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.
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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.
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* 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 distribution • of oil glands (SEM).
5 Upper epidermis of equivalent leaf after PVA treatment (SEM).
6 Lower epidermis of M piperita leaf showing distribution of oil glands (SEM). • 7 Lower epidermis of equivalent leaf after PVA treatment (SEM).
8 Section of young leaf of M piperita illustrating glandular trichome and surrounding tissues (LM).
9 Longitudinal section of young glandular hair viewed by • transmission electron microscopy.
10 Detail of secretory cell and cell wall organisation in glandular hair of M piperita.
11 Detail of secretory cell, cell wall organisation and formation of extraplasmatic cavity in glandular trichome of M piperita. • 12 The effect of 2,4-D and BAP upon callus growth from M piperita shoot explants.
13 Groups of lignified cells from M piperita suspension cultures.
• 14 Open field cultivation of Peppermint in the USA, still a highly efficient process. - 12-
ABBREVIATIONS
2,4-D - 2 ,4 ,-Dichlorophenoxyacetic acid 2-CEPA - 2-chloroethyl phosphonic acid 5-MVAP - 5-phosphomevalonate 5-MVAPP - 5-pyrophosphomevalonate ABA - Abscisic acid ATP - Adenosine triphosphate BAP - Benzylamino purine beta-MCA - Beta-methyl crotonic acid CCC - chlorocholine chloride DMAPP - Dimethylallyl pyrophosphate DMSO - Dimethylsulfoxide FID - Flame ionisation detector FSD - Full scale deflection
g a 3 - Gibberellic acid GLC - Gas liquid chromatography GPP - Geranyl pyrophosphate IAA - Indol-3yl-acetic acid IOFI - International Organisation of the Flavour Industry IPP - Isopentenyl pyrophosphate MV A - Mevalonic acid NPP - Neryl pyrophosphate PAR - Photosynthetically active radiation PVP - Polyvinylpyrrolidine (RMM 40K) Paraquat - 1,l-dimethyl-4,4-bipyridinium dichloride RMM - Relative molecular mass Retinol - Vitamin A, SEM - Scanning electron microscopy TBA - Tertiary butyl alcohol TEM - Transmission electron microscopy TIBA - Triiodobenzoic acid WCOT - Wall coated open tubular (GLC column)
*FAP - Furfurylaminopurine (Kinetin) 1. INTRODUCTION INTRODUCTION
The work presented in this thesis investigates the potential application of plant cell and tissue cultures for the production and biotransformation of monoterpenes. Factors affecting such processes have been studied, and in the light of results obtained, hypotheses regarding future developments in the field made.
The research programme was initiated following a period spent by the author at the laboratories of Bush Boake Allen Ltd, and was carried out with their further support.
As the world population increases, new sources of foodstuff will have to be secured. Frequently these will be less palatable than traditional fare, and the addition of flavouring materials will become increasingly important. The natural flavours of herbs and spices have been used for many centuries, and more recently these have been supplemented with chemically synthesised materials. However, neither is completely satisfactory alone in factory processed foodstuffs.
Natural flavourings are usually a complex mixture of several hundred compounds, the correct proportions of which are critical to ensure a "true to life" and recognisable flavour. Changes in growing conditions affect this balance, and lead inevitably to products of variable quality. Whilst "flavours" reconstituted from chemically synthesised components give a more standard product (Polak 1970), only a small proportion of natural flavour constituents are amenable to cheap chemical synthesis, and these simple blends lack the richness and authenticity imparted by natural flavours. As a result, synthetic compounds are frequently blended with natural materials as a means of reinforcing and extending the authentic flavouring. - 15-
Recently there has been an interest in identifying new sources of supply for both existing and novel natural products. The in vitro culture of cells derived from aromatic plants, to yield a natural product, of predictable quality appears to be a promising area of research.
A summary of flavour producing plants (Hodge 1975, Nicholas 1973, 1973a) and an examination of the natural products of commerce (Erickson, 1976) shows that ESSENTIAL OILS are an important group of materials. Essential oils are complex mixtures of organic compounds which can be isolated from certain higher plants by steam distillation. Lower terpenoids; the mono and sesquiterpenes; are quantitatively the most important components and are largely responsible for the characteristic odour of many plants and flowers (Lawler, 1977).
Monoterpenes are also occasionally found in animals; such as insects (Karlson 1970), alligator (Fester e£ al 1937), and beaver (Walbaum et^ aJL 1927), but in these cases usually represent an accumulation from dietary sources (Renwick et^ al^ 1976). Infrequent reports of the presence of monoterpenes in lower plants (Katayama 1955) and fungi (Collins 1979) have appeared in the scientific literature and a number of halogenated monoterpenes are known to occur in marine algae. (Burreson et^ al^ 1975).
However, monoterpenes are most widely distributed amongst the higher plants, with members of the Labiatae, Rutaceae, Gramineae, Myrtaceae, Umbelliferae, Myristiceae and Pinaceae accumulating significant amounts. Data reviewed by Nicholas (1973a) showed that higher plants tend to predominantly produce the monocyclic para-menthane type of monoterpene. Therefore, it was decided that; using a suitable species; a better understanding of the biosynthesis and metabolism of this fundamental group would be usefully obtained. Several important essential oil crops eg; peppermint, spearmint, basil, lavender, sage, thyme and patchouli are members of the Labiatae. Of these, "mint" oils are most widely used, and contain a high percentage of para-menthane type monoterpenes. The essential oil metabolism of this genus had previously been studied to improve crop husbandry (Steward 1962) for plant breeding purposes (various papers by Murray et al), and most recently, for the isolation of enzymes involved in terpenoid metabolism. (Croteau et^ al 1973, Croteau & Hooper 1978, Martinkus & Croteau 1981). For these reasons Mentha was selected as a model system in which to study monoterpene metabolism.
In this study a number of investigations were carried out using intact plants in an attempt to identify the sites of synthesis and accumulation of essential oils. Microdissection, direct volatilization gas chromatography and the preparation of leaf surface films have been used to determine the oil concentration of various tissues, whilst oil gland structure and distribution has been detailed using light and electron microscopy.
The monoterpene profiles of leaves of various ages were documented for plants grown under constant environmental conditions, and the results taken as indicative of those changes normally occuring during leaf growth and development. Periods of monoterpene biosynthesis and metabolic transformation were identified and related to changes in leaf growth and development. A major obstacle to the study of terpenoid metabolism in vivo has been the apparent inability of plants to incorporate suspected precursors into the final product. This low incorporation of exogenous precursors into the monoterpenes of higher plants is thought to be due in part, to a failure in transporting such material in an appropriate form to the site of biosynthesis. This may be due to problems of permeability, compartmentation of metabolic pools or restricted translocation per se. Since callus and suspension cultures possess a simpler organisation, some of the problems inherent with intact plants can often be overcome (Laetsch 1971, Dougall 1980). Particularly in the case of suspension cultures, precursors and substrates can be applied aseptically to the growth medium, with all cells theoretically receiving equal exposure to the reagent. Moreover, cell cultures can be grown reproducibly under controlled conditions, have relatively short growth cycles and are virtually independent of climatic variation. The rigorous sterility concomitant with tissue culture techniques has the further advantage of ensuring that the observed metabolic processes are due solely to the plant material concerned, and not to the activities of associated micro-organisms. In addition, many plant cell cultures are reported to contain only low levels of phenols and quinones (Overton & Picken, 1977) - compounds which when liberated from intact plant material, can inactivate enzyme systems, including those of secondary metabolism (Loomis, 1974, et_ al^ 1979). Being self sufficient in cofactors and low in phenolics, cell cultures may therefore represent a possible compromise between the "physiologically complex" whole plant, and the technically challenging multistage enzyme systems required for natural product biosynthesis.
The production of secondary metabolites by plant cell cultures has recently been reviewed by several authors. (Alfermann & Reinhard 1978, Butcher 1977, Carew & Staba 1965, Rhodes & Kirsop
1982, Yeoman et_ al 1980, Tabata 1977, Bohm 1977, Lee & Scott 1979, Staba 1980). - 18-
Callus and suspension cultures of the commercial peppermint cultivar; Mentha piperita var "Black Mitcham" were established on a variety of media, and their growth rates determined. Murashige & Skoog medium (1962) was selected for routine use, and the suitability of the culture vessel microenvironment for sustaining monoterpene biosynthesis determined by the examination of cultured plantlets regenerated from sterile nodal explants. The requirement for light and effect of its intensity on monoterpene metabolism was also studied using this system.
The influence of "stress" on monoterpene metabolism was examined since many such phenomena are known to induce the production of secondary metabolites in biological systems. Since there is some evidence to suggest that terpenoid metabolism may be affected in intact plants (Brown & Nix 1975, Audley 1979), the controlled application of "stress" was considered as a possible means of initiating essential oil synthesis in cell cultures. Paraquat, abscisic acid (ABA) and ethephon (2-CEPA) were selected for use because of their previously recorded activites upon the terpenoid metabolism of intact plants. Cell cultures were also exposed to temperatures of 35°C for various periods in order to impose a physical rather than a chemical "stress".
