Aspects of Physiology and Trichome Chemistry in the Medicinal Plant Tanacetum parthenium (L.) Schultz-Bip. by Kevin Bernard Usher B.Sc, Okanagan University College, 1994 A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BOTANY We accept this thesis as conforming to the required standard G.H.N. Towers, Supervisor (Botany, University of British Columbia) .E.P. Taylor, Co^ipen/isor (Botany, University of British Columbia) P.A. Bowen, Committee Member (Pacific Agriculture Research Center, Agriculture and Agri-Food Canada) A.D./vKala^s, Commit$e4v1ember (Botany, University of British Columbia) THE UNIVERSITY OF BRITISH COLUMBIA September 2001 © Kevin Bernard Usher, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date S" Oct , Zoo( DE-6 (2/88) 11 ABSTRACT This study investigated aspects of physiology and terpenoid chemistry in feverfew, a medicinal plant used for migraine therapy. The sesquiterpene lactone parthenolide accumulates in feverfew shoots and is thought to contribute to feverfew's antimigraine activity. The first part of this study examined the effects of nitrogen application and irrigation on shoot yield and shoot parthenolide concentration. Reduced shoot yield was observed under treatments of low nitrogen application and irrigation frequency. Leaf parthenolide concentration increased in plants grown with high nitrogen application rates and decreased with high irrigation rates. In the second part of this study, shoot yield, parthenolide concentration and trichome distribution were examined in response to developmental changes. Days longer than approximately 12 hours induced flowering. Feverfew grown under days shorter than 12 hours for extended periods remained in a vegetative stage and their leaves accumulated parthenolide in concentrations up to 10x that of flowering plants. Yield was lower in vegetative plants grown under short days but leaf to stem ratio was high. Glandular trichomes are the site of parthenolide biosynthesis and storage. Leaf parthenolide concentration is related to glandular trichome densities on leaf surfaces. Young leaves of vegetative plants have high trichome densities while young leaves of flowering plants have low trichome densities. Trichome densities decreased with leaf expansion and as density decreased, parthenolide concentration decreased. The third part of this study investigated the two terpenoid biosynthetic pathways involved in parthenolide biosynthesis. Experiments using 14C and 13C labeled substrates revealed that both the mevalonate (MEV) and methylerythritol phosphate (MEP) pathways contribute isoprene subunits to parthenolide biosynthesis. Two of the Ill three isoprene subunits in parthenolide were enriched after feeding shoots 1-13C- mevalonate and 2-13C-acetate, an enrichment pattern consistent with the MEV pathway. Parthenolide's enrichment pattern after feeding 2-13C-pyruvate and 1-13C-Glucose was consistent with isoprene contributions from both pathways. After feeding 2-13C-pyruvate however, there was a higher proportion of 13C-enrichment from the MEP pathway than from the MEV pathway. TABLE OF CONTENTS Abstract ii Table of Contents iv List of Tables vii List of Figures viii Acknowledgements x Chapter 1: General Introduction 1.1. Plant Natural Products 1 1.2. Terpenoids 4 1.3. Sesquiterpene lactones 10 1.4. Feverfew: Historical use and modern medicine 13 1.5. Objectives 17 1.6. References 18 Chapter 2: Effects of irrigation and nitrogen application on feverfew shoot yield and parthenolide concentration 2.1. Introduction 26 2.2. Materials and Methods 2.2.1. General methods 30 2.2.2. Field irrigation trial 33 2.2.3. Field irrigation and nitrogen application trial 34 2.2.4. Greenhouse irrigation and nitrogen application trial 36 2.3. Results 2.3.1. Field irrigation trial: effects of irrigation frequency on parthenolide concentration and plant growth ... 38 2.3.2. Field irrigation and nitrogen application trial 39 2.3.3. Greenhouse irrigation and nitrogen application trial 42 2.4. Discussion 44 2.5. References 52 Chapter 3: Development and Regeneration 3.1. Introduction 55 3.2. Materials and Methods 3.2.1. General methods 58 3.2.2. Field growing medium and fertigation trial 60 3.2.3. Greenhouse growing medium and fertigation trial 62 3.3. Results 3.3.1. Field growing medium and fertigation trial 63 3.3.2. Greenhouse growing medium and fertigation trial 69 3.4. Discussion 72 3.5. References 82 Chapter 4: Developmental effects on glandular trichomes and leaf chemistry 4.1. Introduction 85 4.2. Materials and Methods 87 4.3. Results 4.3.1. Parthenolide variability during leaf development and flowering 89 4.3.2. Feverfew glandular trichome development, density, and concentration 94 4.4. Discussion 102 4.5. References 108 Chapter 5: Biosynthetic studies using 14C and 13C incorporation into Parthenolide 5.1. Introduction 110 5.2. Materials and Methods 113 5.2.1. 