’s wort, duckweed, bay willow, wormwood, sugar maple and elm(Weatherhead et al. 1978; Weatherhead et al. 1979; Ruuhola and Julkunen-­‐Titto 2003; Gitz et al. 2004; Hu et al. 2011; Jones et al. 2012; Jones et al. 2013; Jones and Saxena 2013; Klejdus et al. 2013). In Grosso lavender, which shows poor growth with no additivies, addition of the adsorbant AC at both concentrations variably reduced browning, however the addition of PVP, the second adsorbing agent had no effect. This could be due to efficacy of these compounds as adsorbants with either insufficient adsorption of phenolics or excessive adsorbtion of growth regulators, minerals or other compounds vital to plant growth that are present in media (Toth et al. 1994; Pan and Staden 1998). Treatment with AIP showed a strong improvement in shoot quality and survival, however, the effect seen from AIP was highly variable. Although AIP inhibits production of phenolics, some of these products of the phenylpropanoid pathway may be required in higher amounts than are able to be produced in the presence of AIP and therefore lead to plant death (Klejdus et al. 2013). Additionally flavonoids, though not considered to be a main player in the regulation of plant growth and development have been found to be involved in finer regulation of the auxin, cytokinin system in plants and modification of downstream compounds via the main entry point at PAL, may have inadvertently had effects on the growth hormone balance in the plant and addition of AIP in downstream regeneration may have a significant effect on rooting capacity of the plant (Peer and Murphy 2007). Ascorbic acid which is a reducing agent which prevents and reverse oxidation of phenolics in media had the highest efficacy in preventing browning and promoting maturation in Grosso. Though this effect is likely only due to the direct effects on phenolics in media, its reducing effect on reactive oxygen species, which have been found to interact extensively with plant signaling cascades, has 38 been reported to also have plant growth regulatory effects, thus the effect seen may be due to either or both of these mechanisms (Mittler et al. 2011; Suzuki and Mittler 2012; Gallie 2013). These results suggest regeneration efficiency of Grosso can be improved by addition of any of the additives with the exception of PVP which had no effect, however the low variability and high response obtained from ascorbic acid makes it the most reproducible additive. The positive effect of the polyamines Spd, Spm and Put have been previously reported to have a positive effect on plant rooting, however, in Grosso lavender they were found to inhibit rooting. Though the mechanism for this inhibition was not investigated all three polyamines Spm, Spd and Put have been reported to have similar browning effects in cultures as has been reported for phenolic compounds (Tang and Newton 2004). In particular it appears that in lavender addition of Spd and Spm in particular and to a lesser extent Put lead to tissue browning, necrosis and growth inhibition. Interestingly, although there was no significant interaction between polyamine treatment and media type in terms of rooting efficiency, there does seem to be a difference in overall shoot health and callus production between media types as shoots treated with polyamines on MS media were generally healthier in appearance than those grown on WPM media. Further studies are required to determine the mechanism of this interaction. In summary we have developed an efficient method for the regeneration of L. x intermedia cv Grosso from leaf tissue. The plant growth regulator TDZ efficiently induces callus formation and shoot initiation. Although rooting, induced by IAA, has highest efficiency under red light when full-­‐strength media is used, shoots grown on half strength MS and WPM media efficiently produce roots. Further, white light promotes healthier (non-­‐etiolated, more robust) shoots with less callus production, and is thus preferred. The 39 inclusion of AA minimizes browning through the culture process, leading to improved shoot quality and survival. In contrast, application of the polyamines Spd and Spm promotes browning and inhibits root formation in Grosso cultures and as the mechanism of this response are unknown further studies are required to investigate this inhibition. 40 Chapter 3. Modification of Essential Oil Composition in L. x intermedia cv Grosso 3.1. Abstract Once a regeneration protocol was established EMS, a powerful alkylating agent was used to induce point mutations and the protocol was used to regenerate viable mutants. EMS was used to make mutations in callus cells, which then have the potential to differentiate into full, mature plants. Using this method plant cells can be mutated at the single cell level and give rise to a plant within which every cell contains this mutation. Due to the non-­‐specificity of the mutations, and possibility of fatal mutations, large numbers of explants were regenerated to increase the odds of obtaining a plant with a desirable mutation, such as one affecting the essential oil phenotype. Although the leaf essential oil from lavender is not of significant medicinal or commercial value it was used as a pre-­‐screening method to identify plants that show potentially valuable mutations in regions of the genome influencing the essential oil composition. Once plants flowered, floral oil was also screened, with emphasis on those identified during pre-­‐screening. 3.2. Methods 3.2.1. Induction of point mutations in L. x intermedia cv Grosso Using the protocol for regeneration of L. x intermedia cv Grosso from leaf tissue described in Chapter 2 wild-­‐type Grosso cells were exposed to the chemical mutagen EMS with the intention of inducing point mutations which resulted in unique oil chemotypes. Briefly leaves were excised from mature wild-­‐type Grosso plants and sterilized for 20 minutes in 20% bleach with 0.1% Triton-­‐X and washed three times with sterile distilled water. Leaves were lacerated at the petiole, and along both edges and placed abaxial side down on MS 41 media with 3% sucrose, 2.35% gellan gum and 9 µM TDZ, cultures were incubated at 25oC in the dark and moved to new media every two weeks. After first callus production (approximately three weeks) explants were removed from MS medium and incubated for 60 minutes in 0.01M EMS in 1% dimethyl sulfoxide for one hour with stirring. Explants were then rinsed three times, for five minutes each in liquid MS medium (as described above, less gellan gum), blotted dry and re-­‐plated on MS medium (as described) and incubated at 25oC, in the dark until shoot formation was observed. Shoots were then excised and moved to MS medium with 3% sucrose, 2.35% gellan gum, 2.9 μMNAA and 10 mg/L AA. Shoots were incubated at 25oC under a 16-­‐hour photoperiod on this medium until mature morphology was observed, at which point they were moved to half strength WPM medium with 2.9 μM IAA, 3% sucrose, 2.5% gellan gum and 10 mg/L AA. Once shoots were rooted they were potted in pro-­‐mix hp potting soil and kept under a humidity cover under a 16-­‐hour photoperiod for 3 days, the cover was then removed and plants were maintained in the greenhouse then the field until they reached maturity. 3.2.2. Screening of essential oil for modified composition Leaf, bud and 30% flower essential oil was extracted by either steam distillation, or sonication into 5 mL pentane (Figure 3.1). The former was used first; however, because the procedure is time-­‐consuming and not suitable for large numbers of samples, sonication was used for the remaining samples. Oil composition from both extraction methods has been shown to be equivalent (Schaneberg and Khan 2002). In both cases approximately 0.4 g of tissue of approximately evenly distributed age was weighed on an analytical balance and extracted into 5 mL pentane, with 100 µL 20 mg/mL menthol for leaf and bud samples and thymol for flower samples added as internal standard to allow for determination of yield. In the case of steam distillation samples were brought to a boil then distilled for 45 42 minutes in a steam-­‐distillation/liquid-­‐liquid extraction set-­‐up, apparatus was then allowed to cool and pentane-­‐containing oil was collected. For sonication tissues were sonicated on ice for 30 minutes, pentane-­‐containing oil was then collected. Samples were diluted 10 times and run on a Varian CP 3800 GC coupled to a Varian Saturn 2200 MS/MS. Peak areas for major constituents and internal standard (menthol for leaves, and bud and thymol for 30% flower) were used to calculate relative percent composition and yield using the below formulae (1 & 2). Leaf and bud samples were analyzed using the following GC method: Sample injection volume, 1 μL; column flow rate, 1 mL/min; injector temperature, 40 oC held for 0.1 min then increased to 100 oC at 20 oC/min and held 16.5 min; column oven temperature, 40 oC held for 5 min ramp to 170 oC at 6 oC/min and held for 4 min then ramp to 230 oC at 30 oC/min and held 5 min. Solvent delay for MS detection was 3.0 min and m/z values from 39-­‐300 were monitored. Flower samples were analyzed using the following GC method: 1 μL of sample injected on, column flow rate, 1 mL/min, injector temperature, 40oC held for 0.1 min then increased to 100oC at 20oC/min and held 16.5 min; column oven temperature, 40 oC held for 2 min, ramp to 100 oC at 20 oC/min held for 5 min, ramp to 220 oC at 10 oC/min held for 1 min then ramp to 300 oC at 40 oC/min and held for 0.75 min. Solvent delay for MS detection 3.6 min and data collected from 39 – 300 m/z values. Quantification ions for terpene constituents included in analysis are given in Tables 3.1 and 3.2. Constituents were identified by comparison of mass spectra to the NIST database and those of authentic standards. Sample chromatograms for leaf, bud and flower samples are given in Figure 3.3. ! ^[!!!! ! ! z!NAP!! ! ! ! ! z!N?P!!!!!J/O&'(![4A4!51%O(:!;<!+%9()-('!<+;X('/)O!N%P!"&-7!N"P!%)1(12(:/:7!N6P!A@f!<+;X('!%)-!N-P![@f!<+;X('4!!!!!!! 44 Table 3.1. Quantification ions for terpene constituents included in analysis of leaf and bud essential oils. Compound Quantification Ion (m/z) Terpinene 93.0 Camphene 93.0 Sabinene 93.0 Carene 93.0 Cineole 93.0 Camphor 95.0 Menthol 81.0 Borneol 95.0 Cadinene 161.0 Cadinol 161.2 Bisabolol 119.0 Caryophyllene 91.0 Table 3.2. Quantification ions for terpene consituents included in analysis of floral essential oils. Compound Quantification ion (m/z) Bisabolol 161.0 Cadinol 160.9 Carene 135.8 Caryophyllene 133.0 Linalool 92.8 Linalyl acetate 92.8 Menthol 81.0 Sabinene 120.8 Terpinene 90.9 Borneol 95.0 Camphor 95.0 Phellandrene 92.8 Cineole 43.0 Lavandulyl acetate 92.8 45 Figure 3.2. Sample chromatograms with (top three panes) and without (bottom three panes) labels of leaf (top), bud (middle) and flower (bottom) chromatograms from wild-­‐type Grosso leaf samples extracted by sonication. To ensure variation in leaf oil phenotype was not due to age of tissue selected oil samples were then taken from young, medium and old leaf tissue from each suspected mutant (Figure 3.3) . Young tissue was considered to be leaves found within 4 nodes of the apical ! ^`!>('/:1(>7!;+-!1/::&(!X%:!6;):/-('(-!1;!"(!+(%9(:!<';>!12(!";11;>!?!);-(:!<';>!12(!"%:(!;<!12(!:1(>7!%)-!>(-/&>!%O(!1/::&(!X%:!6;):/-('(-!1;!"(!12(!+(%9(:!/)!"(1X(()!12(:(!'(O/;):4!*++!:%>V+(:!X('(!(,1'%61(-!/)!-&V+/6%1(!"#!:;)/6%1/;)!%:!-(:6'/"(-!%";9(4!I(+%1/9(!V('6()1!6;>V;:/1/;)!%)-!#/(+-!X%:!12()!6%+6&+%1(-!<;'!-&V+/6%1(!(,1'%61/;):!&:/)O!<;'>&+%(!A!y!?4!!E(%):!%)-!:1%)-%'-!('';'!>%'O/):!X('(!6%+6&+%1(-!%)-!V+;11(-!/)!M'%V2H%-!H'/:>!Q`!NM'%V2H%-!H'/:>!D)647!?@A^P4!!J/O&'(![4[4!51%O(:!;<!+%9()-('!+(%9(:\!N%P!#;&)O7!N"P!>(-/&>!%)-!N6P!;+-!+(%<4!!!!STRTST -<EL?=DEF(EB(bc@?JDFEL((!.::()1/%+!;/+!<';>!`@!O!O';::;!+(%<!1/::&(!X%:!-/:1/++(-!/)1;!A@!>$!V()1%)(4!F/:1/++%1/;)!V';-&61!X%:!12()!+;%-(-!;)1;!A@!O!:/+/6%!/)!%!O+%::!<+%:2!62';>%1;O'%V2#!6;+&>)!%)-!12()!X%:2(-!X/12!(+(9()!6;):(6&1/9(!:;+9()1!'/):(:!NV()1%)(\62+;';<;'>P\!J'%61/;)!NJPA!]!A@@\@7!J?!Y!m@\A@7!J[!]!Z@\?@7!J^!]!e@\[@7!JT!]!`@\^@7!J`!]!T@\T@7!Je!]!^@\`@7!JZ!]![@\e@7!Jm!]!?@\Z@7!JA@!]!A@\m@7!JAA!]!@\A@@4!!J'%61/;):!X('(!12()!-'/(-!-;X)!%)-!"';&O21!&V!/)!A!>$!V()1%)(!12()!'&)!;)!%!Q%'/%)!5%1&')!M=YE5!%1!%!1()!1/>(:!-/+&1/;)!<;++;X/)O!12(!>(12;-!V'(9/;&:+#!-(:6'/"(-!<;'!+(%<!(::()1/%+!;/+!%)%+#:/:4!! 47 The purified unknown was then send to two independent facilities for structural determination by nuclear magnetic resonance spectroscopy (NMR) – Dr. Paul Shipley at the University of British Columbia, Okanagan and Dr. Andrew Lewis at A.R. Lewis and Associated, Simon Fraser University. Both analyses identified the compound as α-­‐cadinol . 3.2.4. Confirmation of essential oil screening method To confirm the accuracy of the relative percent composition method for determining essential oil composition standard curves were created for the following mono-­‐ and sesquiterpene standards: camphor, ∂-­‐carene, 1,8-­‐cineole, borneol, β-­‐caryophyllene and a α-­‐bisabolol. As α-­‐cadinol standard is unavailable for purchase α-­‐cadinol was purified as described in section 3.2.3. This was done by creating eight dilutions from 0.5 mg/mL to 0.002 mg/mL at increments of two times dilution. All dilutions were run in triplicate. Samples were then analyzed using a Varian Saturn GC-­‐MS following the method described for leaf essential oil analysis above. Peak in the extracted ion chromatograms were integrated, and selected characteristic ions used for quantification as given in Table 3.1. Peak areas were then plotted against concentration in Microsoft Excel and a line of best fit was plotted through the linear range. Essential oil composition for wild-­‐type Grosso plant was then determined by both relative percent composition and using the standard curves. 