In order to investigate the control of monoterpene metabolism in cell cultures further, potential "dedicated" precursors such as mevalonate (MVA), dimethylallyl pyrophosphate (DMAPP) and beta-methyl crotonate (beta-MCA) were incubated with cells taken from log phase suspension cultures. It was considered that the application of such compounds in a suitable form might increase the yield of monoterpenes. This approach was extended by supplying beta-Ionone and retinol; since they are thought to activate an early rate-limiting enzyme of terpenoid metabolism (Reyes et al 1964); thereby facilitating better substrate utilisation. - 19-
Since the qualitative composition of essential oils is a crucial determinant of their commercial acceptability, it was considered important to assess the ability of cultured cells to biotransform exogenously supplied monoterpenes into the full diversity of related forms found in natural oils. The results from such studies would indicate whether the system could potentially produce a complete essential oil profile; identical to that of the intact plants; from a single inexpensive feedstock. Additionally, if the factor limiting monoterpene biosynthesis in cell cultures operates at the precursor or early intermediate stage, it may be commercially viable to supply a plant cell system with a cheap terpene feedstock in order to prepare a mixture of the more complex forms. Cells were incubated for varying periods with a number of pure monoterpenes known to occur in natural peppermint oil (Lawrence et al 1972), and several factors affecting the progress of biotransformation identified.
For any new process to find commercial application, product yield and quality must be consistent. This requirement may limit the opportunities of many cell cultures, in which genetic and chromosomal abberations sometimes occur with high frequency. In order to establish the genetic stability of M piperita cell cultures, aliquots of cell suspension were plated out for proliferation on agar medium, and cell clumps subsequently transferred for plantlet regeneration.
To be able to induce the early stages of differentiation in cell cultures may also provide a means of increasing the yield of essential oil by initiating some degree of cellular specialisation, and facilitating localised product accumulation. Preliminary studies have been carried out in which a number of materials known tc induce organogenesis in other plant cell cultures were assessed for their effectiveness with M piperita. The implications of being able to control cellular differentiation in M piperita are wide ranging with respect to the control of secondary metabolism; but additional commercial opportunities can also be seen in the accelerated mutational breeding of new cultivars with novel essential oil profiles and the micropropagation of "high vigour" stock material of M piperita; which is itself a sterile hybrid. 2 MONOTERPENE BIOSYNTHESIS LITERATURE REVIEW MONOTERPENE BIOSYNTHESIS LITERATURE REVIEW
Introduction
Isoprenoids - a class of compounds all based on an integral number of five carbon units - are now known to be widely distributed throughout the plant and animal Kingdom (Weissman 1966). All green plants have the ability to produce at least the linear isoprenoids, as is manifest by the presence of the phytyl side chain of chlorophyll. However, whilst the higher terpenoids; composed of more than 25 carbon atoms; appear universally distributed amongst plants (Swain, 1974); lower terpenes (those between C5 and C25) are mainly restricted to the Tracheophyta although some monoterpenes (CIO) and sesquiterpenenes (C15) have been found amongst the Bryophyta, algae and the fungi. The variety of plant products arising through terpenoid metabolism are summarised in figure 1 .
Advances in separation, structure elucidation and radioisotope techniques have resulted in a rapid increase in the biosynthetic information available this century. Most of the work on the biosynthesis of terpenoids has in fact been derived from studies on the metabolism of higher terpenoids in mammalian tissues and microrganisms (see recent review by Porter & Spurgeon 1981). However, a number of reviews concentrating on monoterpene biosynthesis have been published (Loomis 1967, Francis 1971, Loomis & Croteau 1973, Banthorpe et al 1972a, Banthorpe & Charlwood 1977, 1979, Charlwood & Banthorpe 1978, Loomis & Croteau 1980, Croteau 1980, 1981a). - 23-
Figure 1 - PRODUCTS OF TERPENOID METABOLISM IN HIGHER PLANTS
Mevalonic Acid
I 5-MVAP
i 5-MVAPP Leucine
I Zeatin <— IPP DMAPP Hemiterpenes (C5H8 ) (eg Isoprene)
Geranyl Monoterpenes (C^qH ^ ) Pyrophosphate (eg Essential Oils) (CiqH6)
Sesquiterpenes (C15H24) Farnesyl (eg Essential oils, Pyrophosphate Abcisic Acid)
Diterpenes (C20H32) 'V (eg Vitamin A, Phytol, -11 Geranylgeranyl Gibberellins) Pyrophosphate (c20h32) Tetraterpenes (C40H64) (eg Carotenoids)
Polyterpenes ((C5Hg)n) (eg natural rubbers) - 24-
Composition of M Piperita essential oil
Lawrence et a_I (1972, 1980) have compiled extensive analytical data on the essential oil's of Mentha spp. The following constituents have been reported in natural peppermint oils. (Table 1).
Table 1 - Reported constituents of the oil of peppermint
HYDROCARBONS alpha-thujene humulene alpha-pinene gama-cadinene caraphene calamenene fenchene arooadendrene beta-pinene beta-elemene sabinene alpha-ylangene delta-3-carene delta-bulgarene alpha-phellandrerie delta-cardinene alpha-terpinene alpha-muurolene 1imonene gatsma-muurolene beta-phellandrene eps ilon-muurolene cis-ocimene alpha-maaliene trans-oc imene alpha-bourbonene gamma-terpinene alpha-cubebene para-cyroene bicycloelemene terpinolene alpha-calacorene caryophy1lene guaiene bisbolene delta-guaiazulene delta-cadinene
ALCOHOLS pentanol i6opulegol 3-methyl butanol neoiso pulegol 3-octanol linalool 3-hexenol geraniol menthol menthan-1,3-diol neomenthol menthan-2,3-diol isomenthol trans-p-menth-2-er>-l ,A-diol neoisomenthol beta-betulenol cis-sabinene hydrate caryophyllene alcohol trans-sabinene hydrate clovandiol
ESTERS KETONES octyl acetate 3-octanone 3-octyl formate menthone 3-octyl acetate isomenthone menthyl acetate piperitone menthyl valerate piperitenone pulegone jasmone ALDEHYDES acetaldehyde OXIDES 2-methylpropanal menthofuran 3-me thylbutanal 1,8-cineol trans-2-hexenal piperitone oxide 3-hexenal phenylacetaldehyde PHENOLS thymol ACIDS carvacrol acet ic 3-roethyl butyric oc t enoi c SULPHIDES dimethyl sulphide - 25-
Monoterpene Biosynthesis
The biosynthesis of monoterpenes is thought to proceed via the conversion of mevalonic acid (MVA)-into isopentenyl pyrophosphate (IPP) and 3 ,3-dimethylallyl pyrophosphate (DMAPP), followed by the condensation of IPP and DMAPP to form geranyl pyrophosphate (GPP).
The early stages of the biosynthetic route as presently understood, are summarised in figure 2 .
Figure 2, The biosynthesis of monoterpenes
0 0 0 0 x + I CoA^^ CoA^^ CoA Acetyl Co A Acetyl Co A Acetoacetyl Co A
Acetyl Co A
Mevalonic acid Mevalonate-5-phosphate (MVA) 1
00H
Mevalonate-5-pyrophosphate
Isopentenyl pyrophosphate Dimethylally pyrophosphate (IPP) I ( DMAPP) - 26-
In early experiments, radio-labelled substrates such as (2-^C) MVA and (^C)-acetate were fed exogenously to green plants, and in 1958 the biosynthesis of a monoterpene; alpha pinene; from (2- C) MVA was first demonstrated in Pinus attenuata (Stanley, 1958). However, for many years the hypothesis that MVA was a key precursor in monoterpene biosynthesis remained poorly substantiated, as all of the work published indicated very low incorporation. Only 0. 01.- 0.5% of (2-^C) MVA or labelled acetate were found in newly biosynthesised monoterpenes (Loomis: 1967, Francis 1971, Banthorpe et al 1972a), with the balance of the tracer 14 often assumed lost as CO^.
A number of possible explanations for this low incorporation have been proposed by various authors, (reviewed by Banthorpe et al, 1972a) including:-
1. Poor translocation of precursors from the site of administration to the site of terpene biosynthesis.
2. Lack of penetration to intracellular sites of monoterpene biosynthesis by precursors such as MVA;
3. MVA rarely occurs in its free form in nature, therefore; as such; it may not be able to intervene in the biosynthetic pathway for monoterpenoids;
4. Assuming that exogenous MVA is able to penetrate the terpenoid pathway, much of it may be shunted into the biosynthesis of other compounds such as steroids or carotenoids which appear more physiologically important than monoterpenes.
5. MVA probably occurs in vivo at very low concentrations. The introduction of large quantities may therefore lead to its degradation, including the formation of products which may inhibit the enzymes of terpenoid biosynthesis. - 27-
Undoubtedly, one of the main reasons for the acceptance of the mevalonate hypothesis was the lack of evidence to support any likely alternative. However, in 1969, Francis and O'Connell found that whilst less than 1% of exogenously applied (2-^C) MV A was incorporated into free monoterpenes; approximately 11% was incorporated into the non volatile beta-D-glucosides of geraniol, nerol and citronellol in the petals of Rosa dilecta. They found that both free and bound monoterpene alcohols accumulated when the rose petals started to unfurl. Two years later, Banthorpe and Mann (1971) showed that just after Tanacetum vulgare flowers had opened, the petals contained 0.06% monoterpenes, of which 50% were bound as the beta-glucosides of isothujol, neoisothujol, alpha-terpineol and terpinen-4-ol. They also showed that cell-free extracts from the flower heads were able to convert 14 (4- C)-IPP into geranyl and neryl beta-D-glucosides. Monoterpene glucosides have now been found in a large number of plant species; including Camellia sinensis (Takeo 1981, 1981a), Dicoria canescens (Miyakado 1974), Mentha arvensis var piperascens (Sakata & Mitsui 1975, Sakata & Koshimizu 1978), Mentha piperita (Croteau & Martinkus 1979), Rosa dilecta (Francis & Allcock 1969, 1969a) Thymus vulgaris (Skopp & Horster 1976) Betula alba and Chaenomeles japonica (Tschesche e£ aT 1977). Because of their water solubility they are now believed to play an important role in the physiological process of terpene translocation and accumulation.
