MONOTERPENE METABOLISM OF MENTHA AND ITS CELL CULTURES BY MICHAEL JOHN HUDSON A thesis submitted in part fulfillment for the degree of Doctor of Philosophy of the University of London, and for the Diploma of Membership of the Imperial College. Department of Pure and Applied Biology Imperial College of Science and Technology London, SW7 -2- ABSTRACT The production and modification of monoterpenes has been studied using cell cultures of Mentha piperita, and comparison drawn with # the intact plant whenever possible. In vegetative plants the accumulation of an essential oil; containing many related forms of monoterpenes; was shown to be associated with morphological differentiation; leaf and shoot tissues bearing distinct oil glands. More than 85% of the total plant monoterpenes were found to be located in the leaf epidermal glands. The parenchyma of vegetative plants was shown to contain the 5-carbon compounds; 2-methyl butan-l-ol and 2-methyl butan-l-al; possibly derived from progenitors of monoterpenes; together with trace amounts of alpha-terpinene, 1-menthol, menthyl acetate, and menthyl-glucoside. Light was not a prerequisite for monoterpene biosynthesis since these materials were also detected in etiolated shoots. Qualitative and quantitative changes in the oil occurring during leaf development have been documented, indicating substained active metabolism of these compounds. Callus and cell suspension cultures exhibited a similar "monoterpene profile" as parenchymatous cells of the intact plant. In an attempt to stimulate the accumulation of monoterpenes, the primary growth of cell cultures was restricted by gibberellic acid, chlorocholine chloride and colchicine or exposure to stress factors such as paraquat, ethephon and abscisic acid, all with negative results. Potential precursors of monoterpenes were actively metabolised by cell cultures, but did not lead to an increase in the yield or diversity of monoterpene end-products, even when applied in * conjunction with beta-ionone or retinol. Cultures were capable of interconverting certain exogenously applied monoterpenes, but were unable to accumulate the monoterpene products from this activity. « 4 -3- « Mentha cell cultures proved particularly recalitrant towards shoot regeneration; limiting an assessment of the genetic stability of cultures, and the direct control of differentiation as a means of increasing the accumulation of monoterpenes. The implications of this work have been discussed in relation to the possible commercial exploitation of plant tissue cultures for the production and modification of scarce or complex natural materials. % * ACKNOWLEDGEMENTS The work presented herein owes much to the encouragement and interest shown by my friends and colleagues. In particular I should like to thank Dr. A. Goldsworthy of Imperial College for his lively discussion and attentive supervision, and the Science and Engineering Research Council for supporting my work under the CASE scheme. Bush Boake Allen Ltd sponsored this research and Dr. W.D. Fordham introduced me to the world of flavours and fragrances. My thanks are extended to the Trustees of "The Spencer Industrial Arts Trust", in Coventry, whose support enabled me to make an academic visit to fellow workers in North America. W.D. Loomis (Oregon State University), R. Croteau (Washington State University) P.M. Townsley (University of British Colombia), B.M. Lawrence (R.J. Reynolds Ltd) and R. Carrington (Mint Industry Research Council) have all guided my thoughts at one time or another. Finally, my wife, Diane, who has shown patience and encouragement during my studies. I dedicate this thesis to her and our family. -5- CONTENTS Title ^ Ab s t rac t Acknowledgement s Contents List of tables List of figures List of plates Abbreviations 1. INTRODUCTION % 2. MONOTERPENE BIOSYNTHESIS - literature review 3. MATERIALS AND METHODS 3. 1 Plant material 3.2 Growth conditions • 3.3 Isolation of Terpenoid Compounds 3.3. 1 Steam distillation 3.3.2 Direct volatilisation gas liquid chromatography 3.3.3 Terpene - glycosides 3.4 Analytical Procedures • 3.4. 1 Analysis of reference materials 3.4.2 Gas-liquid chromatography - working method 3.4.2 Data storage system 3.5 Intact Plant Studies 3.3. 