3.3. Results 3.3.1. Production of lavender essential oil for essential oil mutants A total of 313 EMS-­‐treated explants produced 1, 591 shoots of which 259 survived to be rooted. 154 plants were successfully rooted and of those 117 survived acclimatization in the greenhouse and were moved to the field (Table 3.3), though not all regenerants were moved to the field in the same growing season. Approximately two thirds of all explants were moved to the field in the summer of 2013, while the remaining plants were planted in 48 the field in the summer of 2014. Only those transferred to the field in 2013 bloomed in the spring of 2014. Table 3.3. Summary of explants, shoots and mature plants surviving the culture process after treatment with EMS Total explants treated with EMS 313 Shoots regenerated 1,591 Plants moved to rooting medium 259 Plants successfully rooted and moved to soil 154 Plants surviving acclimatization 117 3.3.2. Identified novel leaf EO mutants A screening of leaf, bud and flower essential oil revealed that eight plants produce novel essential oil composition, as compared with a wild-­‐type plant. Though there are eight mutants, they can be grouped into three general categories: mutants with modified camphor composition, mutants with modified cineole composition and mutants with modified linalool composition. Though leaves were readily available from all plants, only plants that were planted in the summer of 2013 bloomed. Mutants 2-­‐6 completed regeneration after this time period and were not able to overwinter (Tables 3.4 – 3.6). As Grosso requires a dormancy period before flowering, flower and bud oil data was not collected for these mutants. An overview of classes of mutants is given in Figure 3.4. 49 Figure 3.4. Overview of identified mutants by phenotype. 3.3.2.1. Mutants with modified camphor composition Mutants 1, 3-­‐6 lack or have greatly reduced levels of the monoterpene camphor and significantly more borneol in leaves, flowers and bud, though the latter two stages were only available for mutant 1. This mutant also has a decreased amount of the sesquiterpene α-­‐cadinol , and has an additional sesquiterpene, alpha-­‐bisabolol present in the leaves. Both α-­‐cadinol and alpha-­‐bisabolol have demonstrated biological activity including anti-­‐inflammatory and anti-­‐fungal activity, additionally alpha-­‐bisabolol has been used in commercial applications making it a potentially valuable addition to the oil (Chang et al. 2008; Kamatou and Viljoen 2009). Leaves of mutants 1, 5 and 6 also show significantly lower cineole content. Bud and 30% flower oil are not significantly different from wild-­‐type except in their camphor and borneol compositions as stated above (Tables 3.4 – 3.6). 3.3.2.2. Mutants with modified cineole composition Mutants 2 and 7 show significantly lower concentrations or complete lack of cineole, however this expression varies with tissue in the case of mutant 7. Unfortunately, as noted above mutant 2 did not overwinter in the field and as such did not produce flowers in the modified cineole contentmodified camphor contentmodified linalool contentmodified growth patternEG e28-8EG 417EG e47-3EG 123-3EG 123-1EG e47-5EG 28EG 254EG 108modified essential oil 50 Spring of 2014. Mutant 7, interestingly showed close to a complete lack of cineole in 30% flower and bud, but cineole concentrations were the same as wild-­‐type in the leaves, while mutant 2 shows decreased cineole concentrations in the leaves. In both cases no other essential oil constituents were significantly affected (Tables 3.4 -­‐3.6) 3.3.2.3. Mutants with modified linalool composition Only one mutant, mutant 8, showed a change in linalool concentration. This plant had wild-­‐type leaf oil, however, bud and flower showed significant decreases in the linalool content without any effects on other essential oil constituents (Tables 3.4 – 3.6) 51 Table 3.4. Mutants identified by oil screening -­‐ time for regeneration from initial explant to soil, proportions of major leaf oil constituents and oil yield, where yield is the averaged across all ages of leaf. Relative percent composition of major oil constituents in leaf (standard deviation) ID Mutant # Time for full regeneration (days) 1,8-­‐Cineole Camphor Borneol Cadinol Bisabolol EG 28 1 154 4.3 (3.7) 0.8 (0.03) 59.0 (3.4) 12.1 (0.9) 3.7 (0.7) EG e28-­‐8 2 221 1.5 (0.5) 40.6 (1.7) 29.7 (2.5) 22.4 (2.1) nd EG e47-­‐3 3 251 16.1 (2.5) 1.2 (0.5) 53.0 (4.9) 15.7 (0.6) 9.2 (0.3) EG e47-­‐5 4 235 12.6 (1.2) 0.28 (0.4) 52.5 (4.0) 16.3 (2.1) 10.1 (1.2) EG e123-­‐3 5 224 2.4 (0.3) nd 89.7 (0.4) 2.3 (0.4) 3.8 (0.4) EG e123-­‐1 6 224 1.3 (0.2) 1.2 (1.0) 89.2 (0.6) 1.2 (0.2) 2.8 (0.7) EG 417 7 169 4.29 10.68 23.71 26.19 nd EG 254 8 141 9.16 (4.26) 17.93 (1.72) 31.49 (6.85) 26.14 (5.24) nd Wild-­‐type Grosso n/a 119 12.7 (0.5) 21.9 (0.1) 18.1 (0.7) 28.9 (0.1) nd 52 Table 3.5. Mutants flowering in spring 2014 identified by oil screening -­‐ time for regeneration from initial explant to soil and proportions of major bud oil constituents. Relative percent composition of major oil constituents in bud (standard deviation) ID Mutant # Time for full regeneration (days) 1,8-­‐Cineole Camphor Borneol Cadinol Linalool Linalyl acetate Phellandrene Lavandulyl acetate Terpineol EG 28 1 154 14.6 (0.4) 1.0 (0.1) 57.3 (1.6) 5.8 (0.2) 8.3 (0.9) 8.1 (0.5) 3.7 (0.8) -­‐ -­‐ EG 417 7 169 2.0 (1.2) 30.1 (0.7) 14.0 (0.3) 13.0 (1.2) 25.1 (3.2) 11.4 (1.9) 1.9 (0.2) 1.6 (0.2) -­‐ EG 254 8 141 29.6 32.7 11.3 8.4 5.3 4.5 3.7 1.2 2.1 Wild-­‐type Grosso n/a 119 23.0 (0.1) 29.7 (0.1) 10.1 (0.3) 8.4 (0.2) 15.5 (0.2) 9.4 (0.1) 2.7 (0.04) -­‐ -­‐ 53 Table 3.6. Mutants flowering in Spring 2014 identified by oil screening -­‐ time for regeneration from initial explant to soil, proportions of major 30% flower oil constituents and oil yield. Relative percent composition of major oil constituents in 30% flower (standard deviation) ID Mutant # Time for full regeneration (days) 1,8-Cineole Camphor Borneol Cadinol Linalool Linalyl acetate Phellandrene Lavandulyl acetate Terpineol Oil Yield (mg oil/g bud) EG 28 1 154 8.4 (1.0) 1.1 (0.1) 51.2 (5.4) 3.3 (0.4) 11.3 (1.1) 16.5 (2.7) 3.7 (0.7) 4.0 (0.3) 1.8 (0.2) 6.1 (3.5) EG 417 7 169 0.92 (0.5) 4.4 (0.3) 16.7 (1.2) 7.6 (2.0) 13.5 (2.8) 32.7 (2.3) 2.4 (1.8) 5.9 (0.9) 2.2 (0.7) 9.4 (1.2) EG 254 8 141 11.3 (3.0) 24.43(6.2) 17.9 (5.3) 2.9 (0.7) 11.3 (5.0) 18.3 (6.3) 3.8 (0.4) 4.7 (0.6) 2.7 (0.5) 10.4 (1.8) Wild-type Grosso n/a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igure 3.6. Average percent composition of major essential oil constituents across young, medium and old leaves from wild-­‐type and mutant Grosso plants, as calculated by equation 1, error bars represent standard error. 56 Figure 3.7. Average total essential oil yield from young, medium and old leaves from mutant and wild-­‐type Grosso plants as calculated using equation 2, error bars represent standard error. 3.3.5. Purification of unknown from wild-­type leaf essential oil The unknown was not found in any of fractions 1-­‐8 resulting from subsequent elutions with 0 – 70% pentane, with different combinations of other mono-­‐ and sesquiterpenes that are normally found in the oil being present in these fractions. Although the unknown was found to be present in fractions 9-­‐11, only fraction 10 showed high purity, while Fractions 9 and 11 had high levels of other mono-­‐ and sesquiterpenes present. Fraction 10 yielded 21 mg of unknown with ≥ 90% purity (Figure 3.8). 57 Figure 3.8. Fraction 10 (10:90 pentane:chloroform) containing primarily α-­‐cadinol. 3.3.6. Confirmation of accuracy of relative percent composition calculations Calculation of absolute percent composition from standard curves (Appendix III) was found to yield a percent composition that was close to or within the range of standard error for all essential oil constituents with none differening by more than 3% (Table 3.7). Table 3.7. Comparison of composition as determined by relative percent composition and absolute quantification from standard curves Relative percent composition Absolute percent composition Constituent Average Standard Error Average Standard Error ∂-­‐carene 3.91 0.39 6.66 0.04 1,8-­‐Cineole 14.75 0.37 16.36 0.11 Camphor 25.52 0.02 26.06 0.79 Borneol 21.07 0.52 22.03 0.08 α-­‐cadinol 33.69 0.23 33.41 1.39 β-­‐caryophyllene 1.05 0.25 4.09 0.23 58 3.4. Discussion Regeneration efficiency for EMS-­‐treated plants was poorer than is seen in wild-­‐type grosso cultures. This could be a result of lethal mutations induced by EMS treatment (Erland and Mahmoud 2014). Lavandula floral oils are commercially harvested and used in all applications, while leaf oil has yet to see a demand. Screening of mutants was limited, however, due to Grosso’s requirement of vernalization for flowering and only plants which were planted in the spring of 2013 flowered the following summer limiting the number of plants that could be screened for floral oil composition. To allow for screening of plants at an earlier stage leaf essential oil pre-­‐screening was performed. Although EO composition is different in leaves and flowers -­‐ as mechanisms regulating production of essential oil constituents in various plant tissues may differ along with oil composition itself -­‐ some of the main constituents are shared between leaves and flowers, including camphor, cineole and borneol. This screening, therefore, provided an indication of potential changes in EO composition, and allowed for investigation of these plants by molecular techniques and minimized loss of time while waiting for the vernalization period. Several mutants were identified by leaf essential oil screening and a further two were identified by floral essential oil screening from the first group of plants to flower. These mutants were then grouped into four categories: those with modified camphor composition, those with modified cineole composition, those with modified linalool composition and those with modified growth patterns. Five mutants were found to have modified camphor composition, these five plants showed roughly the same chemotype in the leaves with all having little to no camphor, greatly 59 increased borneol, slightly decreased cineole, decreased α-­‐cadinol and increased alpha-­‐bisabolol though exact composition varied slightly in each mutant. Only one of these mutants was in the first group of plants that was planted and flowered in the summer of 2014. This mutant (EG 28), also showed decreased camphor and increased borneol in the bud and flower oil as well as increased. It is of interest that such a high proportion of the mutants identified were found to have a similar chemotype with decreased camphor production. The mechanisms behind this are yet unknown, and they merit further investigation. The increase in borneol in conjunction with decreased camphor production supports the postulate that camphor is produced in lavender from the dehydrogenation of borneol and indicates that the the expression or activity of the gene responsible for this step (borneol dehydrogenase) has been affected in this plant. To date a borneol dehydrogenase gene has been cloned and characterized, however, this enzyme had very slow reaction rates, suggesting that this may not be the primary enzyme responsible for this conversion (Sarker et al. 2012). These mutants represent a resource for identifying the gene and enzyme that is primarily responsible for this conversion. Further investigation of these mutants using RNA-­‐seq, differential expression analyis, single nucleotide variant (SNV) identification and knock-­‐out or recovery experiments could confirm the identity of this enzyme. These plants are also potentially of significant economic value as new commercial lavender cultivars. The presence of camphor in lavender and lavandin oils is generally considered to decrease the value of the oil for aromatherapy, cosmetic and fragrance applications. A result English lavender oil, which contains the lowest levels of camphor of the commercially grown species is of highest value. English lavender, however, is a relatively 60 low yielding species as compared to hybrid cultivars, in particular Grosso, which yields up to ten times more oil than English lavender. A Grosso plant, with reduced camphor content has the potential to be commercially valuable, yielding large amounts of fine quality oil for growers. Two mutants were found to have modified cineole production; one with decreased cineole production in the leaves and the other with decreased cineole production in the flowers. This is of interest from a scientific point of view as cineole is found in both leaves and flowers of the wild-­‐type plant and the enzyme which produces this monoterpene is well characterized (Demissie et al. 2012). As the most likely reason for this modification would be a mutation effecting the cineole synthase gene there are several ways in which this may be possible. The first would be through a mutation in the enzyme itself, which would effect all copies of this gene in the plant. If this was the case, Grosso would need to have at least two variants of this enzyme, one highly expressed in the leaves, and the other that is highly expressed in the flowers. The more likely situation is that the mutation is in a regulatory region, which differs between tissue types and not the enzyme itself. Linalool is a highly desired monoterpene, with powerful medicinal properties as well as high economic value, due to its frequent use in the fragrance and cosmetics industries. One mutant showed increased levels of linalool in both bud and flowers. As linalool is considered to improve the quality of oil due to its pleasant scent this plant may also be of potential commercial value as a new cultivar. Very little work has been done with this mutant to date, however, and before it could be considered as such, additional screening and sampling would need to occur. 