Further studies by Banthorpe et^ al (1972d) showed that when cell-free extracts of Tanacetum vulgare, Artemisia annua and Santolina chamaecyparissus were fed labelled precursors, approximately 40% of the tracer was incorporated into water-soluble materials. The enzymes responsible were designated "salvage enzymes" and it was postulated that their function was to regulate the unphysiological levels of exogenously supplied monoterpene precursors by converting them into water soluble products for transport and subsequent degradation. - 28-
Of the tracer in the water soluble fraction, 60% was incorporated into 3-methyl-3,4-oxobutan-l-ol, 9% in 3-methylbutan-l,3,4-triol and the remainder (8%) was spread between a number of products, many of which were based on 3-methyl but-2-en-l-ol. It was concluded that degradation by "salvage enzymes" and the formation of monoterpene glucosides could both contribute to the low incorporation of exogenous substrates often observed in monoterpene biosynthesis.
In 1966, Valenzuela et al investigated the incorporation of 14 CO2 into alpha and beta-pinene in Pinus radiata and found that the needles were important sites of monoterpene 14 formation. Interestingly, CO^ at physiological concentrations was found to be a much more efficient precursor for monoterpenes than (^C)-MVA or (^C)-acetate (Refendehl et al 1967, Loomis 1967, Burbott & Loomis 1969, Croteau & Loomis 1972, Croteau et al, 1972a). 14 In feeding experiments CO^ uptake was extremely rapid, with label incorporated into monoterpenes within two minutes.
Prompted by these discoveries; Banthorpe and Charlwood (1972d), examined whether catabolic pathways were responsible for degrading the exogenous precursors previously studied (e.g. MVA and acetate) into labelled CC^J which might then be available for reincorporation into newly biosynthesised terpenoid compounds. Using (1-^C)- and (2-^C)-MVA; they showed that MVA was actively decarboxylated by plant tissues, but that the C09 arising from the cleavage of 1 . z C did not contribute to the label observed in monoterpenes. They concluded that MVA was therefore incorporated directly into monoterpenes without prior conversion to CC^* Subsequently Machado £t al (1974) showed that linalool and terpinen-4-ol were also biosynthesised in vivo directly from (2-^C)-MVA by Pinus pinaster seedlings. - 29-
However, in 1975 Croteau & Loomis drew attention to the following observations in support of the hypothesis that monoterpenes are naturally catabolised in plants.
Diurnal Fluctuation - a decrease in oil content during the night coupled with increase during the day (Weiss & Fluck 1970).
Ontogentic Fluctuation - at the onset of flowering there is a rapid decrease in the total amount of monoterpenes (Burbott & Loomis 1969).
Incorporation Fluctuation - there is evidence that radio-tracer passes through monoterpene pools without changing the overall pool size (Banthorpe & Wirz-Justice 1969, Croteau e£ al 1972b).
It is now generally agreed that monoterpenes are not inert waste products as was once supposed, though their roles still remain little understood. However, a greater appreciation of their wide-ranging involvement in metabolism, particulary as agents of chemical defence and communication has developed in recent years (Swain 1974).
During the early 1970’s the search for more efficient monoterpene precursors continued. In 1974, Suga e_t al reported that labelled L-leucine was incorporated into the acyclic monoterpene; linalool; in Cinnamomum camphora. This was the first time that an amino acid had been cited as being involved in monoterpene biosynthesis. The following year they reported that both leucine and valine participated in the biosynthesis of DMAPP, which subsequently led to their incorporation into monoterpenes. Over the last ten years, the symmetry of the labelling pattern in the "f irst-formed*' monoterpene; the condensation product of IPP and DMAPP; has also been the subject of much discussion and conjecture (Banthorpe et^ al^ 1970, 1972a, 1972b, Wuu & Baisted 1973, Suga et al 1974). In 1976, Banthorpe & Ekundayo found that only the IPP - derived moiety carried label into the delta -3-carenes when (2^C) - MVA was fed to Pinus palustris and P sylvestris. They similarly showed that geraniol and nerol biosynthesised from (4-^C)-IPP and (2-^C)-MVA by a cell-free extract of Tanacetum vulgare leaves had most of their label in the fraction derived from IPP. They suggested that the non-equivalent labelling pattern could be due to the preferential incorporation of amino acids; such as valine and leucine; into biosynthetically active DMAPP, or alternatively, the existence of complex compartmentation and the segregation of intracellular DMAPP and IPP pools. Allen et al conducted a series of experiments in 1976 to elucidate the origin of DMAPP utilised in monoterpene biosynthesis. Using Tanacetum vulgare, Pelargonium graveolens and Mentha pulegium, various factors causing asymmetric labelling patterns were examined. From their results, two metabolic pools were proposed at the substrate level; one in which DMAPP and IPP were considered to be in the free form (A), and another (B), in which the transient components were bound to 14 protein. They postulated that when CO^ or labelled MVA was fed to a plant at physiological concentrations, the labelled precursor was channelled into pool A and that the t IPP so formed condensed with existing protein bound DMAPP from pool B to yield asymmetrically labelled geranyl pyrophosphate. When terpene biosynthesis was rapid, or longer periods of metabolism were examined, then it was proposed that both pools would assimilate tracer leading to the formation of symmetrically labelled products. - 31-
Whilst it appeared usual for the IPP-derived portion of monoterpenes to be predominantly labelled when physiological 14 concentrations of ( C)-labelled acetate, MVA or CO^ were used in feeding experiments; Banthorpe et al (1975) reported widely varied labelling WITHIN the IPP-derived moiety of pulegone and geraniol when (^C)-acetate and were fed to Mentha pulegium and Pelargonium graveolens respectively. They suggested that separate metabolic pools of acetyl-CoA and/or acetoacetyl-CoA might also exist, and contribute to mevalonate biosynthesis in varying degrees depending upon season, environmental conditions and the prevailing physiological status of the plant. Such problems; inherent in the application of exogenous precursors to a physiologically and structurally complex organism; have limited further discoveries in this field. However, with improved techniques for extraction and stabilisation; cell-free enzyme systems have made a major contribution to our understanding of terpenoid metabolism during recent years.
Cell-Free Systems
It was well known from mammalian and microbial studies; that the formation of geranyl pyrophosphate (GPP) followed a sequential process, each step being enzymatically controlled. However, the isolation of active enzymes from plant material had always been more difficult (Loomis & Battaile, 1966). During the initial maceration of plant material; the release of proteases, non-specific interaction between proteins, their absorption onto cell walls and the liberation of vacuolar phenolics with acidic material can cause denaturation and inactivation of enzymes. Likewise the liberation of phosphatase and pyrophosphatase in cell-free extracts can cause dephosphorylation of intermediates in terpene biosynthesis and lead to the formation of artifacts (Croteau & Karp, 1979a,b). One of the earliest enzyme studies was carried out by Valenzuela et al (1966), who demonstrated the formation of 5-phosphomevalonate (MVAP) from (2-^C)-MVA in Pinus radiata, and proposed that mevalonate kinase may be a regulatory enzyme in the biosynthesis of monoterpenes from MV A. At the same time, Pollard et al (1966) used cell-free extracts of Pisum sativum to characterise the early stages from MVA to geranyl pyrophosphate (GPP) for the first time. However, it was Potty & Brueramer (1970) who demonstrated that the phosphorylation of MVA was dependent upon both adenosine triphosphate (ATP) and kinase concentration. Their results showed that the enzymes required for MVA activation were present in the juice vesicles of oranges, and that these same vesicles also possessed a full compliment of enzymes for the conversion of MVA; via IPP and DMAPP; to linaloyl pyrophosphate (LPP). LPP was hypothesised as an important monoterpene precursor in citrus.
During the early 1970's there was a detailed search and prolonged debate regarding the identity of a "fundamental" monoterpene entity, from which all related forms might be derived. Geraniol, nerol and linalool or their pyrophosphate derivatives were all considered as likely candidates.
3 In 1970, Francis ej: al^; using ( H)-mevalonate in feeding studies with rose petals; found that nerol was actually biosynthesised via geraniol or one of its phosphorylated derivatives. Later, Shine & Loomis (1974) showed that cell-free extracts from both carrot and peppermint, enzymically catalyzed the trans-cis isomerization of GPP and geraniol to NPP and nerol respectively. By 1974, Burbott et al had shown that although geraniol could be phosphorylated by such extracts, nerol was a much more efficient substrate. This was in agreement with the results obtained by Dunphy & Allcock (1972) and Dunphy (1973) who showed that in Rosa dilecta, geraniol was first converted to its phosphorylated aldehyde and then reduced to the isomeric alcohol, nerol. They postulated that this isomerization reaction might be responsible for regulating the relative biosynthesis of cyclic monoterpenes and higher terpenes; with geranyl pyrophosphate as the common precursor.
During their studies on acyclic monoterpene alcohols, Potty & Bruemmer (1970) established the presence of an enzyme in oranges that was capable of mediating the reversible oxidation of geraniol to geranial depending upon available co-factors and pH. This same enzyme was also capable of oxidizing nerol, citronellol and farnesol to their corresponding aldehydes. Dunphy & Allcock (1972) and Dunphy (1973) demonstrated that a similar enzyme preparation; obtained from rose petals; was able to reduce either geraniol or nerol to citronellol, and that the reductase was specific for primary terpene alcohols which possessed a cis- or trans-allylic double bond.