14C feeding experiments 114 5.2.2. 13C feeding experiments 116 5.2.3. Extraction methods, parthenolide isolation, and NMR analysis 117 5.3. Results 5.3.1. 14C labeling of parthenolide 119 5.3.2. 13C enriched parthenolide 120 5.4. Discussion 126 5.5. Conclusion 136 5.6. References 137 vi Chapter 6: General Discussion 6.1. Overview 139 6.2. Future Research Directions 143 6.3. References .' 146 Appendix 148 vii LIST OF TABLES Table 2.1. Parthenolide concentration of feverfew leaves 43 days and 87 days after transplanting. 38 Table 2.2. Average whole plant and organ dry weights of field grown feverfew. 39 Table 2.3. Leaf water status in field grown feverfew measured at 3 am (night), 12 pm (mid-day), and 6 pm (evening). 40 Table 2.4. Dry leaf parthenolide content measured over a 3 month period in the field. 41 Table 2.5. Leaf and flower parthenolide concentrations and total parthenolide content per plant of stems, leaves and flowers. 42 Table 2.6. Leaf parthenolide concentration in feverfew leaves grown in the greenhouse under irrigation and nitrogen treatments. 43 Table 3.1. Field experiment treatments and abbreviations. 61 Table 3.2. Treatments in the greenhouse trial and abbreviations. 63 Table 3.3. Dry weight and dry to fresh weight ratios of feverfew plants grown in the field and greenhouse. 64 Table 3.4. Leaf water potential and osmotic potential of greenhouse and field grown plants measured at 3 p.m. (light) and 4 a.m. (dark). 66 Table 3.5. Average leaf parthenolide concentration in greenhouse and field-grown plants. 67 Table 3.6. Parthenolide content at harvest in leaf, stem, and flower tissues, based on subsample analysis. 68 Table 4.1. Parthenolide concentration in leaves of different ages from vegetative and reproductive shoots. 92 Table 4.2. Trichome density and parthenolide concentration of leaves measured from the apex to the base ofthe stem in vegetative and reproductive plants. 100 Table 5.1. Incorporation of 14C labeled substrates into parthenolide. 119 Table 5.2. 13C-NMR assignments and enrichment of parthenolide after feeding 13C-enriched substrates. 124 viii LIST OF FIGURES Figure 1.1. Mevalonate and methylerythritol phosphate routes to isoprene Biosynthesis. 5 Figure 1.2. The diversity of terpenoid biosynthesis showing examples of the compounds derived from this pathway 8 Figure 1.3. Major skeletal types of sesquiterpene lactones showing the common pathway through the germacranolides. 9 Figure 1.4. Scanning electron micrograph of non-glandular and glandular trichomes on the abaxial leaf surface of Tanacetum parthenium. 12 Figure 4.1. A glandular trichome derived from an epidermal cell with a subcuticular extracellular space where secretory cells secrete non-polar compounds for storage. 86 Figure 4.2. Greenhouse-grown feverfew leaf parthenolide concentration during development from vegetative to reproductive growth. 89 Figure 4.3. Leaf parthenolide concentration of greenhouse-grown feverfew overtime. 91 Figure 4.4. HPLC chromatograms of leaf and flower extracts from feverfew shoots in the vegetative and reproductive stages. 93 Figure 4.5. HPLC chromatograms of a composite disk flower trichome extract and a receptacle trichome extract. 94 Figure 4.6. Scanning electron micrographs of feverfew leaf surface showing glandular and non-glandular trichomes. 96 Figure 4.7. Scanning electron micrographs of feverfew flower glandular trichomes on the floret petals of the inflorescence. 97 Figure 4.8. Increased visibility of trichomes on slide preparations of leaf epidermal peals after drying. 99 Figure 4.9. Trichomes before and after treatment with dichloromethane. 101 Figure 5.1. The methylerythritol phosphate pathway and the mevalonate pathway to terpenoid biosynthesis. 111 Figure 5.2. 1H-NMR spectra of parthenolide. 121 Figure 5.3. Carbon numbering of parthenolide and the predicted conformation of the three isoprene units. 121 Figure 5.4. 13C NMR of parthenolide isolated from feverfew shoots fed with 2-12C-mevalonolactone or 2-13C-mevalonolactone. 122 Figure 5.5. 13C enrichment patterns in parthenolide after feeding enriched substrates. 125 Figure 5.6. Glucose catabolism. 127 Figure 5.7. 1-13C-D-glucose feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.8. 2-13C-acetate feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.9. 2-13C-mevalonolactone feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.10. 2-13C-pyruvate feeding experiment. Observed and predicted patterns of 13C enrichment. ACKNOWLEDGEMENTS I extend my gratitude to Professor G. H. Neil Towers for his guidance and support throughout this thesis.