61 Only one mutant showed a modified floral growth pattern, though the essential oil of this mutant showed no difference from wild-­‐type oil. This creeping morphology is unique among lavenders and may represent commercial potential as a new ornamental. New and desirable growth patterns are valued by the ornamental industries and the production of this trailing lavender has already invited interest from lavender breeders for its use in hanging baskets and other floral displays (personal communication). Lavender essential oil composition varies significantly with both age and tissue, and as leaf essential oil was distilled from all ages of leaves it was desirable to confirm these unique chemotypes were not merely the result of developmental stage. This was done by screening the oil of all mutants at young, medium and old leaf ages and in all cases the mutant oil composition was found to be consistent across all samples (Figure 3.6). Yield is another important characteristic in determining the value of new cultivars, increased yield is equal to increased profit for the lavender grower. Although yield of the commercially used floral oil is of greatest interest, yield was also determined for leaves as this is a poorly documented oil source. Leaf oil was found, as is the case in flowers, to vary with age, however, while oil yield is highest in flower at antethesis to 30% flower, oil yield in leaves was found to be highest in young leaves. Four mutants were found to have increased oil yield in leaves (e47-­‐3, e123-­‐1, 417 and 254), however, of the two of these mutants from which flowers were able to be harvested neither showed increased oil yield. No mutants have been identified as high yielding individuals to date, though screening of mutants that have yet to flower is still required. 62 The accuracy of relative percent composition calculations is dependent on similar fragmentation of all constituents in the mixture during ionization in the MS. To confirm that the relative percent compositions calculated in this thesis were accuracte, and not the result of differential fragmentation of particular constituents standard curves were constructed for all major leaf essential oil constituents. This allowed for the determination of the absolute percent composition of these oil samples. It was found that both calculations produced percent oil compositions that were not significantly different from each other and confirmed that relative percent composition is a valid method for determining lavender essential oil composition. One of the sesquiterpene constituents found in both leaf and floral oil of Grosso could not be identified due to poor matching to the NIST database and lack of an authentic standard for purchase. This constituent could, however, be purified with relative ease and was therefore sent for identification by NMR by two independent sources which both confirmed this constituent to be the sesquiterpene α-­‐cadinol . This purified sample was then used as a standard for future analysis. 63 Chapter 4. Identification of mutation targets in novel essential oil mutants 4.1. Abstract A draft transcriptome for lavender was assembled from wild-­‐type Grosso and Lady leaves and flowers, mutant Grosso and ESTs from L. angustifolia and L. x intermedia cDNA libraries to yield with 17,003 contigs with an average length of 804 bp. As a parallel sequencing strategy Illumina reads for each individual sample were separately aligned to the pre-­‐existing cDNA library. Mutant and wild-­‐type samples were then mapped back to this transcriptome and these individual transcriptomes were screened for differentially expressed transcripts that may be responsible for observed variations in essential oil composition between wild-­‐type species and mutants. Several promising transcription factor, terpene synthase and 3-­‐hydroxy-­‐3-­‐methylglutaryl co-­‐A reductase proteins were identified as showing unique expression patterns between species and tissues and in mutant plants as compared to wild-­‐type. These candidates are valuable lead genes in identifying new terpene synthase enzymes and in studies investigating the as yet poorly understood regulation of terpene biosynthesis in plants. 4.2. Methods 4.2.1. Identification of gene targets through next-­generation transcriptome sequencing Putative mutants (based on leaf EO profile) identified in the previous section were selected for further analysis. In all cases four biological replicates and two technical replicates were used for qPCR experiments. RNA was extracted from the following mutants: EG 28, EG e123-­‐1, EG e123-­‐3, EG e47-­‐5 and EG e47-­‐3. RNA was extracted using the Qiagen RNeasy Plant Mini Kit (Qiagen, USA) and treated with the Qiagen RNAse-­‐free DNAse I (Qiagen, USA) !! 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Transcriptome Assembly Transcriptome assembly was performed in two ways, first by alignment to an existing EST library, performed by the staff at Plant Biosis, and second by de novo assembly in the CLC Genomics workbench (Qiagen, USA). 4.2.1.2. Transcriptome alignment to existing Lavandula EST library Transcriptome alignment to the L. angustifolia EST library was performed at Plant Biosis. In summary, base calling and demultiplexing was performed using Illumina© CASAVA 1.8.1 with default parameters and according to the manufacturer’s manual. Adapter and base quality trimming were performed using cutadapt software with options specified to search for Illumina© RNA PCR primers and adapter sequences anywhere in the reads, trim detected adapters and low quality bases. Quality of the sequencing reads was assessed before and after trimming using FastQC software. Bowtie aligner was then used to match short sequencing reads to lavender ESTs collections: Lavandula x intermedia and Lavandula angustifolia. Preliminary alignment results showed that in case of L. x intermedia only 6% of sequences could be aligned to ESTs, while in case of L. angustifolia about 13% of reads could be aligned, therefore the L. angustifolia library was used for further analysis. Bowtie alignment files were then parsed using an ad hoc Perl script to determine number of reads aligning to each EST. 4.2.1.3. De novo transcriptome assembly Illumina© reads (27 bp) from RNA sequencing were quality checked and trimmed to remove adapter regions and low quality sequence reads (limit 0.05 and length >20bp) in the CLC genomics workbench prior to assembly. Sequences from all EST libraries (L. angustifolia flower and leaf and L. x intermedia cv Grosso glandular trichomes) and the 46,153,023 Illumina© reads were assembled in the CLC genomics workbench multiple 66 times to attain largest possible contigs with a word sizes of 16 and 18 and minimum contig size of 300 bp. Reads were mapped back to contigs with: mismatch cost = 2, insertion cost = 3, deletion cost = 3, length fraction = 0.5 and similarity fraction =0.8. 4.2.1.3.1. BLAST annotation and GO mapping Sequences were then annotated using blastx part of the BLAST+ command line program (NCBI, USA) on the Jasper facility of Compute Canada’s Westgrid Network. Gene ontology (GO) mapping, Interpro scan, annotation and ANNEX annotation augmentation was then performed in Blast2Go (Myhre et al. 2006). 4.2.2. Differential Expression Analysis Differential expression analysis was performed on both the contigs aligned to the EST library and those assembled de novo. The former was done using the bioconductor package in Degseq and statistical analysis and normalization were performed according to developer guidelines, with contigs with less than five reads aligned to them being excluded from the analysis. For de novo assembled data reads from each individual plant were mapped against the newly assembly de novo trasncriptome library and differential expression analysis was performed in the CLC Genomics Workbench (Qiagen) using reads per kilobase of transcript per million reads mapped (RPKM) and Benjamin-­‐Hochberg false discovery rate (FDR) corrected p-­‐values. The de novo assembled library was run through the Mercator pipeline to create a mapping file with all sequences binned by function using comparison against: 3 BLAST, 2 RPSBLAST and Interproscan and databases (Lohse et al. 2013). Heatmaps of differentially expressed genes were generated and overlayed on metabolic pathways in Mapman software and comparisons between groups were performed using the Wilcoxon test, with an e-­‐value 67 cutoff of 1 and using FDR corrected p-­‐values using the Pageman function of Mapman to identify groups overrepresented in the differential expression analyis. Expression levels of gene targets identified as being differentially regulated in RNA-­‐seq results were then confirmed by real-­‐time quantitative PCR (qPCR) by the “delta delta” CT method using the BioRad CFX96 Platform in the Ghosh Lab (UBC, Okanagan) for candidates identified by comparison to the EST library and using the Applied Biosystems StepOne Plus system for all remaining samples. RNA was extracted as described in section 5.2.1 in quadruplicate and biological replicates were run in duplicate during qPCR analysis. cDNA (comlementary DNA) was synthesized using the iScript cDNA Synthesis Kit (BioRad) following manufacturer’s recommended instructions. Quantitative real-­‐time PCR reactions were performed using Sso Fast Eva Green Supermix (BioRad) for the Bio-­‐Rad CFX Manager 2.0 and Syber Select MasterMix (Life Technologies, Canada). All primers were synthesized by Life Technologies, Canada. Primer efficiencies were calculated using LinReg PCR verified according to the minimum information for publication of quantitative real-­‐time PCR experiments (MIQE) guidelines (Bustin et al. 2009; Ruijter et al. 2009) (Appendix IV). Primer sequences are given in Table 4.1. Expression of the beta-­‐actin gene was used to normalize gene expression and quantification of gene expression was carried out using the CFX manager software version 1.6.541.1028 (Bio-­‐Rad) for data collected on the BioRad CFX96 platform. For data acquired with the Applied Biosystems instrument data collection, reference genes were chosen from four previously reported reference genes. These reference candidates were screened for expression levels and stability across tissue types and samples by visual inspection of amplification plots (Appendix V). Additionally Life Technologies Data Assist 68 software was used to score candidate reference genes and the top two genes were selected as reference genes. In all cases the two reference genes used were 18S rRNA and beta-­‐actin (Table 4.1) (Soto et al. 2011; Wu et al. 2012; Demissie et al. 2012; Julio et al. 2014). All samples were run with four biological replicates and two technical replicates on an Applied Biosystems StepOnePlus System using Life Technologies Sybr Select Mastermix. Complementary DNA was synthesized from RNA extracted from four individual tissue samples from a single plant using the BioRad iScript cDNA synthesis kit. Relative quantitative expression was determined by the delta delta Cq method in Data Assist. Due to quantity of samples references were run separately from genes of interest and interrun calibrators were included on all plates along with ‘no template controls’ for quality control. As Grosso leaf samples were present on all plates grosso leaf biological replicate three was used as the calibrator in the delta delta Cq calculation. For mutant samples all samples were manually normalized to the average of the wild-­‐type samples either leaf or flower depending on tissue to give expression relative to wild-­‐type in Microsoft Excel. For wild-­‐type comparisons no further normalization was undertaken and relative expression values calculated using Data Assist. Plots were generated and means and standard error were calculated in GraphPad Prism, and all further statistical analysis for difference expression studies was performed in GraphPad Prism. An ANOVA was run to compare all wild-­‐type samples (Grosso leaf (GrL), Grosso flower (GrF), L. angustifolia cv Lady (Lady) leaf (LaL) and Lady flower (LaF)) using a full factorial design. In cases where variance was significantly different in groups the Kruskal-­‐Wallis nonparametric test was used. Significant difference is indicated by different letters using alpha = 0.05. 69 An ANOVA was again used to determine significant changes in relative expression values between mutant samples (28 flower (28F), 28 leaf (28L), e47-­‐3 leaf (e47-­‐3L), e47-­‐5 leaf (e47-­‐5L), e123-­‐1 leaf (e123-­‐1L), e 123-­‐3 leaf (e123-­‐ 3L), 417 leaf (417L), and 417 flower (417F)) and wild-­‐type, however, the multiple comparisons model quantified difference between each mutant and wild-­‐type only . Significance level is indicated by ns – no significant difference/ * p 0.05/ ** p 0.01/*** p 0.001. For wild-­‐type comparisons where variance was significantly different the non-­‐parametric Kruskal-­‐Wallis test was used. All statistical analyses were performed in GraphPad Prism. In all cases where there were only two groups being compared a student’s t-­‐test was used with significance levels as stated for comparisons between wild-­‐type and mutants. The Mann-­‐Whitley non-­‐parametric test was used in cases where groups showed significantly different variance. 