In 1967, Loomis postulated that the cyclization of neryl pyrophosphate (NPP) to alpha-terpineol was probably a key step in the biosynthesis of cyclic monoterpenes; and later, Cori (1969) reported that alpha-pinene was formed from (2- C)-NPP with cell-free extracts of P. radiata. He further suggested that because of its configuration and results of acid hydrolysis, NPP was the most likely precursor of cyclic monoterpenes, and may occur as an intermediate between GPP and the latter. However, in 1973, it was Croteau et al who demonstrated that a cell-free extract of M piperita would catalyze the direct cyclization of NPP to alpha-terpineo1. Battaile e£ al (1968) found that after the removal of phenolics, endogenous substrates, and air, a cell-free extract of M piperita would metabolise pulegone. Using such an extract with NADPH2 and ATP; (r)-pulegone was enzymatically reduced to 1-menthone, (l)-menthol and (r)-isomenthone. Later, this same group (Burbott e£ al 1974) demonstrated that a cell-free extract of M piperita was able to reduce the isopropylidene bond in piperitenone to yield piperitone, and that this enzyme exhibited specificity, since no reduction of the 8,9-double bond in carvone, was observed*
Croteau & Karp (1976) used a purified cell-free extract of Salvia officinalis to investigate whether 1,8-cineole, alpha-terpineol and limonene were formed independently from an acyclic precursor; such as NPP; or by the modification of a single monocyclic intermediate such as alpha-terpineol. In 1977 they demonstrated that under anaerobic conditions a partially purified, soluble enzyme extract was capable of converting NPP into 1,8-cineole; and that alpha-terpineol was not an intermediate in the reaction. This was the first experimental evidence of the enzymatic formation of a cyclic monoterpene directly from NPP. At the same time they demonstrated that a soluble enzyme preparation obtained from S officinalis cyclised NPP to bornyl pyrophosphate, which was then hydrolysed to borneol. In the presence of NAD, the latter was dehydrogenated to camphor.
Banthorpe et^ jil (1972a) have proposed that within monoterpene biosynthesis there are highly specific enzymes, whose activities are under rigid genetic control. - 35-
These enzymes; which they suggested might additionally be restricted to certain plant tissues; would be responsible for the formation of the main terpenoid skeleta and the introduction of key functional groups. It was proposed that other modifications to terpene structures might arise as a result of relatively non-specific enzymes or possibly even non-enzymatic processes. For example, Burbott et al (1974) reported that monoterpene hydrocarbons were always found during their enzyme studies of M piperita, M spicata and Daucus carota. However, in the case of M piperita and D carota, terpinolene was always the major hydrocarbon, whereas with M spicata extracts, limonene was the usual result. These authors suggested that the variation in monoterpene hydrocarbons was fundamentally related to the source of the enzyme extract. They postulated that limonene was the specific precursor of carvone in M spicata and terpinolene the specific precursor for piperitenone in M piperita. The genetic control of monoterpene biosynthesis has been investigated in great detail by other authors during recent years and has provided a valuable complement to other studies.
Genetic Studies
In 1958, Reitsema proposed a biogenetic arrangement of mint species based upon an emerging understanding of the biochemical characteristics of various cultures.
In 1954, Murray and Reitsema examined the genetic basis for menthone and carvone production in Mentha crispa, and designated the dominant gene "C" as being responsible for the production of carvone from a nominal precursor. The control of menthone formation took longer to unravel, but in 1971 Murray concluded from a cross between M arvensis var. piperascens x M gattefossei, that more than one gene was involved in controlling menthone production. It was found that there were a number of modifying genes which could influence the amount of a number of precursors, which in turn would directly influence the amount of end product formed. Two years later, Hefendehl & Murray obtained results which seemed to indicate that limonene was the precursor for both 2- and 3- substituted p-menthane compounds in Mentha spp.
Other genetic studies on Mentha have also been described in the literature (Lawrence, 1977). For example, in 1972 Hendriks & van Os examined a number of laboratory crosses between M suaveolens and M longifolia and found that the formation of dihyrocarvone was dominant over the formation of piperitone oxide. This finding was in agreement with Murray (1960) who found that in general, the formation of oxidized 2-substituted compounds (spearmint, carvone-type) takes precedence over the formation of oxidised 3-substituted compounds (peppermint, menthone-type).
Major genetic studies relating to monoterpene composition of various Mentha species are summarised in Table 2 and a scheme showing the relationships between various components of Mentha oils based upon biosynthetic, genetic and biochemical data is presented in figure 3. MAJOR GENETIC STUDIES IN MENTHA
Table 2
GENE HYPOTHESIS CROSSES EXAMINED REFERENCE
C precursor -- carvone M spicata x M piperita Murray and Reitsma (1954)
A piperitenone -- pulegone Fi(M spicata x M spicata) x itself Murray (1960) C and A independently inherited
R menthane -- menthol M arvensis var. piperascens sel fed Murray (1960a)
I precursor -- linalool M aquatica x M x citrata Murray and Lincoln (1970)
M aquatica x M x citrata Lm precursor -- limonene lmc precursor -- 3 Fi (M aquatica x substitution M x citrata) Lincoln et al (1972) lmC precursor -- 2 Selfed or x M spicata substitution
FF pulegone -X- menthofuran M aquatica x M spicata Hefendehl and Murray (1972) ff pulegone -- menthofuran Fi aquatica x M spicata) x M aquatica
Ff pulegone -- menthofuran M arvensis var. piperascens x M aquatica oxidation takes Fi (M arvensis var. Murray and Hefendehl (1972) precedence over piperascens x M aquatica) reduction x M aquatica
R carvone -- carveol Fi (M aquatica x M spicata) Hefendehl and Murray (1973) x M spicata
00 piperitone -X- piperitone oxide 00 piperitone -- piperitone M aquatica x M longifolia Murray et al (1972) oxide - 38-
Figure 3, MONOTERPENE INTERCONVERSIONS IN THE GENUS MENTHA 3 MATERIALS AND METHODS MATERIALS AND METHODS
PLANT MATERIAL
Hybridization occurs regularly between members of the genus Mentha. Peppermint itself (M piperita) is a sterile hybrid, thought to have arisen through crosses between M spicata and M aquatica. (Murray e£ aT 1972.)
Culture collections of the genus Mentha are located at the Royal Botanic Gardens, Kew (UK), US Department of Agriculture - Oregon State University (USA) and the Department of Agriculture and Natural Resources - Delaware State College (USA).
Peppermint plants - M piperita var "Black Mitcham", were obtained from the collection of Oregon State University; and used for experimental purposes, including the initiation of tissue and cell cultures. Upon receipt of the material a chromosome count was made in actively growing root tips.
Fresh root tips (1cm) were fixed in Carnoys fluid (3 parts ethanol: 1 part acetic acid) for approximately one hour at room temperature. After rinsing briefly in 70% ethanol, a standard Feulgen staining procedure was performed (Gomori 1952). An average count of 72 was obtained, in agreement with Harley & Brighton (1977). PLANT GROWTH CONDITIONS
M piperita plants were maintained under conditions previously shown to sustain good vegetative growth and oil yield (Battaile and Loomis 1961),
Cuttings with 2 leaf pairs were rooted in water, and the plantlets maintained in a perspex growth chamber, 60cm x 60cm x 120cm, with all vertical walls lined externally with reflective aluminium foil. After 10 days, experimental material was transferred to a mixture of John Innes loam and a medium grade peat (50:50 v/v), and each plant received a weekly mineral supplement of 25 ml of a dilute solution of liquinure (Fisons Ltd). During the course of growth, fresh water was supplied daily to the trays in which the plants were arranged.
Lighting was provided by five 80 Watt white fluorescent tubes with supplementary "red light" from 2 centrally-placed 200 Watt incandescent bulbs. Both lighting systems were mounted 30 cm above the top of the cabinet to allow air circulation. The light intensity delivered to midstem leaves of 7 week old mint plants was approximately 20 Wm- (photosynthetically active radiation, PAR), measured by means of an Lambda LI 185 quantum meter (Lambda instruments corporation, Lincoln, Nebraska). The photoperiod was fixed at 14 hrs.
The temperature of the chamber was maintained at 25°C + 3°C by continuous (14-17K rpm) small (110 mm) extractor fans (plannette fan, Plannair Ltd., Leatherhead) mounted in the vertical end walls.
After 7-8 weeks growth, plants were used for experimental purposes. For ease of reference leaf pairs have been numbered in chronological order of appearance, i.e. leaves 1 and 2 were present on cuttings and the highest numbered leaves refer to the youngest, most recent tissues. 3.3 ISOLATION OF TERPENOID COMPOUNDS
3.3.1 STEAM DISTILLATION
Monoterpenes and* other essential oil components were isolated from plant material (0.1-lOg) by the use of an all-glass, steam distillation-extraction apparatus, (Likens & Nickerson,
1964) modified for microscale work (Godefroot et aJL 1981) and fabricated at Imperial College. (See figure 5). The apparatus was installed in a fume cupboard for safety, and to reduce draughts which might cause condensation in the solvent circulation and vapour transfer lines.
Figure 4. Likens-Nickerson Micro Distillation Unit
From Codefroot et al (1980) Calibration
The extraction efficiency was investigated experimentally by carrying out a series of timed distillations. The recovery of natural peppermint oil additionally provided a means by which artifacts in the oil profile; arising as a consequence of distillation; could be identified by comparison with starting material of known composition.
Recovery of Standard Terpenes
Flask (A) was charged with 10 ml distilled water and 1 ml dichloromethane (DCM) containing 1 mg of authentic peppermint oil. The central "condensation chamber" contained 1 ml DCM and 1 ml distilled water, whist the reflux and sample collection flask (B) contained 0.5 ml DCM. Iced water was continuously circulated through the cold finger, and flask (B) heated to 85°C, via a boiling water jacket, for five minutes, until a continuous reflux was established.