1 Development of essential oil during ontogenesis 3.5.2 Leaf surface topography - scanning electron microscopy 3.5.3 Localisation of essential oil 3.5.4 Oil gland ultrastructure and development - Light microscopy - Vital staining - Transmission election microscopy 3.5.5 Ultrastruetural localisation of beta-D-glucosidase -6- 3.6 Plant Tissue and Cell Culture Studies 3.6.1 Initiation and maintenance 3.6.2 Basal terpenoid metabolism 3.6.3 Environmental effects 3.6.4 Inhibition of primary growth 3.6.5 Chemical and physical stress 3.6.6 Substrate availability 3.6.7 Chemical regulation 3.6.8 Enzymatic potential 3.6.9 Cellular compartmentation 3.6.10 Morphological differentiation 4. RESULTS 4.1 Development of Plant Material 4.2 Isolation of Terpenoid Compounds 4.2.1 Steam distillation 4.2.2 Direct volatilisation gas liquid chromatography 4.2.3 Quantification of terpene - glycosides 4.3 Analysis of Terpenoid Compounds 4.3.1 Reference materials 4.3.2 Gas liquid chromatography - working method 4.4 Intact Plant Studies 4.4.1 Development of essential oil during ontogenesis 4.4.2 Leaf surface topography - scanning electron microscopy 4.4.3 Localisation of essential oil 4.4.4 Oil gland ultrastructure and development - Light microscopy - Vital staining - Transmission electron microscopy 4.4.5 Ultrastructural localisation of beta-D-glucosidase 4.5 Plant Tissue and Cell Culture Studies 4.3.1 Growth kinetics 4.3.2 Basal terpenoid metabolism 4.3.3 Environmental effects 4.3.4 Inhibition of primary growth 4.3.5 Chemical and physical stress 4.3.6 Substrate availability 4.3.7 Chemical regulation 4.3.8 Enzymatic potential 4.3.9 Cellular compartmentation 4.3.10 Morphological differentiation 5. DISCUSSION 6. SUMMARY 7. BIBLIOGRAPHY 8. APPENDICES * TABLES Reported Constituents of the Oil of Peppermint Major Genetic Studies in Mentha Characterisation and identification of essential oil components in Willamette and Yakima Peppermint Oils. Components isolated from M piperita essential oil by GLC working method. Development of leaf monoterpenes in M piperita Main terpenoids of isolated glands and leaf tissue identified by direct-volatilisation GLC. Main terpenoids of stem epidermis and internal tissues identified by direct-volatilisation GLC. The effect of PVA treatment on recovery of essential oil from leaf tissues of M piperita. Differential staining of M piperita tissues by toluidine blue. Basal terpenoid metabolism of M piperita suspension cultures during the growth cycle. The effect of plant cell culture conditions on the essential oil yield of regenerated M piperita plantlets. -8- 12. The effect of light intensity upon the essential oil yield of M piperita plantlets regenerated under plant cell culture conditions. 13. Monoterpene composition of essential oils from light and dark grown M piperita plantlets. 14. Biotransformation of monoterpenes by M piperita cell cultures. -9- FIGURES 1 Products of terpenoid metabolism in higher plants. 2 The biosynthesis of monoterpenes. • 3 Monoterpene interconversions in the Genus Mentha. 4 Likens - Nickerson micro distillation unit. 5 Direct-volatilisation GLC injection unit. 6 Synthesis of 1-menthyl glucopyranosides. 7 The data storage system input channel. 8 The data storage system output channel. 9 Histochemical localisation of beta-glucosidase. • 10 The effect of distillation period upon recovery of monoterpenes. 11 The effect of direct-volatilisation period upon recovery of monoterpenes. 12 Essential oil profile of midstem leaves, by direct-volatilisation GLC. 13 The effect of acid pretreatment on release and recovery of bound menthol from midstem leaves of M piperita. 14 Essential oil profile of M piperita (Yakima) by high resolution GLC. • 15 Essential oil profile of M piperita (Yakima) by working method. 16 Changes in the total monoterpene content of M piperita leaves during their development. 17 Concentration of monoterpenes during leaf • development. 18 Variation in major monoterpenes during leaf development. • Variation in "Free" and "Bound" menthol during leaf-development. Comparison of volatiles recovered from M piperita stem epidermis and internal tissues by direct-volatilisation GLC. Schematic representation of M piperita glandular triehome. The effect of 2,4-D and BAP upon initiation and dry weight gain of M piperita callus. Growth cycle (cell density mg ml“^ dry weight) of M piperita suspension cultures. Growth cycle (conductivity mMhos) of M piperita suspension cultures. The effect of PVP upon dry weight gain of M piperita suspension cultures. The effect of CCC, GA3, colchicine and regulator removal on the growth of M piperita suspension cultures. The metabolism of beta-methyl crotonic acid and dimethylallyl alcohol by M piperita suspension cultures. The metabolism of beta-ionone by M piperita suspension cultures. The biotransformation of geraniol by M piperita cell cultures. The biotransformation of pulegone by M piperita cell cultures. The biotransformation of menthone by M piperita cell cultures. The biotransformation of menthol by M piperita cell cultures. Products of geraniol biotransformation. - 11- PLATES • 1 Data storage system - recording 2 Data storage system - computation 3 Lower epidermis of M piperita illustrating variety of epidermal appendages (SEM). 4 Upper epidermis of M piperita leaf showing