70 Table 4.1. qPCR primer sequences, amplicon length and melting temperature used in qPCR experiments. Gene Function Database/ Contig ID Putative ID Forward Primer (5’ -­> 3’) Reverse Primer (5’ -­> 3’) transcription factor 17057 transcriptional factor wrky i CTCCCTTTGGCTTTGTGAGG GTTCGGTTTTAGGGACCACG transcription factor 14504 ethylene response factor erf6 GAAAGTGGAGCAGAACCCAC CATTTCCCCCATTTCCGCTG transcription factor 10411 ap2-­‐like protein TTAACTTGCTGCCGCTTCTG GCAACTGCGATTACGAGGAG transcription factor 17452 ap2-­‐type transcription factor CTTCCTTCGATTGGCCTTCC TGGCTGCATTGAAGTATTGGG transcription factor 5015 transcription factor bhlh71 AAGGACGGTGAGGAAGACAG TATCCGAATCCTCTCACGGC transcription factor 4954 WRKY14 CGACCCGACAATGCTAATCG GATGGCGTGATTGTGGTCTC transcription factor 6102 wrky transcription factor ATCGATCGTTTTCCGTTGGG TCACTTTCTTCACGCGCTTC isoprenoid biosynthesis 10341 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase TACAACCGATCAAGCAGGGA CCAGCGTGGTTGTGATCTTC isoprenoid biosynthesis 10355 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase GTACAACCGATCAAGCAGGG CCAGCGTGGTTGTGATCTTC isoprenoid biosynthesis 15232 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase 1 ATGCCCTCTACCTCACCAAC GATCTTCTCTCGCCATCGGA isoprenoid biosynthesis 2579 mevalonate diphosphate decarboxylase TTCAACCGTGTCACGCATTC TCGCTGATGAAAAGCACTGG isoprenoid biosynthesis 2580 mevalonate diphosphate decarboxylase TACCTCCTTTCCGTTGAGCC GGGGGAAGAGGGATGAAGAC 71 Table 4.1. qPCR primer sequences, amplicon length and melting temperature used in qPCR experiments. isoprenoid biosynthesis 9781 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase GGTTCTTCCCAGCAATAGCG TTGTGAGGTTTTCGTCTGCG isoprenoid biosynthesis Primary assembly HMGS TGGACAGGCTGAATGGATGT TGTTCTCGTATGGCAGTGGT isoprenoid biosynthesis Primary assembly HMGR AGCTCGTCTGAGGATGATGG TCCCCTTCTTCTCATGCTCG terpene synthase 1894 terpene synthase 6 CATCGGAGCTCACGGAAATC CGTTGGATTGCATCGACGAG terpene synthase 2929 linalool synthase GCGATATGAACCGTGTCTCG TCTCTCCACCTCAGCCAATG terpene synthase 2521 linalool synthase CCGCTTCCATTTCTTCCTCC GTACTCGACAAGACACGCTG terpene synthase 7138 bicyclogermacrene synthase GGGGCTCAACAAACACCATC ACCGGAGACGATTGGTATGG terpene synthase 3866/EST library linalool synthase TAACCCAGGGCATGACCAAC TTGCTCCCAACCTAGTCAGAG terpene synthase 7139 bicyclogermacrene synthase TAACATAGAGCCCCCGATGG ATGACCTAGTGGGCGATGAG terpene synthase 7765 copalyl diphosphate synthase partial ACAAGTCATGTGAGCATCGC AACGTGGAATGTTGATGGCG terpene synthase 1895 terpene synthase 6 GATGCCATGGATCAGTTGCC TCGACGGCGTATGACTTTCT terpene synthase 2279/EST library Cadinol Synthase AGATTATGAACGCGACGCAG TAAGCCCTCGCAAGTTGTTG terpene synthase 2520 monoterpene synthase-­‐like protein AGTTTCCGGAGCGTCGAATA CTACAACAACATCCGCCACT terpene synthase 2522/EST library linalool synthase CGATTCCGACTCCTCAGACA ATCGTCAGCAAGGTTAGGCT terpene synthase 4905 limonene synthase AACGTCATTCGAAATGGGGC GTAACACTCAACCGCCTTCG terpene synthase 1742 linalool synthase GCAAATCCACCCACGATCTC AACTTTGTTTCCGAGGTGGC 72 Table 4.1. qPCR primer sequences, amplicon length and melting temperature used in qPCR experiments. terpene synthase 7411 linalool synthase AGCTTGCGATAGTACCGGAG CGAGGTGGCTTACGACATTC terpene synthase 1740 monoterpene synthase-­‐like protein TTAATGTGTTCGCGTGCCTC GCTGTTAAGGCTTCCCGATG terpene synthase 1741 monoterpene synthase-­‐like protein AGTTGCAATCATCGGGAAGC TCTTGACAACGGGCAGATTTC terpene synthase Cloned gene borneol dehydrogenase CCTACCCCTACATCTGCTCG GTCCCTCGTCATCGGAGTAG terpene synthase Cloned gene Cineole synthase (CINS)a CCAAGCCTCAGCCATGATAGA TTGCACATCGATGCTTATCGTA terpene synthase EST library Unknown monoterpene synthase 1 (UTPS1) TACTCAACAAGGCCCGACAT AAATTGGGCTTCAACGACCC terpene synthase EST library Unknown monoterpene synthase 3 (UTPS3) CGGTGTTCATCTGCATCGAC AGGTGATGTGCCAAAAGCAG reference gene Actinb TGTGGATTGCCAAGGCAGAGT AATGAGCAGGCAGCAACAGCA reference gene 18S rRNAc GTGACGGGTGACGGAGAATT GACTCAATGAGCCCGGTATTG candidate reference gene 26S rRNAd GTCCTAAGATGAGTCCAA GTCCTAAGATGAGTCCAA candidate reference gene Ubiquitine CTCTTCATTTGGTGTTGAGGCTTC TGCCTTCACATTATCGATGGTGTC aAs described by Demissie et al. 2012 bAs described by Soto et al. 2011 cAs described by Lane et al. 2010 dAs described by Julio et al. 2014 eAs described by Wu et al. 20124.3. Results 4.3.1. Transcriptome assembly Two to eight million 27 bp reads were obtained per sample, and total assembly of all samples lead to 10% of reads being assembled into contigs, minimum contig length was 15 bp and maximum contig length was 8,455 for a total of 17,671 contigs. All contigs less than 300 bp in length were then removed to give a total of 17,003 contigs (Table 4.2 -­‐ 4.3). After read mapping to de novo assembly (with minimum contig length of 300) 68-­‐75% of reads per sample were able to be mapped back to the de novo library while approximately 12% of reads per sample could be mapped back to the EST library (Table 4.4.) Table 4.2. Contig measurements for de novo assembly Length N75 575 N50 804 N25 1,088 Minimum 15 Maximum 8,455 Average 699 Count 17,671 Table 4.3. Summary statistics for de novo assembly Count Average length (bp) Total bases Reads 41,287,265 29.47 1,216,742,275 Matched 10,576,177 30.86 326,377,646 Not matched 30,711,088 28.99 890,364,629 Contigs 17,671 699 12,353,273 74 Table 4.4. Summary statistics for read mapping to L. angustifolia EST library (Lane et al. 2010) and de novo assembled library ID Source Tissue Total reads Percent Reads Mapped to EST Library Percent Reads Mapped to de novo Library e47-­‐5 Leaf 4958018 12.63 71.53 e47-­‐3 Leaf 3557352 12.57 71.03 wild-­‐type Grosso Leaf 8392462 12.44 70.59 Provence Leaf 6660845 12.3 69.77 Lady Leaf 6284997 14 75.79 28 Leaf 3242039 12.16 75.25 Lady Flower 4877352 13.26 67.97 e123-­‐3 Leaf 2548990 12.44 71.69 wild-­‐type Grosso Flower 2226272 12.67 68.36 e123-­‐1 Leaf 3404696 12.86 71.84 4.3.2. Annotation and functional classification Functional annotation was assigned using blastx search against the NCBI NR (non-­‐redundant) database with an e-­‐value cutoff of 1e-­‐10 followed by KEGG (Kyoto encyclopedia of genes and genomes) analysis and Interproscan, functional annotations and after ANNEX augmentation. Putative functional annotations were assigned to 60%. Distribution of GO annotations and KEGG pathways is given in Figure 4.2. 75 Figure 4.2. Percentage distribution of gene sequences after GO mapping for GO cellular components, biological processes and molecular function at GO level 2 and top metabolic pathways with sequences representing enzymes from the KEGG database. 4.3.3. Differential expression analysis Differential expression analysis was performed with three different platforms: Mapman, the CLC Genomics Workbench and Degseq, with the latter being applied only to the alignment to the EST library while the former two were applied only to the de novo assembled library. The top 100 differentially expressed genes were mapped and analyzed by hierarchial clustering to show the relationship between all plants sequence. Alignment 76 to the EST library gave seven genes putatively involved in terpene biosynthesis that were differentially expressed. This expression was then confirmed by real time PCR analysis with all candidates showing significantly different expression as compared to wild-­‐type in at least one mutant with the exception of HMGR and HMGS, however all genes showed a trend towards differential expression (Figure 4.3). Of particular interest is that UTPS1 was not expressed in e123-­‐3 as well as CADS showing very minimal expression in this same plant, while all other mutants showed the opposite trend. UTPS 2-­‐4 showed significant trends towards increased expression. HMGR and HMGS both showed a trend towards increased expression in all mutants. !! ..!!V'(<1&!@BDB![TLQ!V$+3G;)2,(&4!,$1*2+'\&3!%$!#'+3G%60&!&>01&44'$,!+&E&+4!7$1!3'77&1&,%'2++6!&>01&44&3!%)&!(&,&!;2,3'32%&4!DG)631$>6GDG*&%)6+(+<%216+!L$C!1&3<;%24&!F_OUQH5!DG)631$>6GDG*&%)6+(+<%216+!L$C!46,%)24&!F_OUKH5!<,I,$#,!%&10&,&!46,%)24&!F`"TKH!8G@!2,3!;23',$+!46,%)24&!FLCSKH5!4&+&;%&3!71$*!2+'(,*&,%!%$!%)&!JK"!32%2/24&5!',!%)&!+&2E&4!$7!*<%2,%4!&8-DGD5!&@.G=5!&@.GD!2,3!-?5!24!#&++!24!#'+3G%60&!U1$44$!+&27B!a2+<&4!21&!;$*021&3!%$!#'+3G%60&!<4',(!K%<3&,%b4!%G%&4%!#)&1&!4'(,'7';2,;&!E2+<&4!21&!24!7$++$#4!c!2+0)2!d!9B9=5!cc!2+0)2!d!9B985!ccc!2+0)2!d!9B998!2,3!,4!d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he normalized expression values for the de novo assembled transcriptome calculated in the CLC Genomics Workbench as reads per kilobase of target per million mapped reads (RPKM) are given in Table 4.5 for genes from three groups of interest: (1) transcription factor families implicated in the regulation of isoprenoid biosynthesis including the AP2-­‐EREBP family, ethylene response factors (ERF), WRKY family and bHLH family, (2) isoprenoid biosynthetic genes and (3) terpene synthases. To identify candidate genes which may be responsible for unique chemotypes in two wild-­‐type plants (Grosso and L. angustifolia cv Lady) and among mutants differential expression analysis by proportions analysis in CLC Genomics Workbench then yielded candidates for which expression values were then confirmed by qPCR. These candidates are those with RPKM values provided in Table 4.5. Five transcription factors were identified as having expression values that were significantly different by proportions analysis, though a proper statistical analysis could not be performed due to lack of replication in RNA – Seq samples sent, in the mutants as compared to wild-­‐type while there were six that showed different expression in wild-­‐type samples. Two genes involved in the pathways producing IPP and DMAPP were identified – HMGR and MVAPP decarboxylase. Both these genes appeared to show differential expression patterns between species and tissue types and all but one isoform of MVAPP decarboxylase were found to have differential expression in mutants as compared to wild-­‐type. Terpene synthases were by far the best represented in this analysis with sixteen contigs putatively identified as terpene synthases. All candidates were found to show differential expression in wild-­‐type samples while all but three appeared to have differential expression in at least one of the mutants. Expression values results of qPCR of leaf and flower of both wild-­‐type species are given in Table 4.6. No transcription factor candidate was found to have significant results which 80 matched the trends observed in the RPKM expression results in fact for some of the significant qPCR results the observed expression values are opposite to what is expected, as is the case for contig 17057, a putative member of the WRKY transcription factor family where RPKM expression values show no expression in Lady and high expression in Grosso while qPCR results show low expression in Grosso and higher expression in Lady. Both contigs 17452 (AP2), and 5015 (bHLH) show significantly higher expression in Grosso leaf than in Lady leaf while there is no difference in either contig 14504 a putative ethylene response factor or 10411 a putative member of the AP2 transcription factor family. Two isoforms of HMGR were found to be differentially expressed. The first – contig 10341 -­‐ was differentially expressed between tissue types: leaf and flower while the second, contig 9781 was found to be differentially expressed between species. One isoform of MVAPP decarboxylase was found to have differential expression between leaves of Grosso and Lady. The majority of terpene synthase candidates tested showed differential expression in wild-­‐type tissues. Nine of these candidate genes had significant results which agreed with the results obtained from transcriptome analysis: contig 7138 and contig 7139 which are both putative bicyclogermacrene candidates, contigs 1895, 2520, 1740 and 1741 unidentified terpene synthases, contig 2279 which is an already characterized cadinol synthase from Lavandula angustifolia (Jullien et al. 2013), and three linalool synthase candidates; contigs 2521, 7411 and 1742. Contigs 2520, 2521 and 2522, contigs 7138 and 7139, and contigs 1740, 1741, 1742 & 7411 showed very similar expression patterns and so an additional assembly and alignment of these contigs was performed and it was found that these contigs assembled to form three individual contigs that were putatively identified as a linalool synthase, a germacrene synthase and another linalool synthase respectively. In the case of 81 all members of the two linalool synthase meta contigs differential expression was seen between tissues with flowers having significantly higher expression values than leaves for all gene candidates. Both germacrene synthase members showed significant difference between grosso flower and lady leaf. Contig 2929 which is homologous to the EST library member UTPS4 was found to show differential expression between tissues, with expression levels being highest in lady flower. Results from qPCR of gene candidates in the leaves and flower of mutants are given in Tables 4.7 and 4.8 respectively. Only one transcription factor showed significant differential expression in leaves of mutants – an AP2 type transcription factor candidate, contig 10411, which had significantly higher expression levels in mutant e47-­‐5 leaf (e47-­‐5L), however, this was the opposite of what was predicted by transcriptome analysis which gave an RPKM value of zero for this mutant. The flowers of the cineole mutant EG 417, for which no RNA-­‐Seq data was available, had two transcription factors which showed significantly increased expression levels – the first a WRKY family transcription factor candidate (contig 17057) and the second another AP2 family transcription factor family candidate (contig 17452). One HMGR isoform candidate was found to have significant differential expression in mutant leaf samples with the leaves of e47-­‐3 showing no expression of contig 15232. Another HMGR isoform candidate contig 10341 was shown to have half the expression of wild-­‐type plants in the flowers of EG 28. No other isoprenoid precursor genes showed significant differential expression in mutant leaves or flowers as compared to wild-­‐type tissues. Though there were no tepene synthase candidate genes with significant differential expression in any of the mutant flower samples tested, in leaves the terpene synthases, as 82 was observed in wild-­‐type samples, had the greatest number of differentially expressed candidates. Two linalool