Flask (A); containing the experimental oil sample; was then rapidly heated to 140° C by immersion in a paraffin oil bath for periods ranging from 5 to 90 minutes. After the timed distillation; Flask (B) was allowed to continue under reflux for a further 20 minutes, and finally the entire apparatus was left to cool for approximately 5 minutes.
During the course of distillation the lipophilic materials present in the steam condensate partitioned into the DCM phase, and were continually concentrated in Flask (B). When cool, an internal standard (0.5 ml of a 1 mg ml ^ solution of n-tetradecane in DCM) was thoroughly mixed with the contents of Flask (B). The combined contents were then reduced in volume to approximately 250 jil under nitrogen, at room temperature. -44-
The control sample was undistilled. 1.5 ml DCM, 1 ml of 1 mg ml ^ M piperita oil in DCM and 0.5 ml of 1 mg ml-^ n-tetradecane in DCM were mixed, reduced in volume under nitrogen and analysed as for other distillations. Recovery was calculated as a percentage of the undistilled control.
Recovery of Essential Oil from Plant Material
The time course for recovery of essential oil from plant tissues was also determined experimentally. Midstem leaf pairs (numbers 8 and 9) were collected from 7 week old chamber grown plants of M piperita and 6 g (fw) transferred to a laboratory pestle. The tissues were immersed in liquid nitrogen, broken into small fragments (2-3 ram diameter) and thoroughly mixed. 1 g samples were sequentially withdrawn for distillation, the remainder being stored at -17°C until required.
The plant material was placed in Flask A, together with 10 ml distilled water and several clean, glass boiling chips. 1 ml DCM and 1 ml distilled water were placed in the condensation chamber, and 0.5 ml DCM was placed in flask B. After equilibration of flask B at 85°C the contents of flask A were distilled for various periods as described previously, and n-tetradecane added as an internal standard once distillation was complete. Recovery was calculated as a percentage of that obtained after 120 minutes (designated 100%).
/ * Working Method
The following procedure was selected for optimum recovery of monoterpenes from plant tissues, and was employed in all distillations of experimental material.
1. 5 minutes pre heating (Flask B; 80°C) 2. 60 minutes distillation (Flask A; 140°C (oil bath), Flask B; 80°C) 3. 20 minutes sovent reflux (Flask B; 80°C)
The material was placed in Flask A together with 10 ml distilled water and 5 or 6 clean glass boiling chips. Immediately prior to distillation 0.5 ml of a 2 mg ml ^ solution of n-tetradecane in dichloromethane (DCM) was added to Flask A as an internal standard. 1 ml each of DCM followed by distilled water were loaded carefully into the central condensation chamber. Flask B initially contained only 0.5 ml DCM.
3.3.2 DIRECT VOLATILISATION GAS LIQUID CHROMATOGRAPHY
The Likens-Nickerson apparatus was suitable for determining the essential oil content of 0.1-10 g samples of plant material. However, in order to study the distribution of lower terpenoids within the cells and tissues of natural botanical material and small samples of plant cell cultures (e.g. 1-5 mg) a more sensitive and rapid microanalysis was required. Using direct - volatilisation gas liquid chromatography; (von Rudloff 1965, 1969, Svendson 1966, Swan 1966, Harley & Bell 1967, Roberts 1968, Malingre et al 1969, Henderson et al 1970), volatile components were driven off from the sample by controlled heating, and the vapours transferred directly on to the analytical column of the gas liquid chromatograph (GLC). Samples of excised leaf, shoot, root, sepal; epidermis, parenchyma, glandular appendages and cell cultures were examined by this technique. -46-
The samples were each placed in a glass precolumn, and held in place by a small quantity of the material used to pack the GLC column (acid-washed/DMCS-rinsed chromosorb W (80-100 mesh) carrying 10% w./w SP 2100 stationary phase). The latter served to limit the loss of volatiles to the external environment whilst loading the sample into the GLC, and also restricted the transfer of high molecular weight, pyrolysis products onto the analytical column.
At the start of an analysis the sample, contained within a precoluran, was placed within the heated injector unit of a Perkin Elmer Fll GLC and the external septum closed. The flow of nitrogen carrier gas through the injector was responsible for the transfer of terpene vapours onto the analytical column, which was programed to rise in temperature from 70°C-200°C at 2.5°C per minute. After the volatilisation period the precolumn was quickly removed from the instrument in order to minimise pyrolysis of the plant residues. The arrangement is illustrated in figure 5.
Figure 5. Direct volatilisation GLC injection unit -47-
Recovery of Monoterpenes
The recovery of monoterpenes was assessed by volatilising a standard solution of 1-menthol in hexane, and also by the double-injection of plant samples. The latter proved to be the most useful guide, since the ultimate resolution of individual components by GLC also depended upon having a very short volatilisation period. Initial volatilisation at 200°C was performed for 15, 30, 45, 60, 90, 120 and 180 seconds prior to sample withdrawal. On the second injection all samples were left for 120 seconds. The recovery of the first volatilisation was then expressed as a proportion of the combined total.
Working Method
In order to improve the analysis of small samples the sensitivity of the GLC was increased by a factor of 10 from its normal working range. As a result the detection limit was also improved.
Using a single volatilisation for 60 seconds at 200°C, it has been possible to quickly screen fragments of plant cell cultures for the presence of essential oil, and also to examine various plant tissues, including the contents of epidermal glands isolated with glass capillaries.
3.3.3 ISOLATION OF TERPENE-GLYCOSIDES
Essential oil, in its characteristic volatile form has been isolated from plant material by steam distillation and direct-volatilisation GLC. However, certain components; including monoterpenes; often form glycosidic derivatives in vivo (Francis & Allcock 1969, Banthorpe & Mann 1971, Sakata & Mitsui 1975, Croteau & Martinkus 1979). These products are not steam volatile, and as a result usually remain "tissue bound" following the normal extraction procedures. -48-
In order to develop an extraction procedure for the monoterpene glycosides of M piperita, a sample of 1-menthyl alpha and beta-D-glucopyranosides was prepared with minor modifications to the method of Sakata & Iwamura (1979) (see figure 6).
STAGE I Condensation of 1-menthol and beta-D-glucose penta acetate
10 g 1-menthol and 25 g beta-D-glucose penta acetate were dissolved in 60 ml of dry benzene in a round-bottomed flask. 5 g zinc chloride catalyst was added, and the reaction allowed to proceed under reflux for 30 minutes at 85°C.
The reaction mixture was then transferred to a separating funnel, washed with an equal volume of cold distilled water and the aqueous fraction discarded. The residue was then steam distilled for 90 minutes to remove residual benzene and unreacted 1-menthol. A clear, light yellow solution remained; which upon addition of 20 ml anhydrous ethanol; immediately formed a dense, cream-coloured floculant precipitate.
The mixture was shaken gently for 5 minutes and a further 20 ml aliquot of anhydrous ethanol added in order to completely solubilize the yellow pigment (assumed to be caramelised glucose). 20 ml of cold distilled water was then added and the mixture gently shaken once again to complete the precipation of the beta-isomer. The solvent was discarded and the off-white precipitate filtered and dried at 25°C prior to storage at -17°C in a dessicator. The reaction product was a mixture of 1-menthyl 2,3,4,6 tetra-o-acetyl alpha -D glueopyranoside and 1-menthyl 2,3,4,6 tetra-o-acetyl beta -D glueopyranoside. STAGE II Deacetylation of 1-menthyl 2,3,4,6 tetra-o-acetyl alpha/beta glucopyranoside
1 g of the anhydrous reaction product from Stage I, was dissolved in 10 ml of dried ethanol to give a white, milky suspension and 1 g of activated molecular sieve (0.5 nm) added to absorb the water produced during the subsequent reaction. Sodium ethoxide was prepared by dissolving 0.2 g sodium metal in 5 ml anhydrous ethanol to form a gel which was then dispersed throughout the reaction mixture. The reaction was then allowed to proceed under constant agitation at room temperature.
It was noted that in the presence of water, the reaction products rapidly caramelised due to the production of highly concentrated sodium hydroxide and elevated reaction temperatures.
After 2 hours the suspension was poured from the flask, separated from the molecular sieve, and neutralized by the addition of 4.4 millimoles of chilled sulphuric acid whilst being continually stirred. The pH was adjusted to approximately 7.5 as the glycosidic linkage in the products is acid- labile. After washing with a minimal quantity of cold distilled water, the solids were filtered from the reaction mixture and recrystallised as semipure 1-menthyl alpha/beta-D glucopyranosides. -50-
Zj-.gure 6« SYNTHESIS OF 1-MENTHYL GLUCOPYRANOSIDES
L-menthyl 2,3,4,6, tetra-o-acetyl oc/B-D glucopyranoside
* -51-
Recovery of menthol from menthyl-glucopyranosides
Menthol was released from the sample of 1-menthyl alpha/beta D glucopyranosides by steam-acid distillation, in which the aqueous phase normally employed with the Likens-Nickerson apparatus was replaced with 3N HC1 (pH 0.1). Good recovery was achieved after 60 minutes distillation, reaching approximately 80% of theoretical yield. Rather than prolonging the distillation period to improve recovery, a pretreatment in 3N HC1 at 60°C was considered as a less aggressive technique.