synthase candidates were found to be upregulated in mutant leaves, contig 2929 (UTPS4) in e123-­‐1 and contig 2521 in e123-­‐3 with UTPS4 showing 8 times the expression level of wild-­‐type plants and contig 2521 having almost three times the expression levels. One bicyclogermacrene synthase candidate was found to be expressed at eight times wild-­‐type expression levels in the leaves of 28, although no difference in expression was seen in the flowers. Three unknown (mono)terpene synthases were found to be significantly differentially expressed in mutants: contigs 3866 and 1895 in e123-­‐3, and contig 2520 in e123-­‐1. A copalyl diphosphate synthase candidate was found to be differentially expressed in e47-­‐5. The last group of genes screened were two genes that have been previously cloned and the enzymes functionally characterized: cineole synthase and borneol dehydrogenase, both from Lavandula angustifolia (Demissie et al. 2012; Sarker et al. 2012) (Tables 4.7 – 4.8). Borneol dehydrogenase expression levels were assayed only in plants with modified camphor levels. Expression was not found to be downregulated in any mutants and was only found to have significantly different expression in mutant e47-­‐5 in which was upregulated four fold. Cineole synthase was tested only in plants with modified cineole levels and was found to be significantly decreased in flowers with almost no expression as compared to wild-­‐type. 83 Table 4.5. Genes identified as showing differential expression during expression analysis in CLC, normalized expression values are given as reads per kilobase of target per million mapped reads (RPKM). Samples for which there is no RNA-­‐Seq data but expression was still studied by qPCR are noted by “qPCR” and results that are supported by significant qPCR results are given in bold Gene Function Contig Putative ID Lady leaf Lady flower Grosso leaf Grosso flower e123-­‐1 leaf e123-­‐3 leaf e47-­‐3 leaf e47-­‐5 leaf 28 leaf 28 flower 417 leaf 417 flower transcription factor contig 17057 transcriptional factor wrky i 0 0 187.59 341.37 0 0 qPCR qPCR transcription factor contig 14504 ethylene response factor erf6 275.12 275.12 208.31 379.06 0 0 qPCR qPCR qPCR transcription factor contig 10411 ap2-­‐like protein 0 0 83.8 0 0 0 0 0 0 qPCR qPCR qPCR transcription factor contig 17452 ap2-­‐type transcription factor 0 0 220.11 400.54 0 qPCR qPCR qPCR transcription factor contig 5015 transcription factor bhlh71 97.45 0 73.78 0 qPCR qPCR transcription factor contig 4954 WRKY14 135.04 245.73 0 qPCR qPCR transcription factor contig 6102 wrky transcription factor 0 0 101.89 qPCR qPCR isoprenoid biosynthesis contig 10341 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase 0 90 79.18 0 0 0 0 0 0 qPCR isoprenoid biosynthesis contig 10355 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase 0 104.1 91.58 0 0 0 0 0 0 qPCR isoprenoid biosynthesis contig 15232 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase 1 0 127.22 111.92 0 0 0 0 0 0 qPCR isoprenoid biosynthesis contig 2579 mevalonate diphosphate decarboxylase 130.54 114.84 0 0 0 84 Table 4.5. Genes identified as showing differential expression during expression analysis in CLC, normalized expression values are given as reads per kilobase of target per million mapped reads (RPKM). Samples for which there is no RNA-­‐Seq data but expression was still studied by qPCR are noted by “qPCR” and results that are supported by significant qPCR results are given in bold Gene Function Contig Putative ID Lady leaf Lady flower Grosso leaf Grosso flower e123-­‐1 leaf e123-­‐3 leaf e47-­‐3 leaf e47-­‐5 leaf 28 leaf 28 flower 417 leaf 417 flower isoprenoid biosynthesis contig 2580 mevalonate diphosphate decarboxylase 170.01 146.31 128.72 0 isoprenoid biosynthesis contig 9781 3-­‐hydroxy-­‐3-­‐methylglutaryl-­‐coenzyme a reductase 0 101.9 0 0 0 qPCR terpene synthases contig 1894 terpene synthase 6 0 170.98 150.42 273.72 terpene synthases contig 2929 linalool synthase (UTPS4) 0 0 136.43 0 0 0 terpene synthases contig 2521 linalool synthase 0 0 131.54 0 0 terpene synthases contig 7138 bicyclogermacrene synthase 0 0 119.62 217.69 0 0 0 0 0 qPCR terpene synthases contig 3866 terpene synthase 3 0 0 111.35 0 0 terpene synthases contig 7139 bicyclogermacrene synthase 0 0 107.55 0 0 0 0 0 0 qPCR terpene synthases contig 7765 copalyl diphosphate synthase partial 0 0 89.48 0 0 0 0 0 0 qPCR terpene synthases contig 1895 terpene synthase 6 0 47 41.35 75.24 0 terpene synthases contig 2279 Cadinol Synthase (CADS) 0 38.89 34.21 62.26 0 terpene synthases contig 2520 monoterpene synthase-­‐like protein 220.24 189.54 0 303.44 241.74 0 230.22 199.4 275.29 qPCR terpene synthases contig 2522 linalool synthase (UTPS2) 204.25 0 154.64 0 terpene synthases contig 4905 limonene synthase 143.68 0 108.79 0 0 85 Table 4.5. Genes identified as showing differential expression during expression analysis in CLC, normalized expression values are given as reads per kilobase of target per million mapped reads (RPKM). Samples for which there is no RNA-­‐Seq data but expression was still studied by qPCR are noted by “qPCR” and results that are supported by significant qPCR results are given in bold Gene Function Contig Putative ID Lady leaf Lady flower Grosso leaf Grosso flower e123-­‐1 leaf e123-­‐3 leaf e47-­‐3 leaf e47-­‐5 leaf 28 leaf 28 flower 417 leaf 417 flower terpene synthases contig 7411 linalool synthase 0 78.35 0 125.44 terpene synthases contig 1740 monoterpene synthase-­‐like protein 0 79.01 0 126.49 100.77 95.96 83.12 114.75 qPCR terpene synthases contig 1741 monoterpene synthase-­‐like protein 0 197.52 0 316.23 239.91 207.81 terpene synthases contig 1742 linalool synthase 0 41.86 0 346.29 Cloned genes Cineole synthase qPCR qPCR qPCR qPCR qPCR qPCR Borneol dehydrogenase qPCR qPCR qPCR qPCR qPCR qPCR qPCR qPCR 86 Table 4.6. Expression levels of genes identified as differentially expressed candidates in wild-­‐type plants expressed as mean fold change as determined by qPCR analysis. Different letters indicate statistical significance by ANOVA with alpha = 0.05. Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID Grosso Flower Grosso Leaf Lady Flower Lady Leaf transcription factor contig 17057 transcriptional factor wrky i 1.93a 7.60b 8.24b transcription factor contig 14504 ethylene response factor erf6 0.08a 0.39a 0.04a 0.36a transcription factor contig 10411 ap2-like protein 0.29a 0.57a 0.27a 0.62a transcription factor contig 17452 ap2-type transcription factor 1.10a 0.60ab 0.10b transcription factor contig 5015 transcription factor bhlh71 0.20ab 0.94a 0.20b 0.28b isoprenoid biosynthesis contig 10341 3-hydroxy-3-methylglutaryl-coenzyme a reductase 9.11b 2.62a 8.80b 1.53a isoprenoid biosynthesis contig 10355 3-hydroxy-3-methylglutaryl-coenzyme a reductase 3.41a 1.82a 5.14a 1.28a isoprenoid biosynthesis contig 15232 3-hydroxy-3-methylglutaryl-coenzyme a reductase 1 0.59a 2.41a 0.77a 0.70a isoprenoid biosynthesis contig 2579 mevalonate diphosphate decarboxylase 0.49a 1.27a 0.97a isoprenoid biosynthesis contig 2580 mevalonate diphosphate decarboxylase 0.54ab 2.01a 1.32ab 0.28b isoprenoid biosynthesis contig 9781 3-hydroxy-3-methylglutaryl-coenzyme a reductase 2.04a 0.20b terpene synthases contig 1894 terpene synthase 6 263.81a 28.56a 2492.21b 186.33a terpene synthases contig 2929 linalool synthase (UTPS4) 2.88b 3.16b 23.54a 0.12b 87 Table 4.6. Expression levels of genes identified as differentially expressed candidates in wild-­‐type plants expressed as mean fold change as determined by qPCR analysis. Different letters indicate statistical significance by ANOVA with alpha = 0.05. Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID Grosso Flower Grosso Leaf Lady Flower Lady Leaf terpene synthases contig 2521 linalool synthase 0.55a 1.42b 0.30a 0.03a terpene synthases contig 7138 bicyclogermacrene synthase 34.50ab 1.21a 1.94ab 0.48b terpene synthases contig 3866 terpene synthase 3 5.43a 1.18ab 0.93ab 0.13b terpene synthases contig 7139 bicyclogermacrene synthase 55.52a 1.28ab 3.26ab 0.88b terpene synthases contig 7765 copalyl diphosphate synthase partial 10.89a 0.54b 1.53b 0.26b terpene synthases contig 1895 terpene synthase 6 1.38a 0.37ab 0.21ab 0.01b terpene synthases contig 2279 cadinol synthase (CADS) 0.34ab 0.69a 0.16ab 0.02b terpene synthases contig 2520 monoterpene synthase-like protein 128.0b 2.04a 57.89a 2.61a terpene synthases contig 2522 linalool synthase (UTPS2) 0.79a 1.16a 0.05b 0.03b terpene synthases contig 4905 limonene synthase 0.28b 1.61a 0.77ab 0.00b terpene synthases contig 7411 linalool synthase 437.71a 1.56b 519.02a 0.99b terpene synthases contig 1740 monoterpene synthase-like protein 487.27a 0.79b 102.20a 0.05c terpene synthases contig 1741 monoterpene synthase-like protein 2294.02a 3.00b 0.37b 0.02b terpene synthases contig 1742 linalool synthase 202.10a 0.62b 123.70a 0.26b 88 Table 4.7. Expression levels of genes identified as differentially expressed candidates in leaves of mutant plants expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID e123-1 leaf e123-3 leaf e47-3 leaf e47-5 leaf 28 leaf transcription factor contig 17057 transcriptional factor wrky i 1.68 2.99 transcription factor contig 14504 ethylene response factor erf6 3.63 0.95 transcription factor contig 10411 ap2-like protein 1.80 1.83 1.40 8.60* 0.48 transcription factor contig 17452 ap2-type transcription factor 1.85 transcription factor contig 4954 WRKY14 0.26 transcription factor contig 6102 wrky transcription factor 2.27 isoprenoid biosynthesis contig 10341 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.64 0.42 0.64 0.99 0.76 isoprenoid biosynthesis contig 10355 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.93 0.86 1.39 1.64 1.14 isoprenoid biosynthesis contig 15232 3-hydroxy-3-methylglutaryl-coenzyme a reductase 1 0.20 0.73 0.00* 0.46 1.14 isoprenoid biosynthesis contig 2579 mevalonate diphosphate decarboxylase 0.44 0.70 isoprenoid biosynthesis contig 2580 mevalonate diphosphate decarboxylase 0.26 isoprenoid biosynthesis contig 9781 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.08 1.83 1.02 terpene synthases contig 2929 linalool synthase (UTPS4) 8.02* 4.62 terpene synthases contig 2521 linalool synthase 2.67* terpene synthases contig 7138 bicyclogermacrene synthase 0.40 1.13 2.09 3.23 7.94** 89 Table 4.7. Expression levels of genes identified as differentially expressed candidates in leaves of mutant plants expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID e123-1 leaf e123-3 leaf e47-3 leaf e47-5 leaf 28 leaf terpene synthases contig 3866 terpene synthase 3 2.12** terpene synthases contig 7139 bicyclogermacrene synthase 0.52 1.07 2.24 4.51 0.86 terpene synthases contig 7765 copalyl diphosphate synthase partial 0.71 2.11 1.50 3.06* 2.40 terpene synthases contig 1895 terpene synthase 6 3.31* terpene synthases contig 2279 Cadinol Synthase (CADS) 0.45 terpene synthases contig 2520 monoterpene synthase-like protein 8.39* 4.73 4.17 3.34 1.52 terpene synthases contig 4905 limonene synthase 1.33 terpene synthases contig 1740 monoterpene synthase-like protein 142.30 49.49 51.26 69.71 18.02 terpene synthases contig 1741 monoterpene synthase-like protein 5.94 8.64 Cloned genes Cineole synthase 12.75 Borneol dehydrogenase 1.54 2.18 1.63 4.32* 1.86 90 Table 4.8. Expression levels of genes identified as differentially expressed candidates in tissues/plants for which there is no RNA-­‐Seq data available expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID 28 flower 417 leaf 417 flower transcription factor contig 17057 transcriptional factor wrky i 2.28 2.28* transcription factor contig 14504 ethylene response factor erf6 1.45 0.58 4.20 transcription factor contig 10411 ap2-like protein 1.17 0.89 0.34 transcription factor contig 17452 ap2-type transcription factor 1.55 1.34 2.62* transcription factor contig 5015 transcription factor bhlh71 1.05 1.73 transcription factor contig 4954 WRKY14 1.39 2.93 transcription factor contig 6102 wrky transcription factor 1.61 3.98 isoprenoid biosynthesis contig 10341 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.47** isoprenoid biosynthesis contig 10355 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.72 isoprenoid biosynthesis contig 15232 3-hydroxy-3-methylglutaryl-coenzyme a reductase 1 1.63 isoprenoid biosynthesis contig 9781 3-hydroxy-3-methylglutaryl-coenzyme a reductase 0.91 terpene synthases contig 7138 bicyclogermacrene synthase 0.02 terpene synthases contig 7139 bicyclogermacrene synthase 0.33 91 Table 4.8. Expression levels of genes identified as differentially expressed candidates in tissues/plants for which there is no RNA-­‐Seq data available expressed as mean fold change normalized to wild-­‐type Grosso leaf as determined by qPCR analysis. Statistical significance as compared to wild-­‐type Grosso leaf by ANOVA is indicated by * (p < 0.05)/** (p < 0.01). Results that significantly support results obtained from transcriptome profiling are bolded. Gene Function Contig Putative ID 28 flower 417 leaf 417 flower terpene synthases contig 7765 copalyl diphosphate synthase partial 1.18 terpene synthases contig 2520 monoterpene synthase-like protein 0.53 terpene synthases contig 1740 monoterpene synthase-like protein 0.79 Cineole synthase 1.12 0.10 0.02* Cloned genes Borneol dehydrogenase 0.64 92 4.4. Discussion 5. To date the mechanisms governing regulation and biosynthesis of the mono-­‐ and sesquiterpenes, which comprise the majority of plant essential oils, are still not fully understood. Though many terpene synthases have been identified across essential oil producing species and in lavender, there still remain unanswered questions in their biosynthetic pathways. In some cases the enzymes leading to production of specific compounds are not yet known, as is the case for linalyl acetate, which is hypothesized to be produced from linalool using a linalool acetyl transferase though this gene is yet to be identified. In other cases an enzyme has been cloned and functionally characterized which performs a specific conversion but has low efficiency or poor specificity suggesting that there may be another version of this enzyme which is primarily responsible for the conversion and the identified gene is only a minor contributor. This is thought to be the case for the dehydrogenation of boreol to camphor, which is hypothesized to be catalyzed by a borneol dehydrogenase in lavender, however, the only gene cloned to date was slow indicating that other borneol dehydrogenases may also be present in lavenders (Sarker et al. 2012). 