Recovery of Bound-Menthol from M Piperita
Midstem leaf pairs (numbers 8 and 9) were collected from 7 week old, chamber grown plants of M piperita and 6 g (fw) transferred to a laboratory pestle and mortar. Samples were immersed in liquid nitrogen, broken into small fragments (2-3 mm diameter) and thoroughly mixed. The tissues were then stored at -17°C until required. Samples of 1 g were then transferred to a 50 ml round-bottomed distillation flask, and steam distilled to remove free monoterpenes as outlined in section 3.3.1. After cooling, the contents of the flask were acidified with an equal volume of 6 N HCL to give an effective concentration of 3N; and either immediately subject to steam-acid distillation for one hour, or incubated in acid at 60°C for periods of 15, 30, 45, 60 or 90 minutes prior to steam-acid distillation.
The recovery of menthol as determined by GLC was expressed as a percentage of that obtained after 90 minutes pretreatment (nominally designated 100%)
Working Method
All extractions of experimental material included an acid pretreatment for 60 minutes at 60°C prior to steam-acid distillation using the Likens-Nickerson apparatus. ANALYTICAL PROCEDURES
Because of their intrinsic volatility, essential oils are aptly suited to fractionation by gas liquid chromatography (GLC). Burchfield & Storrs (1962), Humphrey (1970), Lawrence (1971), and Jennings & Shibamoto (1980); have reviewed the analysis of essential oils with special reference to GLC.
For the purpose of this study gas chromatographs were equipped with a flame ionsation detector (FID) , since this responds to virtually all compounds; with the exception of inert gases, air, carbon mon-and dioxide, oxides of nitrogen, ammonia, water and silicon; has high sensitivity and a wide linear range (McNair & Bonelli 1969). These features make it eminently suitable for the analysis of trace components of volatile organic compounds in biological materials.
The FID is a differential mass flow detector, and gives a response proportional to the total mass of component present in the eluted zone. However, it does not respond equally to all compounds, and therefore for quantitative analysis it was necessary to correct the integrated peak areas obtained directly from the GLC, by the use of response factors which had previously been determined for each component in the laboratories of Bush Boake Allen.
Analysis of reference materials
Samples of two natural mint oils, commercially distilled and collected by the author at Yakima (Washington State) and Willamette (Oregon State) in the USA, were subjected to extensive analysis at the laboratories of Bush Boake Allen Ltd. The composition of the two peppermint oils; both harvested in 1980 and obtained from clones of the same stock plants; is presented in section 4.1. -53-
1) Gas Chromatography - Mass Spectrometry (GC-MS)
The use of GLC coupled with mass spectrometry is a well known but highly specialised technique used for the structural identification of volatile components in complex mixtures. Its use in relation to natural product chemistry was first reviewed by Duffield in 1969. In this study the preliminary GLC fractionation was carried out on 0.02 ul samples, injected onto a 25 metre long, fused silia, wall coated open tubular (WCOT) column containing SP 2100 (Methyl silicone fluid) as the stationary phase, housed within a Pye 104 chromatograph unit. The injector temperature and carrier gas (helium) were ft maintained at 200°C, and 12 psi respectively, whilst the oven was programmed to rise from 60 to 200°C at 3 degrees per minute. The GLC column was coupled directly to the inlet of the mass spectrometer (VG instruments model 12F). Operating under electron ionisation mode with a source ft pressure of 10 ^ millibars and accelerating voltage of 4 kilovolts, the instrument generated an electron energy of 70 electron volts.
GC-MS revealed the presence of a number of components within each peak separated by GLC alone, and by this means a large number of compounds naturally present in peppermint oils were accurately identified.
ii) High resolution Gas chromatography
The high resolution gas chromatographic analysis of flavour and fragrance compounds has been reviewed by Teranishi (1970), and Jennings & Shibamoto (1980). In order to determine accurate retention indices for the various components identified by GC-MS, the American mint oils were examined by ft high resolution GLC using a Hewlett Packard 5710A instrument equipped with a 50 metre long, fused silica WCOT column containing SP 2100 as the stationary phase.
ft -54-
Injection temperature was constant at 200 C, with the column oven programmed to rise from 60 to 200°C at 3 degrees per minute. The presure of the carrier gas (helium) was maintained at 34 psi, and the 0.02 ul samples of oil were subjected to a 40:1 split ratio within the injector assembly.
Provisional identification of components was made by electronic comparison with a library of standard retention times previously determined for samples of known authenticity (GC-MS). Quantitative data was prepared using a Pye Unicam DP101 integrator; pre-programmed with response-factors for known terpenes.
Gas Liquid Chromatography - Working Method
The "standard method" for the gas chromatographic investigation of flavouring substances; established by the International Organisation of the Flavour Industry (IOFI) in 1976; was followed in selecting a suitable system for use in this study.
Reference to standard retention indices for monoterpenes on SP 2100 stationary phase (BBA research laboratory database) indicated that n-tetradecane was a suitable internal standard, which would elute independently of other components in peppermint oils and appear during the latter half of the chromatogram. In order to reduce error, n-tetradecane was added to all samples, and the retention times of other constituents expressed relative to this for each analysis. Routine qualitative and quantitative analysis of essential oils was performed using a Perkin-Elmer model Fll gas chromatograph fitted with a single 2.75 m long; stainless steel column of 3 mm internal diameter. The column was deactivated by rinsing internally with dimethyl dichlorosilane (DMCS) and then packed with acid-washed/DMCS-rinsed, chromosorb W (80-100 mesh) carrying the stationary phase; (SP 2100) at 10% (w/w) loading. Prior to use, columns were conditioned for 72 hours at 250°C with a nitrogen pressure of 5 psi.
Steam distilled oil samples; 0.5 ul in DCM; were injected into the glass-lined injector of the GLC at 200°C. Oxygen-free nitrogen at 20 psi was the carrier gas and the analytical column was temperature programmed from 70°C to 200°C at 1.5 degrees per minute.
Excellent resolution was obtained using a flame ionization detector, and the analysis was complete within 25 minutes. The GLC output (10 mV fsd) was either fed directly to a Hewlett Packard 3390A reporting integrator, or was frequency modulated and recorded on magnetic tape for subsequent processing.
Data previously obtained from GC-MS and the high resolution GLC analysis of reference materials was used to facilitate the provisional identification of compounds eluting during the routine GLC analysis, and the integrated peak areas corrected to mass percentages on the basis of known response factors for the GLC flame ionisation detector. -56-
3.4.2 DATA STORAGE SYSTEM
During the course of this investigation the need arose for a portable data storage system which could permanently record basic chromatographic data pertaining to a destructive analysis, in a form suitable for electronic processing. Using a commercially available microchip (RS 307 070); whose ti mode of operation is determined partly by the use of associated circuitry; a unit was designed in which the variable analog signal from a GLC, was linearly converted into a frequency modulated (FM) signal which could then be recorded on magnetic tape. Subsequent replay of the tape via complementary circuits was used to convert the FM signal back to a DC voltage for input to the Hewlett Packard HP 3390A computing integrator.
Method of Construction
The various components were obtained from RS Components Ltd, (13-17 Epworth Street, London, EC2P 2HA). Catalogue numbers of the major components are given. High tolerance (1% where possible) and high stability components were used throughout so as to minimise the need for routine maintenance and adjustments during use. All potentiometers were cermet-type.
Each separate circuit (input and output channels) was constructed on copper track stripboard (RS 434 116) cut to 10 x 10 cm and mounted vertically within an earthed aluminium case 25.4 x 20.3 x 15.9 cm (RS 509 888).
% -57-
Internal electrical shielding (1 ram aluminium sheet) was provided between the encapsulated power supply (+/- 15 volt RS 591 124) and other circuits. Connections between the circuit boards and to the panel meters (supplied by JEE Distributors Ltd, 244, Bath Road, Hayes, Middlesex, Model OBN 0102 lOv) were made using screened cable so as to reduce mains induced variations (hum).
Principle of Operation
I) Input Circuit (see figure 7)
The output signal from the GLC (0-10 millivolts) was taken directly as the input to the Data Storage System circuit described in figure 9. The input signal was monitored using a comparator referenced to 10 millivolts, whose output was used to drive an audible warning device (AWD) in the event of an input signal exceeding the range of the data system.
The input signal was amplified one thousand-fold by the use of two sequential, non-inverting operational amplifiers. A 10 volt cut off zener diode was connected across the circuit at this point to prevent an overload signal from damaging the integrated circuit. The signal was then frequency modulated, by the integrated circuit (RS 307 070); which was calibrated by the use of associated circuitry. The response ratio of the voltage to frequency conversion was determined by
variation of R. and V c (see diagram and 'calibration') m ret such that for zero volts input the minimum output
frequency » an<^ for 10 millivolts input, the maximum ouput
IUciDC frequency ^ out^» were set within the frequency response of the tape recorder. % In practice the output frequency range was set between 0.4 and 12 KHz.
F™ln = 0.4 KHz F ™ * = 12 KHz out out
The output waveform from the integrated circuit was a 14 volt, square wave signal, which was reduced to 0.4 volts (peak) prior to recording.
The circuitry was repeated for the dual input system.
Calibration
1. All operational amplifiers were first set to zero and then their gain adjusted by use of the accessory potentiometers.
2. To set to 400 Hz, an input voltage of 10 millivolts was applied directly to the integrated circuit and R£n adjusted.
3. To set to 12 KHz, an input voltage of 10 volt was applied directly to the integrated circuit, and V ^ (pin 6) adjusted.
4. The output frequency was reduced from a 14 volt peak signal to 0.4 volts prior to recording. -59-.
Figure 7. THE DATA STORAGE SYSTEM INPUT CHANNEL
% II) Output Circuit (see figure 8)
Chromatographic data was retrieved by replaying the tapes from earlier GLC analyses. An initial 37-fold preamplification was required in order to increase the output signal from the tape recorder to a level which could be handled by the RS 307 070 integrated circuit. In the circuitry illustrated (figure 8), the RS 307 070 was used to convert the FM signal back to a DC voltage.
For inputs ranging between 0.4 and 12 KHz the RS 307 070 was set to generate an output of 5.3 to 10.3 volts (see calibration section). This signal was then taken as the inverting input to an 'Adder', the non-inverting input of which was referenced to -5.3 volts. The output from this component was adjusted to vary between zero and minus 5.0 volts, but following a subsequent 2-fold inverting amplication, the net output lay on a range between zero and ten volts. The output was taken across a 10 volt zener diode.
Calibration
1. All operational amplifiers were first set to zero and their gain by adjusted by the accessory potentiometers.
2. To set the minimum output voltage a signal frequency of
0.4 KHz was applied and the 1 mega ohm potentiometer (V ref, pin 6) adjusted to give an output of 5.3 volts.
3. To set the maximum output voltage, V a signal frequency of
12 KHz was applied, and the 500 K ohm potentiometer (R^nt, pins 3-12) adjusted to give an output of 10.3 volts).
4. The gain of the 'adder' was adjusted by variation of the 20 K ohm potentiometer such that an input signal between 5.3 and 10.3 volts gave an output between 0 and 5 volts. -61-
Figure 8. DATA STORAGE SYSTEM OUTPUT CHANNEL
% -62-
Plate 1 The data system recording from two independent Fll GLC modules simultaneously
Plate 2 The data system allows electronic computation of prerecorded results at a convenient time. 3.5 INTACT PLANT STUDIES
Prior to investigating the monoterpene metabolism of Mentha cell cultures, it was considered important to pay attention to these same processes as they occur in the intact plant. As well as providing valuable reference material for the development of analytical techniques; data on various aspects of the biosynthesis and accumulation of monoterpenes was obtained for subsequent comparison with cell cultures.
In order to determine which tissues were most suitable for further study, monoterpenes were isolated by steam distillation from the leaf, shoot and root fractions of 7 weeks old, chamber-grown plants. The oil yield of each tissue was calculated as a percentage of their initial dry weight. Monoterpene glycosides were also recovered by steam-acid distillation of the plant residue.
3.5.1 DEVELOPMENT OF ESSENTIAL OIL DURING ONTOGENESIS
It has been assumed that; for plants growing under a constant environmental regime; the sequence from young to old tissues represents a time course of monoterpene biosynthesis and interconversion. Qualitative and quantitative changes in the terpenoid profile of leaves during their development were therefore documented by isolating both free and tissue-bound monterpenes from variously aged leaf-pairs of seven week old, chamber-grown plants. -64-
3.5.2 LEAF SURFACE TOPOGRAPHY
Scanning Electron Microscopy (SEM)
Scanning electron microscopy was used to study the surface topography of M piperita leaves, with particular reference to the density and condition of epidermal appendages. In addition, SEM was used to quantify the number of glandular structures before and after treatment of leaves with PVA solution (Section 3.5.2. pt (iii)).
Prior to examination by SEM, samples of leaf material were subjected to a general aldehydic fixation and then dehydrated into acetone. Critical point drying (CC^ exchange) afforded excellent preservation of the 3-dimensional structure at the final stage.
1. Fixation
Samples were incubated at 4°C in covered glass embryo dishes containing a solution of 4% (w/v) para-formaldehyde and 0.5% (w/v) ultrapure glutaraldehyde in 0.1M cacodylate buffer (pH 7.2) for 14 hours.
2. Rinse
The fixative was removed by three, 10 minute changes of 0.2 M sucrose solution in 0.1 M cacodylate buffer (pH 7.2). -65-
3. Dehydration
Tissues were passed through a 10% graded ethanol series, concluding with three, 60 minute changes in absolute ethanol. Specimens were then transferred from ethanol; through a graded series; into 100% acetone.
4. Critical Point Drying
Samples were transferred under acetone into the sealed chamber of a commercially available apparatus (Polaron Ltd) and "critical point" dried. In this process the acetone was replaced by flushing the sample chamber with liquid CO^, which was subsequently volatilised by slight warming. The low surface tension associated with CO^ volatilisation afforded excellent preservation of Mentha leaf material, especially the easily damaged epidermal appendages.
5. Viewing
In order to increase the electron opacity of the material prior to viewing by SEM, samples were sputter coated with a 30 nanometer layer of gold and palladium. Vaporisation of the precious metals was carried out under an argon atmosphere with 0.1 Tor vacuum.
Material was examined using a Philips scanning electron microscope, model 500; with an accelerating voltage of 25 kilovolts and a scanning electron beam diameter of 32 nanometers. -66-
3.5.2 LOCALISATION OF ESSENTIAL OIL
Visual examination of M piperita plants revealed that aerial surfaces are glabrous and bear large numbers of glandular appendages. Since the leaves proved to be a rich source of essential oil, they were the subject of a more thorough examination, with particular reference to the localisation of essential oil within the epidermal glands.
i) Fine glass capillaries with a tip diameter of approximately 25 microns, were prepared using a hot wire, electromagnetic device designed for the manufacture of microelectrodes. Working with plant material under x25 binocular magnification it was possible to identify large glandular "sacs" on the surface of leaves. When pierced with the tip of a capillary the contents of individual "sacs" were immediately withdrawn into the tube by capillarity. The capillary tip; containing the sample; was then placed within a GLC glass precolumn and broken-off. The contents of ten glands were collected, column packing material added and samples subjected to direct-volatilisation GLC for analysis of monoterpenes. The results were compared with those obtained by volatilisation of small (2mm diameter leaf discs from different regions of the same leaf (number 7).
ii) The presence of specialised epidermal oil glands raises the question as to whether these are the site of monoterpene biosynthesis and accumulation or merely their accumulation. The monoterpene content of epidermal tissues was therefore examined and comparison drawn with those materials found within the underlying parenchyma. However, the isolation of intact epidermis; with its associated glandular appendages; proved virtually impossible to achieve with M piperita leaves of any age. -67-
Thus, young shoots were cut transversely into sections approximately 1.5 cm long. Using forceps, the epidermis was peeled from all 4 faces, weighed; and then subjected to direct-volatilisation GLC for the analysis of monoterpenes. Making a clean cut, the terminal 0.5 cm at each end of the shoot sample was then removed in order to reduce the chance of cross contamination of components between the epidermis and parenchyma. Small samples of the parenchyma amounting to some 5 mg dry weight, were collected using glass capillaries, and analysed by direct- volatilisation GLC. iii) In order to quantify the distribution of essential oil between the glandular "sacs" and other tissues, adhesive films were applied to the upper and lower surfaces of leaves. A 20% (w/v) aqueous solution of Polyvinyl alcohol (PVA) was brushed gently onto the surfaces of one leaf from each of six leaf pairs at different stages of development. The film was dried under ambient conditions for 60 minutes and the resulting thin films were then carefully peeled off using forceps. The residual leaf material was steam distilled to recover monoterpenes. The untreated leaves from the six leaf pairs were similarly distilled and the proportion of the total essential oil removed with the PVA films calculated by difference. Direct distillation of the PVA films was used to confirm the results. PVA treated and untreated leaves from the same leaf pair were examined by Scanning electron microscopy in order to quantify the density of epidermal appendages, and the efficiency of their removal by PVA films. iv) In order to determine whether morphological differentiation in the form of epidermal glands; was a prerequisite for essential oil accumulation, PVA films were applied to a number of young leaves and then peeled off to remove the complement of glandular triehomes.
The leaves were left attached to the parent plant for a further 18 days growth, prior to analysis for free and bound terpenes and visual examination for the development of new glands. The untreated leaves from each leaf pair were examined for comparison.
OIL GLAND ULTRASTRUCTURE AND DEVELOPMENT
Light Microscopy
M piperita plants have been investigated and anatomical features associated with the accumulation of essential oil documented.
Excised shoot meristems and young leaves were fixed in formalin - acetic acid - alcohol (FAA) for 4 hours and then dehydrated through an ethanol/tertiary butyl alcohol (TBA) series. (Jensen 1962). Small samples were then transferred to embedding dishes containing molten paraffin wax at 60°C, for gradual impregnation. After three, 12 hour immersions in fresh wax, the samples were incubated at 60°C for a further 12 hours before chilling to solidify the embedded materials.
Sections; 200 nanometers thick; were prepared on a 'Cambridge' rocking microtome, and transferred to clean glass slides which had previously been coated with Haupts adhesive (see appendix). The sections were then stained with safranin; counterstained with malachite green and examined using a Reichert optical microscope. -69-
Vital Staining
The so-called "vital" stains, exhibit differential staining reactions with cells and tissues depending upon their physiological status (Stadelmann & Kinzel 1972). Glandular, epidermal and mesophyll tissues of M piperita were stained with toluidine blue and examined in order to identify any differences which might relate to monoterpene metabolism.
Sections of fresh and fixed M piperita tissues were examined after staining with an aqueous staining solution containing 0.05% of toluidine blue in 0.1 M phosphate buffer at pH 6.8. The positively charged chromophore of toluidine blue was absorbed onto the negative charges within cell walls, resulting in a variable shift in its spectrum (metachromany).
Tissue element Colour reaction
lignified walls green, blue-green collenchyma red-purple parenchyma red-purple sieve tubes red callose, starch unstained
Toluidine blue may also be absorbed into the cell vacuole, where it affords a means of differentiating between cells containing tannins and flavones, or those containing primarily organic acids and inorganic ions.