6. There is very little known about the mechanisms which regulate the production of mono-­‐ and sesquiterpenes. In fact regulation of the terpenes is the most poorly studied of all the secondary metabolites, though it is well known that this is a highly controlled process as they are produced only in very specific tissues and in response to specific triggers which include developmental, abiotic and biotic cues (Quaglia et al. 2012; Vranova et al. 2012; Patra et al. 2013). As yet 93 three families of transcription factors have been implicated in transcriptional regulation of terpene biosynthesis: WRKY, bHLH and AP2-­‐EREBP/AP2-­‐ERF (Pérez-­‐Rodríguez et al. 2010; Hong et al. 2012; Yang et al. 2012; Patra et al. 2013). The majority of the work done has focused on the transcription factor families involved in production of larger terpenes with even less known about regulation of monoterpene production. These include studies which have elucidated some of the mechanisms regulating the production of the anti-­‐malarial artemisinin, an important medicinal sesquiterpene in sweet wormwood (Artemisia annua), which involve the AP2 family, and the production of paclitaxel in Taxus species by WRKY and AP2-­‐EREBP family transcription factors for latex in the rubber tree (Hevea brasiliensis) (Patra et al. 2013). This leaves a gap in knowledge that is important to investigate as a greater understanding of regulation of biosynthesis of important medicinal, commercial and ecologically valuable compounds such as the monoterpenes hold the potential to improve efforts for commercial production of compounds, for production of improved commercial cultivars, and for species conservation. 7. There were three main objectives for this chapter, (1) create a draft transcriptome for lavender, (2) to perform RNA sequencing on wild-­‐type and mutant lavender plants, and (3) to use the results from objectives one and two to identify gene candidates which may be responsible for the different essential oil chemotypes seen between two wild-­‐type lavender species and mutants of Grosso lavandin. The gene candidates identified in objective three can then be 94 used in future studies to examine their potential roles as novel terpene synthases or as regulatory elements implicated in the resultant chemotype. 8. 9. Despite short (27 bp) Illumina© reads, de novo assembly showed adequate assembly with 10% of reads assembled into contigs, to give a total of 17,003 contigs After read mapping to de novo assembly (with minimum contig length of 300) the de novo library was found to give much better mapping with approximately 60% more reads mapped than was seen in the EST library (Table 4.4). This suggests that this a good quality draft transcriptome but the low number of reads assembled indicates that there is still room for improvement. This could be accomplished by additional sequencing of samples using longer reads and acquiring paired end data, which have both been shown to improve assembly. The read length used in these experiments for sequencing is exceptionally short, due to restrictions in available resources, with Illumina sequencing being capable of acquiring reads as long as 1 kb. The assembly of a lavender genome would also significantly improve assembly allowing for its use as a scaffold against which to assemble the transcriptome. GO mapping was used to visualize distribution of gene families (Figure 4.2) to ensure no gene families showed unexpectedly high representation. GO mapping by cellular component, biological process and molecular function as well as KEGG pathways showed that the functions with greatest representation had essential roles such as structural and energy acquisition and utilization. 95 10. Annotation with blastx was successful in annotating 60% of the sequences in the draft transcriptome. 11. Differential expression analysis performed for camphor mutants on the transcriptome data of individual mutant samples aligned to the EST library yielded seven differentially expressed candidates, which were relevant to terpene biosynthesis including two precursor enzymes – HMGR and HMGS, CADS and four unknown terpene synthases (UTPS 1-­‐4) for which no cloning and functional characterization has been performed. UTPS1 was found not to be expressed in e123-­‐3 but to have increased expression in e47-­‐3 and 28 suggesting that it may not be a gene primarily responsible for the chemotypes shown as all three of these plants have the same oil profile and it is unlikely that one gene acts differently in these closely related plants. UTPS2, a linalool synthase candidate was found to have significantly increased expression levels in leaves of e47-­‐5 and e47-­‐3 and therefore may be a gene that merits further investigation to determine the identity of this EST library member. Increased expression of UTPS3 and UTPS 4 was also seen in e47-­‐5 and e123-­‐3 respectively. Cadinol synthase (CADS) expression was found to be decreased in e123-­‐3 but again was found to be upregulated in e47-­‐3, e47-­‐5, and 28, which is unexpected as cadinol levels are the same in all of these plants. 12. Differential expression proportions analysis performed in CLC Genomics Workbench using the de novo assembled draft transcriptome yielded several very promising candidates for future investigation as both terpene synthases and transcription factors that may be responsible for the varying chemotypes 96 seen in the mutant plants as well as several terpene synthase candidates that are as yet uncharacterized in wild-­‐type plants. Unfortunately, due to lack of replication this data cannot stand alone, and in fact some of the results from this proportions analysis contradict the qPCR data. This thesis will accept the qPCR data, which was performed with proper replication as more accurate than transcriptome analysis, which had no replication in all cases. In addition to determine if certain groups had unusually high numbers of members with differential expression in mutants a test for over-­‐ or under-­‐representation of groups was performed in MapMan. All mutants for which differential expression analysis was performed were those possessing the camphor reduced phenotype, with one exception – EG 417 the mutant which possessed decreased cineole content in the floral oil. The Mapman analysis revealed 50 gene categories which were under or over represented. Of particular interest were seven of which were implicated in secondary metabolism or regulation. Genes in the secondary metabolism and isoprenoids bins were both found to be over represented in mutant 28 along with bZIP family transcription factors in e47-­‐5. Genes in co-­‐factor and vitamin metabolism and MYB transcription factor bins were found to be under-­‐represented in e123-­‐1. C3H family transcription factors were found to be underrepresented in e123-­‐3 as well as Pseudo ARR family transcription factor families in e47-­‐3. The overrepresentation of differentially expressed secondary metabolism and isoprenoid bins in mutants is expected due to the observed modification in chemotype. The overrepresentation of bZIP family transcription factors suggests that members of this family may be associated 97 with this modification in metabolism, which decreases in other bins may be due to a lack of effect on these groups due to mutagenesis. Two transcription factors one WRKY and one bHLH family member showed decreased expression in the leaves of grosso as compared to lady, while one AP2-­‐type transcription factor showed the opposite trend, however, this trend was not seen in flowers. This suggests that these transcription factors, if they are in fact responsible for the different oil composition, may be involved in sesquiterpene production as the primary compounds found in the leaf oil versus the flower oil are sesquiterpenes such as cadinol. The trend of differential expression of sesquiterpene related genes between leaf samples in Lady as compared to Grosso is also reflected in two enzymes in the MVA pathway – HMGR and MVAPP decarboxylase (contig 9781 and 2520 respectively), suggesting that different isoforms of these enzymes may be used in different species. One HMGR candidate, contig 10341 shows significantly different expression between leaves and flowers, with significantly higher expression in flowers versus leaves, since sesquiterpenes are generally more abudndant in leaves this also suggests perhaps different isoforms are active in different tissues. The leaves and flowers of mutant plants showed the most promising transcription factor candidates. In particular the cineole deficient mutant EG 417, which has significantly reduced expression of cineole synthase in the flowers and corresponding decreased cineole levels in the flower essential oil shows two transcription factors with increased expression in the flowers. In contrast significant differences in expression of transcription factors and cineole 98 synthase was not observed in the leaves of 417. This is as expected as the leaf essential oil of this plant does not differ from wild-­‐type oil composition. Contig 17057 a WRKY types transcription factor and contig 17452 an AP2 type protein are both expressed at more than double the levels found in wild-­‐type Grosso. As both cineole synthase and these transcription factors are differentially expressed, these transcription factors are good candidates to investigate possible interaction between them and the promoter region of cineole synthase. If these proteins were to function as suppressors of cineole synthase transcription this could explain the difference in cineole expression in the flowers as opposed to leaves in this mutant. One transcription factor was found to be upregulated in the leaves of e47-­‐5, a camphor leaf oil mutant which has very little camphor content in the leaves, as well as have increased expression of two terpene synthases in the leaves, copalyl diphosphate synthase (contig 7765) a diterpene synthase and cineole synthase. Cineole content, however, is not significantly modified in this mutant suggesting that perhaps carbon supply is limiting cineole production, that cineole is being broken down or that some other mechanism is responsible for this lack of difference in cineole content. No diterpenes were identified in the leaf oil, therefore it is likely the enzyme encoded by contig 7765 catalyzes a different reaction. Mutant e47-­‐5 also shows no expression of one HMGR candidate (contig 15232) which may be related modified sesquiterpene content in the leaves, with decreased cadinol and increased bisabolol, though overall content is still less than the total amount in the wild-­‐type plant. 99 13. Of greatest interest is the differential expression patterns of genes identified as assembling into two linalool synthase contigs which show differential expression in leaves and flowers in both species, with high expression in flowers and almost no expression in leaves. As linalool concentrations are very high in flowers, being one of the major flower oil constituents and is generally absent in leaves this is strong evidence for these genes (contigs 2520, 2521, 2522 and contigs 1740, 1741, 1742) encoding linalool synthase genes though BLAST homology shows only 94% and 86% homology to linalool synthases in L. latifolia and L. angustifolia (Landmann et al. 2007). These genes may therefore represent new isoforms of linalool synthase in lavender, however, identity can only be confirmed by cloning, expression and functional characerization of the proteins encoded by these genes. Two contigs (7138 and 7139), which assembled into a putative germacrene synthase were differentially expressed between leaves of Grosso and Lady. As germacrene is a minor sesquiterpene constituent of Grosso leaves this is not unexpected and though this compound is not of great interest in lavender functional characterization of this candidate is likely to confirm this identity or to identify this gene as a related sesquiterpene synthase. Cadinol synthase was found to have highest expression in Grosso leaves, which is again as expected as it is found in high quantities in Grosso leaves but is not documented to be present at high levels in any other tissue. 14. 100 15. Only one terpene synthase was found to be differentially expressed in leaves of mutant 28, the putative germacrene synthase in contig 7138. This is in combination with decreased expression of only one gene in the flowers, an HMGR candidate (contig 10341). As these are both related to sesquiterpene biosynthesis one or both may be responsible for the decreased sesquiterpene content in this mutant. Though germacrene synthase expression is not effected in any of the mutants, the sesquiterpene bisabolol shows increased expression – it is therefore possible that this enzyme may be a related sesquiterpene synthase that catalyzes the production of bisabolol instead of germacrene. 16. Interestingly though camphor has been reduced in five of the mutant: e123-­‐1, e123-­‐3. E47-­‐3, e47-­‐5 and 28 expression of the known borneol dehydrogenase, previously reported from L. angustifolia, was not effected except in e47-­‐5 in which it was upregulated by more than four fold. Coupled with the high borneol content in these mutants, these results strongly implicate an alternative borneol dehydrogenase being present in these plants, which is primarily responsible for the reduction of borneol to camphor, though a differentially expressed dehydrogenase enzyme was not identified in this study, improvement of the draft transcriptome may lead to identification of this enzyme. 17. Mutant e123-­‐3 had the greatest number of differentially expressed terpene synthases with contig 2521 (linalool synthase), 3866 (TPS) and 1895 (TPS) all showing significantly increased expression. As neither of the latter candidates have good homology to known terpene synthases these contigs would be good candidates to explore as novel enzymes contributing to the unique oil 101 composition of this plant. Interestingly though this set of experiments did not find statistically significant overexpression of contig 2929 (UTPS4) in this mutant, the expression levels are approximately five times that of wild-­‐type and further experiments to decrease variability in replicates may lead to significant results as was seen in previous experiments (Figure 4.3), this gene may therefore also be a good candidate for a causal mutation site. Mutant e123-­‐1 also showed differential expression of two linalool synthase-­‐like candidates (contig 2929 and 2520), however, since linalool is not present in the leaf oil of either of these mutants it is more likely that these enzymes are catalyzing the biosynthesis of a related monoterpene. No terpene synthase or transcription factor candidates were found to be differentially expressed in e47-­‐3. Though CADS expression levels were not found to be significantly different from wild-­‐type in e123-­‐3 in these experiments, it was found to have a fold change of 0.45, which is in agreement from the previous set of qPCR experiments on EST members which found CADS to be significantly decreased in this same mutant. This makes sense as cadinol content is decreased in this plant but is not in agreement with what is seen in mutants e47-­‐3, e47-­‐5 and 28 which all also show decreased cadinol content but have increased cadinol synthase expression. It is possible that these mutants, though having increased cadinol synthase expression may have a mutation present in an important structural motif or the active site of an enzyme which reduces its efficacy. 18. 102 19. These results provide a good starting point for further exploration of the molecular mechanisms dictating the chemotypes of these mutants, however, much work has yet to be done before the oil composition of wild-­‐type plants or mutants can be explained. In particular cloning and functional characterization of candidate terpene synthases is required as well as investigation of interaction of the differentially expressed transcription factors with the promoter regions of differentially expression terpene synthases or precursor pathway enzymes. After identification of these enzymes the next step is to confirm that these specific proteins are responsible for the observed chemotype, which would require either knock-­‐down by RNAi or rescue by viral induced or Agrobacterium transformation of wild-­‐type or mutant plants. As not all mutations will be due to a difference in expression levels, single nucleotide variant (SNV) analysis will also need to be performed on mutant plants as difference in essential oil composition could equally be due to a mutation effecting structure, folding or active site chemistry of the enzyme. Identification of genes harbouring high numbers of SNVs will help identify candidate genes, which may have been effected in this way. 103 Chapter 5. Screening of lavender and mutant essential oil for modified biological activity 5.1. Abstract Commonly known as spotted wing Drosophila (SWD) Drosophila suzukii was first found in the North Okanagan in 2009, and in the past 5 years has spread rapidly throughout the valley and is now a serious pest of tree fruit and berry crops in British Columbia (Thistlewood et al. 2012). With currently available prevention protocols requiring significant and costly application of pesticides the potential activity of Grosso oils may have significant commercial and agricultural importance (Beers and Van Steenwyk 2011). The biological activity of lavenders are most commonly attributed to their essential oils, therefore any modifications to the oil composition of a particular plant has the potential to have a significant impact on its biological activity. Three different cultivars of lavender and two different tissue sources with differing oil composition as well as different commercially available essential oils were tested. For insecticidal activity, both fumigant and contact toxicity at concentrations ranging from 0 -­‐25 % will be tested against the invasive agriculture pest Drosophila suzukii. 5.2. Methods 5.2.4. Rearing Field collected adult D. suzukii were received in mixed culture from Susanna Acheampong at the Canadian Food Insepction Agency, Kelowna, British Columbia, in August of 2013. Adult D. suzukii were identified, sexed and moved to vials containing a yeast based fly media containing 10 % sucrose, 5 % baker’s yeast, 2 % 104 agar, 0.8 % potassium sodium tartarate, 0.1 % potassium phosphate monobasic, 0.05 % sodium chloride, 0.05 % iron (III) sulfate, 0.05 % magnesium chloride, 0.05 % calcium chloride, 0.07 % methylparaben, 0.45 % propionic acid and 0.03 % o-­‐phosphoric acid. Flies were kept at ambient temperature under a 16-­‐hour photoperiod and after an initial population was established at sufficient density (approx. 40 flies/vial) flies were moved every three days to new medium. 5.2.5. Optimization of a method for testing fumigation toxicity and fumigation toxicity testing To test the susceptibility of D. suzukii adults to lavender essential oils in the volatile state thirty, four to seven day old adult flies were chilled at 4oC and counted on ice then added to 500 mL canning jars along with either: (a) a 35 mm petri plate containing approximately 2 mL of Drosophila medium, (b) a filter paper circle moistened with deionized water or (c) nothing and covered by organza mesh and allowed to recover for approximately 20 minutes. Mortality was then recorded after 24 hours. For essential oil testing the above method was used with addition of a 35 mm petri plate containing approximately 2 mL of Drosophila medium. Oil was then directly applied to a 25 mm square of Whattman filter paper in a 35 mm petri plate affixed to the lid of the canning jar and which was placed on top of the mesh (Figure 5.1). Oils tested are given in Table 5.1. Eight concentration levels were tested: 0, 1, 2, 3, 4, 5, 7.5 and 15 μLoil/L air in triplicate. Mortality was then recorded at 24 hours. Half FE$!6+*,)6!.'/.+/*4)*0'/!CUh$EG!3)65+1!>+4+!.)6.56)*+(!78!<4'70*!4+24+110'/!)/)68101!510/2!DLDD!3##!C@Wg=!iDNG%!Y0254+!$%F%!V\<+40-+/*)6!1+*:5<!9'4!'<*0-0A)*0'/!)/(!*+1*0/2!95-02)*0'/!*'\0.0*8!'9!+11+/*0)6!'061!)2)0/1*!)(56*!95)'+<+=&&%!!&)76+!$%F%!e061!51+(!9'4!*+1*0/2=!1'54.+!*0115+!)/(!.'-<)/8%!e06Xh'/1*0*5+/*! &0115+!D'54.+! g)/59).*54+4XW4)/(!N3'.)('!Y450*! h,'1+/!Y''(1!@/.%!CD)/!b0+2'=!hNG!h0/+'6+! D*)/()4(! D02-):N6(40.,!Ch)/)()G!m:.)4+/+! D*)/()4(! D02-):N6(40.,!Ch)/)()G!U0/)6''6! D*)/()4(! D02-):N6(40.,!Ch)/)()G!R5B50!/5*! ^5*! V11+/*0)6!e061!&4)(0/2!L'1*!CiDNG!4(/(*2+1()(*7+'#&631&()Y6'>+4! eB)/)2)/!U)3+/(+4!T+47!Y)4-!CR+6'>/)=!WhG!4(/(*2+1()1(#&631&(!.3!g+(0B51! Y6'>+4! Y%!L%!@%!D)6+1!Cb+6*)=!WhG!4(/(*2+1(!\!&*#$"%$2&(!.3!_4'11'! Y6'>+4! Y%!L%!@%!D)6+1!Cb+6*)=!WhG!4(/(*2+1(!\!&*#$"%$2&(!.3!_4'11') U+)9! b01*066+(!4(/(*2+1(!\!&*#$"%$2&(!.3!L4'3+/.+) Y6'>+4! b01*066+(!4(/(*2+1(!\!&*#$"%$2&(!.3!L4'3+/.+) U+)9! b01*066+(!g).)()-0)!/5*! ^5*! U09+:96'!CiDNG!^++-! R+4/+6! +7)8!CiDNG!2">"D" +)*/-'/#/)C%'%/;#&'!*+1*!*,+!151.+<*07060*8!'9!95)'+<+=&&)*'!(04+.*!+\<'154+!*'!6)3+/(+4!+11+/*0)6!'061!*,04*8=!9'54!*'!1+3+/!()8!'6(!)(56*!960+1!>+4+!.,066+(!)*!`'h!)/(!.'5/*+(!'5*!'/!0.+! 106 and transferred to oil exposure chambers (250 mL baby food jars lined with blotting paper to create an ~100 mL exposure chamber) (Figure 5.2). Oil was diluted to the 8 test concentrations: 0, 0.5, 1, 2.5, 5, 10, 20 and 40% oil in acetone and 0.5 mL of each solution was applied to flies in exposure chambers using a piston style manual pump sprayer (modified from Beers et al. 2011). Flies were then transferred to vials with approximately 15 mL of fly media and mortality was recorded after 24 hours. Each treatment was repeated in triplicate. Oils tested were the same as for fumigation toxicity assays (Table 5.1). Half lethal concentration (LC50) values were calculated by probit regression analysis using SPSS v22 (IBM, USA) Figure 5.2. Exposure chambers used for contact toxicity testing of lavender essential oils against adult D. suzukii. 107 5.2.7. Oviposition deterrent activity The ability of lavender essential oils to prevent oviposition of D. suzukii in blueberries was determined by the following procedure (adapted from Beers et al. 2011). Cotton balls were dipped in sterile 5% sucrose solution and then placed in the bottom of Drosophila vials. Blueberries (store bought, organic) were then washed, allowed to dry and then dipped for 3s into 3 mL of oil solutions of 0, 1, 5, 10 or 20%, using acetone as a solvent. Blueberries were then allowed to dry on blotting paper and once dry were placed in tubes (1 berry/vial). All oil concentrations were performed with five replicates. Five female and three male four to seven day old D. suzukii adults were then added to each vial which was sealed with mesh using an elastic band. Vials were then kept in a controlled environment chamber at 25oC, 16 hour photoperiod for 48 hours. Flies were then chilled and berries were removed from vials. The number of oviposition marks on each berry was counted under a dissecting microscope and recorded. Oils tested were the same as for toxicity assays (Table 5.1). Average number of oviposition marks and standard error was plotted and calculated in GraphPad Prism 6 (USA) (Figure 5.6). 108 Figure 5.3. Experimental set-­‐up for testing oviposition deterrent activity of lavender essential oils. 5.3. Results To optimize survival of flies under control conditions three possible experimental apparatuses were tested – control with no additions, control with Drosophila medium added and control with increased humidity, accomplished by added a moistened filter paper to the bottom of the jar. While the control jar with no additions showed poor survival both of the modified controls showed close to 100% survival (Figure 5.4). FE"!Y0254+!$%`%!L+4.+/*!154303)6!'9!DPb!5/(+4!.'/*4'6!.'/(0*0'/1!C/'*,0/2!)((+(G=!>0*,!9"3'3:;&1(!-+(05-!)((+(!C.'/*4'6!>0*,!9''(G!)/(!>0*,!()-<!906*+4!<)<+4!)((+(!C.'/*4'6!>0*,!,5-0(0*8G%!e06!>)1!(01*066+(!94'-!*,+!96'>+41!)/(!'9!4(/(*2+1(!\!&*#$"%$2&(!.3!L4'3+/.+!)/(!6+)3+1!'9!4(/(*2+1(!\!&*#$"%$2&(!.3!_4'11'!78!1*+)-!(01*066)*0'/!)/(!)/)68A+(!78!_h:gD!)/(!4+6)*03+!<+4.+/*!.'-<'10*0'/!>)1!.)6.56)*+(!C&)76+!#G%!&,+!'06!.'-<'10*0'/!9'4!)66!'*,+4!'061!01!4+)(068!)3)06)76+!+0*,+4!0/!*,+!60*+4)*54+!'4!'/!*,+!<).B)20/2!'9!*,+!'06!CP'4'/5B!+*!)6%!#EFEJ!b4+,+4!)/(!b)3+/<'4*!#EFMG%!N1!*,+!.'-<'10*0'/!'9!*,+!6+)9!+11+/*0)6!'06!94'-!*,+!*>'!0/*+4-+(0)!1<+.0+1!51+(!0/!*,01!1*5(8!,)3+!/'*!7++/!<4+30'5168!4+<'4*+(!*,+!+11+/*0)6!'06!<4'906+!>)1!(+*+4-0/+(!78!_h:gD!)/)68101%!&,+1+!4+156*1!1,'>+(!*,)*!*,+!'06!94'-!_4'11'!6+)9!>)1!,02,!0/!*,+!-'/'*+4<+/+1!7'4/+'6!Ca:Fa!dG=!.)-<,'4!C#E:##!dG!)/(!F=a:.0/+'6+!CFM:#]!dG!)1!>+66!)1!*,+!1+1;50*+4<+/+!.)(0/'6!C#F:#"!dG%!&,+!<4'906+!'9!L4'3+/.+!6+)9!+11+/*0)6!'06!>)1!10-06)4!75*!,)(!-5.,!6'>+4!1+1;50*+4<+/+!.'/*+/*%!&,+!-)?'4!.'/1*0*5+/*1!0/!L4'3+/.+!+11+/*0)6!'06!>+4+!7'4/+'6=!.)-<,'4!)/(!F=a:.0/+'6+%! 110 Table 2. Relative percent composition lavender essential oils tested (ISO) (Woronuk et al. 2010) Lavandula angustifolia Lavandula latifolia Lavandula x intermedia cv Grosso Lavandula x intermedia cv Grosso Lavandula x intermedia cv Provence Lavandula x intermedia cv Provence Flower Flower Flower Leaf Leaf Flower Borneol 1.0-­‐4.0 -­‐ 1.5-­‐3 8-­‐18 24.1 -­‐ Cadinene -­‐ -­‐ -­‐ 1-­‐5 -­‐ -­‐ Cadinol -­‐ -­‐ -­‐ 21 -­‐ 29 0.9 -­‐ Camphor trace 12-­‐16 6-­‐8 20 -­‐ 22 9.22 6-­‐8 Carene -­‐ -­‐ -­‐ 1-­‐3 1.5 -­‐ Caryophyllene -­‐ -­‐ -­‐ 0-­‐2 -­‐ -­‐ 1,8-­‐Cineole trace 22-­‐27 4-­‐7 13-­‐27 53.3 4-­‐7 Linalool 25-­‐38 27-­‐41 24-­‐35 -­‐ trace 25-­‐35 Linalyl acetate 25-­‐45 trace 28-­‐38 -­‐ -­‐ 26-­‐38 Ocimene 3-­‐4 trace 0.5-­‐1.5 -­‐ trace trace Sabinene -­‐ -­‐ -­‐ 0-­‐2 trace -­‐ Terpinene -­‐ -­‐ -­‐ 0-­‐2 -­‐ -­‐ Terpineol 4-­‐5 trace 1.5-­‐5 -­‐ -­‐ trace The most active compounds for fumigation assays were linalool (LC 50 1.85) and cineole (LC50 10.71) while the most active whole oils were Lavandula latifolia (LC50 3.79) and Lavandula x intermedia cv Provence (6.71). Macadamia nut, neem, avocado and kukui nut oils had no activity (Table 5.3). Cineole (LC50 0.54%) and ∂-­‐carene (LC50 2.38%) were the most effective individual constituents in contact toxicity assays, and avocado (LC 50 0.54%), L. latifolia (LC50 0.69%) and L. x intermedia cv Provence (LC50 1.23%) were the most effective whole oils (Table 5.4). Kukui nut oil showed no activity. 111 Oviposition repellency was determined by counting the number of oviposition (Figure 5.5) marks on blueberries that had been dipped in either acetone or oil made up to desired concentrations in acetone. Blueberries were allowed to dry before being placed in vials with flies. No compounds tested showed complete repellency at low concentrations; however, many of the oils assayed showed a decreasing trend in oviposition with increasing oil concentration (Figure 5.6). Macadamia nut oil had the strongest repellent effect with total repellence at 5% oil and whole oils generally showed a stronger repellent effect than individual constituents lone and avocado, neem, provence and Grosso all showing significant reductions in oviposition (Figure 5.6) Table 5.3. Fumigation toxicity of lavender essential oils against 4-­‐7 day old D. suzukii adults, half lethal concentrations (LC50) and confidence intervals (CI) calculated by probit analysis (alpha = 0.05) Oil Source LC50 (uL oil/L air) (95% Confidence Interval) Avocado commercial no activity Cineole standard 10.71 (8.44, 13.01) ∂-­‐carene standard 9.47 (8.36, 10.58) Linalool standard 1.85 (1.48, 2.12) Kukui nut commercial no activity Lavandula angustifolia flower 8.14 (7.65, 8.59) Lavandula latifolia flower 3.79 (3.28, 4.21) Lavandula x intermedia cv Grosso leaf 8.22 (7.40, 9.01) Lavandula x intermedia cv Provence flower 6.71 (6.14, 7.26) Lavandula x intermedia cv Provence leaf 5.68 (5.22, 6.14) Macadamia nut commercial no activity Neem commercial no activity Table 5.4. Contact toxicity of lavender essential oils against 4-­‐7 day old D. suzukii adults, half lethal concentrations (LC50) and confidence intervals (CI) calculated by probit analysis (alpha = 0.05) Oil Source LC50 (%) (95% Confidence Interval) Avocado commercial 0.54 (0.03, 1.40) Cineole standard 0.67 (0.13, 1.41) ∂-­‐carene standard 2.38 (1.57, 3.43) Linalool standard 9.85 (6.56, 17.83) Kukui nut commercial no activity Lavandula angustifolia flower 2.10 (1.55, 2.69) Lavandula latifolia flower 0.69 (0.29, 1.12) Lavandula x intermedia cv Grosso flower 7.21 (4.22, 12.58) Lavandula x intermedia cv Provence flower 1.23 (0.86, 1.64) Macadamia nut commercial 11.17 (7.67, 19.39) Neem commercial 14.83 (10.08, 21.49) !! HH)!!*-.+8&!'('(!UVW!%#86#&!?#C=!06-E09-/-01!,#8O9!?$C!#17!&..!?3C!6-&P&7!+17&8!'B2!,#.1-5-3#/-01(!!! 114 Figure 5.6. Oviposition deterrent activity of lavender essential oils against 4-­‐7 day old D. suzukii adults after 48 hours. 5.4. Discussion As would be expected oils containing high amounts of volatile compounds, including the monoterpenes, showed the greatest effect against SWD. Lavandula latifolia (spike lavender) showed the strongest effect of all oils tested (LC50 3.79 μLoil/L air), while the floral essential oil constituent linalool showed the greatest activity of all samples tested (LC50 1.85 uL/L air). Spike lavender oil is high in linalool and 1,8-­‐cineole. The presence of linalool but also of the less potent, but still effective monoterpene 1,8-­‐cineole, could explain the increased activity of this oil as compared with other oils. As would be expected the aromatic lavender essential oils and their constituents showed the highest activity as the volatility of these compounds facilitates 115 fumigation. The primary hindrance to the use of essential oils as pest control agents has been their cost, however, at the levels reported here this would likely not be a significant factor limiting their use for the control of SWD. Additionally the volatility of these essential oils means that they will not persist in the environment or on food crops, minimizing the concern of pesticide residues for human consumption and their impact on the surrounding environment making them safer alternatives to the existing pesticides available. The trend observed in the fumigation toxicity assays in which volatile oils showed strongest activity was not observed in contact toxicity assays further confirming that the difference in activity is most likely due to the ability of those oils to volatilize. The commercially available avocado oil showed the greatest activity with LC50 of 0.54%, though this is still greater than the reported active concentrations for commercially available control agents such as spinosad which is active at concentrations of 0.01% (Beers and Van Steenwyk 2011). Significant activity was also seen from 1,8-­‐cineole (0.67 %) and spike lavender essential oil (0.69%), which in combination with the potent activity of spike lavender essential oil in the fumigation assays suggest the combine activity of spike lavender could hold good potential for control of SWD in a field setting. It is also of interest to note that the presence of a different primary active constituent in L. latifolia oils for each mode of toxicity indicates that these monoterpenes have different mechanisms for toxicity. Additionally the combined use of the non-­‐volatile avocado oil with the highly effect whole oil of L. latifolia could significantly improve each of the individual activities. Additionally as current control methods for stone fruits such as cherries recommended by Agriculture Canada requires the use of harsh insecticides such as diazanon or malathion the use of these oils as part of an integrated pest management program could reduce the environmental impact of using exclusively synthetic pesticides while still maintaining effective control. Extensive field trials are 116 required, however, before these products would be ready for application in this environment. Leaf essential oils were not tested in the contact bioassay due to difficulty in obtaining sufficiently large amounts and due to the poor activity in the fumigation toxicity assays they were deemed unlikely to be feasible control agents. Lastly the oviposition deterrent activity of these oils was tested in an assay adapted from Beers et al. 2011. Though none of the oils showed powerful repellent activities, some repellency is suggested based on the results from all four floral essential oils tested as well as the commercial oils (Figure 5.1). Though these activities are not significant, in conjunction with the reported toxicities this may augment the efficacy of oils such as spike lavender oil. 117 Chapter 6. Conclusion The objectives of this research were to (1) develop an efficient protocol for regeneration and mutagenesis of L. x intermedia cv Grosso (2) generate unique essential oil mutants using this protocol (3) identify candidate genes effected by mutagenesis that lead to the unique chemotypes observed and (4) evaluate the biological activities (as related to insect control) of essential oils from wild type and improved lavender plants. Objective one was met through exploration of different growth parameters for tissue culture conditions including: light conditions, media type and concentration, growth regulators present, and reduction of explant browning. I hypothesized that by modifying these conditions regeneration efficiency of Grosso could be significantly improved. Chapter two identifies ideal culture conditions as using half strength WPM medium for rooting, and full strength MS for all other conditions, under white light. Addition of ascorbic acid a powerful antioxidant to all media types was found to significantly decrease browning. Other conditions tested such as addition of PVP or polyamines were found to decrease regeneration efficiency of Grosso. Objective one was therefore met and could be applied to achieve success in objective two. Using the protocol described in Chapter Three ten unique mutants were identified – nine essential oil mutants, and one with a unique growth pattern. RNA Sequencing performed in Chapter Four identified numerous candidate genes in mutants which showed unique expression patterns, indicating that the observed chemotypes were likely the result of mutations that affected the expression of these genes. There is much research that needs to be done to follow up with these 118 results. The work that has been done thus far has merely identified candidate genes for which only putative identifications are available based on closest homology to already known proteins. Follow up experiments are required to (1) confirm the identity of these proteins (2) confirm whether these differentially expressed genes do in fact cause the observed chemotype and (3) to explore other mutation types and targets. To address the first of these concerns, these genes must be cloned, expressed and functionally characterized using a microbial system such as E. coli or yeast. Purification and protein assays of these expressed genes can confirm the identity of the encoded proteins, and potentially identify new terpene synthase enzymes or transcription factors. Use of the yeast one-­‐hybrid system would allow investigation of potential interactions between transcription factors or interest and the differentially expressed terpene synthase enzymes. To confirm whether these identified candidates are responsible for the in vivo observed changes, an in vivo system must be used for confirmation. In the case of genes that have been knocked down RNAi silencing experiments could be used to knock these genes down in wild-­‐type plants and if the plant assumes the mutant chemotype then this causal mutation could be confirmed. In the case of over expressed genes Agrobacterium mediated or viral induced transformation to increase copy number of the gene of interest in wild-­‐type could mimick this situation and again allow for confirmation. Although differential expression analysis gives one tier at which to observe possible differences in the transcriptome, mutant chemotypes could also be due to a mutation at the protein or enzyme level. If the amino acid sequence of a protein is modified this may not effect expression of the transcript but could lead to an inactive (or a less active) version of the enzyme if the mutation is in an important structural region effecting folding or at the active site of the enzyme. One way to explore this possibility would be to do SNV analysis of the mutant transcriptome and look for genes with high incidence of SNV sites in and apply analysis to 119 determine which of these SNVs are more likely to be chemically induced versus those seen naturally. The last objective of this experiment aimed to look at the effect of essential oil composition on insect repellence and insecticidal properties of the oils. I hypothesized that as essential oil composition changes, there will be associated changes in biological activity with certain key constituents being responsible for greater activity. Chapter Five outlines these results, it was discovered that the essential oil constituents linalool and cineole are potential insecticidal agents and that oils high in these constituents have greater activity than those with lower levels. In general the whole oils had lower activity, whether this is due to dilution of the active constituents or is due to antagonistic interactions with other essential oil constituents could be determined through further experimentation testing combinations of several individual constituents present in an oil and comparing these to the activity of the whole oil and of the individual constituents. Some complex oils such as avocado oil were identified as potent insecticidal agents. Though the composition of avocado oil is quantified as it is consumed in food products, the exact composition is not well known and further investigation is required to determine the active constituents in this oil. No significant repellency was observed from the oils tested. In conclusion all four objectives of this study were met. However, much work has yet to be done to follow up on the results obtained from the generation and sequencing of the essential oil mutants. This study represents a starting point for the improvement of the draft transcriptome assembled in Chapter Four and for the investigation of the regulation of terpene biosynthesis in lavender by 120 the differentially expressed transcription factors identified in Chapter Four and well as identifying several likely candidates for new terpene synthase enzymes. 121 References Abe S, Maruyama N, Hayama K, et al. (2003) Suppression of tumor necrosis factor-­‐alpha-­‐induced neutrophil adherence responses by essential oils. 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H)I!!*-.+8&!])(!D1/&8#3/-019!E%0/!508!E+/8&93-1&!/8&#/,&1/!#/!50+8!3013&1/8#/-019!B=!HB=!HBB!#17!HBBB!+S!#17!/P0!,&7-#!/4E&9=!SU!#17!VRS(!R#8#%%&%!%-1&9!-17-3#/&!10!9-.1-5-3#1/!-1/&8#3/-01=!P>-%&!#!9>-5/!-1!9%0E&!-17-3#/&9!#1!-1/&8#3/-01!-9!E8&9&1/(!50556065707580Mediamean of Rooting.EfficiencyMS WPM Concentration0101000100!! H)K!C@@*57"D$,$E$%91571(7$)'(:*>$6&($1G>&#'9*$L'159"6")19"&5$&6$*>>*59"1#$&"#$>1F@#*>$$!*-.+8&!AH(!U/#17#87!3+86&!508!#%E>#;$-9#$0%0%!9/#17#87(!!!! H:B!!*-.+8&!AM(!U/#17#87!3+86&!508!$081&0%!9/#17#87(!!!! H:H!!*-.+8&!A)(!U/#17#87!3+86&!508!v;3#7-10%!!9/#17#87(!!!! H:M!!*-.+8&!A:(!U/#17#87!3+86&!508!3#,E>08!9/#17#87(!!!! H:)!!*-.+8&!A'(!U/#17#87!3+86&!508!3#8&1&!9/#17#87(!!!! H::!!*-.+8&!AL(!U/#17#87!3+86&!508!3#840E>4%%&1&!9/#17#87(!!!! H:'!!*-.+8&!A<(!U/#17#87!3+86&!508!H=I;3-1&0%&!9/#17#87 146 Appendix D – Primer Efficiencies Table D1. Mean primer efficiency across all tissue types as calculated by linreg analysis Gene Function Contig ID Mean Efficiency (% ) transcription factor 17057 91 transcription factor 14504 72 transcription factor 10411 71 transcription factor 17452 89 transcription factor 5015 90 transcription factor 4954 89 transcription factor 6102 80 isoprenoid biosynthesis 10341 84 isoprenoid biosynthesis 10355 88 isoprenoid biosynthesis 15232 88 isoprenoid biosynthesis 2579 83 isoprenoid biosynthesis 2580 85 isoprenoid biosynthesis 9781 83 isoprenoid biosynthesis HMGS 99 isoprenoid biosynthesis HMGR 99 terpene synthase 1894 78 terpene synthase 2929 89 terpene synthase 2521 89 terpene synthase 7138 90 terpene synthase 3866 93 terpene synthase 7139 93 terpene synthase 7765 89 terpene synthase 1895 90 terpene synthase 2279 90 terpene synthase 2520 84 terpene synthase 2522 94 terpene synthase 4905 88 terpene synthase 1742 89 terpene synthase 7411 89 terpene synthase 1740 81 terpene synthase 1741 81 terpene synthase CINS 86 terpene synthase BDH 89 terpene synthase UTPS1 99 terpene synthase UTPS3 99 reference gene actin 80 reference gene 18SrRNA 87 !! 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Enhancement of specialized metabolism, regeneration efficiency and biological activity in lavandin (Lavandula x intermedia cv 'Grosso')
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