Cell Component Colour reaction
Polyphenols blue-green nucleic acids purple flavone-glycosides green-blue organic acids violet -70-
Transmission Electron Microscopy (T.E.M.)
In order to study the organisation and fine structure of tissues involved in essential oil accumulation, transmission electron microscopy was employed for the examination of young leaf primordia.
Fixation was firstly required to preserve the spatial relationship between organelles within a cell; at a molecular level. Primary fixation with volatile aldehydes (glutaraldehyde and para-formaldehyde) was used to rapidly cross-link proteins and some carbohydrates, whilst subsequent treatment with Osmium tetroxide (OSO^) was used to stabilize lipids through the formation of electron-dense, addition-complexes with unsaturated acyl chains. (Gray 1973).
3 Small fragments of plant material (2-3 mm ) were prepared according to the general procedure of Sabatini e£ al (1963).
1. Primary Fixation Samples were incubated at 4°C in covered, glass embryo dishes containing 4% w/v para-formaldehyde and 0.5% w/v ultra-pure glutaraldehyde in 0.1M cacodylate buffer (pH 7.2) for 6 to 8 hours.
2. Rinse The fixative was removed by three, 10 minute changes of 0.2 M sucrose solution in 0.1 M cacodylate buffer (pH 7.2).
3. Secondary Fixation Samples were transferred and incubated for 2 hours at 4°C in 1% OSO^ (prepared in 0.1 M cacodylate buffer (pH 7.2) containing 0.2 M sucrose). -71-
4. Dehydration Tissues were passed through a 10% graded ethanol/buffer series, concluding with three 60 minute changes in absolute ethanol.
5. Solvent Transfer The samples were then transferred from ethanol into epoxy-propane in 25% steps, and finally left in covered embryo cups for 12 hours in 100% epoxy-propane.
6. Resin Impregnation i) A limited number of suitable specimens were transferred into a mixture of epoxy-propane and epon resin (3:1) for 12 hours impregnation in an open embryo dish at 35°C. ii) The specimens were then transferred to a plastic embedding dish covered with fresh, warm resin and left for 24 hours at 35°C. Three further resin changes at 24 hour intervals were carried out before curing.
7. Curing A small selection of samples in fresh resin were incubated at 60°C for 48 hours.
Sections were cut to a thickness of approximately 60 nm using an LKB ultramicrotome equipped with a glass knife. The sections were floated on double-distilled water and collected on formvar-coated copper grids. Sections were double-stained, first for 30 minutes with 2% w/v aqueous uranyl acetate (pH 4,60°C) and subsequently 10 minutes in aqueous lead citrate (pH 11, 20°C). (Reynolds 1963). Specimens were stored within a dessicator and air-dried sections viewed using a Phillips transmission electron microscope (model 301). -72-
3.5.4 ULTRASTRUCTURAL LOCALISATION OF BETA-D-GLUCOSIDASE
It has previously been suggested that monoterpene glycosides may be the biosynthetic precursors of "free" terpenes; (Skopp and Horster, 1976). It was therefore considered that glycosidase activity might be associated with the sites of "free" essential oil accumulation in M piperita.
Based upon the method of Smith and Fishman (1969) a simultaneous-coupling azo-dye technique with naphthol AS BI beta-D-glucopyranoside as substrate; was developed for the localisation of beta-glucosidase activity (see fig 11). Histochemical examinations were carried out on transverse sections of young M Piperita shoots and leaves, cut by hand under chilled and buffered 5% methanol. Sections were passed through a series of 10%, 15% and 20% buffered methanol, allowing 5-10 minutes in each solution in order to minimize osmotic damage (Ashford, 1970a). Mcllvaine buffer (pH 5.0); prepared by combining equal volumes of 0.2 M Na2HP0^ and 0.1 M citric acid was used throughout.
Equal volumes of freshly prepared, diazotized para-acetoxymercuric aniline (2 mg ml ^) and the substrate; naphthol AS BI beta-D-glucopyranoside (0.2 mg ml ^) were combined, the solution stirred and filtered. The tissue sections were immediately added to the reaction medium and incubated for 30 minutes at 35°C.
Initial examination of the sections was carried out by light microscopy, with sections mounted in buffered, 20% methanol. Although the water solubility of the reaction product is very low ( < 1 ug ml ^) (Burstone, 1962), the sections were finally immersed in a buffered solution of 1% thiocarbohydrazide for 30 minutes at 35°C. The latter acts as a mordant, the formation of mercuric sulphide both intensifying and stabilising the staining. -73-
In addition to sections of experimental material, controls were examined to assess the specificity of the technique.
CONTROLS
i) There was no endogenous enzyme activity. - Enzymes had been inactivated by steam. * ii) There was no added substrate. - The diazo salt and mordant were applied in the absence of substrate. (No naphthol ASBI beta-D-glucopyranoside). iii) The substrate was hydrolysed chemically and was freely available for reaction and therefore not localised to sites * of endogenous enzyme activity. - Hydrolyzed substrate was supplied in conjunction with the diazo salt and mordant.
Details regarding the preparation of reagents are documented in * the appendix.
« -74-
Figure 9, HISTOCHEMICAL LOCALISATION OF B-GLUCOSIDASE
B-glucosidase -75-
3.6 PLANT TISSUE AND CELL CULTURE STUDIES
One of the main purposes of this study was to develop novel approaches which might lead to an improved understanding; and ultimate control of the factors regulating monoterpene metabolism in plants and their derived cell cultures.
® Callus cultures; relatively slow growing and somewhat limited in their suitability for experimental manipulation; were first initiated from leaf and stem sections of M piperita. However, the majority of physiological experiments have been performed using cell suspension cultures derived from the agitated liquid culture * of callus explants. Suspension cultures have the advantage of relatively predictable growth cycles, and also, when chemical additions are made to the media, all cells receive a similar exposure to the reagent.
• 3.6.1 INITIATION AND MAINTENANCE
For the purposes of this study no attempt was made to optimise the complete formulation of the culture media, explant pretreatment or culture conditions. Whilst realising that these can be important factors in determining the growth of plant cells in culture, it was considered that existing defined media would sustain adequate growth, and improvements above this would be likely to be marginal in relation to the time and effort required. Defined media contain the correct amounts and proportions of inorganic nutrients to satisfy the nutritional as well as the physiological needs of many types of plant cells in culture (Gamborg et al 1976). The use of defined media was also preferred since they offer a means of sustaining predictable and reliable growth kinetics for the production of experimental material. - 76-
Callus Cultures
Healthy midstem leaves and young shoots were cut from vegetative plants of M piperita and surface sterilized on a laminar flow bench according to the following procedure.
1. Plant material was initially rinsed in 70% ethanol for 1 minute to 'wet' the cuticle and remove microbial spores.
2. Tissues were then rinsed in sterile distilled water for approximately 3 minutes to remove any remaining traces of ethanol. »
3. The material was then immersed in sodium hypochlorite solution containing 5-7% free chlorine for 5 minutes, in order to effect surface sterilisation.
4. Finally, the tissues were given four, successive 5-minute rinses in sterile distilled water in order to thoroughly remove all traces of chlorine.
Using a pre-sterilized cork borer, 0.5 cm diameter discs were aseptically removed from the surface sterilised leaves, rinsed briefly in sterile distilled water to remove the phenolic materials released from damaged cells, and used as explants for callus initiation. When shoot sections were employed, 1 cm sections of young shoots were treated identically to leaf material.
Two defined media were assessed for their ability to support the growth of M piperita cell cultures - M&S (Murashige and Skoog, 1962) and B5 (Gamborg, Miller & Ojima, 1968).
9 -77-
M&S medium has a relatively high nitrate, potasium and ammonium content, whilst B5 contains a lower level of ammonium; a nutrient that may repress the growth of batch cultures. (Gamborg & Shyluk 1970). Detailed formulations for both media are included in the appendix. Sucrose was supplied at 3% (w/v) as the carbon source in both formulations.
A solidified medium was used for the initiation of callus cultures. Difco "Bacto" agar was dissolved in the media by heating, to give a final concentration of 1% w/v. The hot medium was dispensed; 20 ml in 100 ml erhlenmeyer flasks, or 10 ml in 25 ml pyrex glass boiling tubes. The culture vessels were loosely » capped with aluminium foil and sterilized by autoclaving (15 mins, 15 psi, 121°C).
Once the medium was cool, sterile explants were transferred to individual culture vessels for callus initiation and the flasks transferred to darkness for the first 36 hours of culture. This procedure was adapted from work by Paul & Bassham (1977) who found that a short recovery period in darkness improved the viability of leaf protoplasts in culture. Callus cultures were subsequently maintained at 25°C — 2°C under constant low intensity —2 illumination (1.6 Wm PAR). The efficacy of various plant growth regulators to induce callus formation was assessed visually, and subsequently by measurement of fresh and dry weight gain. Combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), indol-3yl-acetic acid (IAA), 6-benzylaminopurine (BAP) and 6-furfurylaminopurine (FAP) were evaluated over a concentration range of 0.001-10 mg 1 \ with triplicate samples examined for each experiment. If the medium was suitable for growth, callus cell divisions could usually be seen around the vascular elements at cut surfaces of the explant within one week of initiation.
9 After approximately four weeks of growth a small amount of new tissue was excised aseptically from the most actively growing cultures and transferred onto fresh medium for proliferation. In order to minimise the carry over of metabolites from the parent plant, the callus was subcultured monthly for at least two periods prior to experimental use. The growth of established callus cultures was determined by measurement of fresh and dry weight after a period of 40 days.
Media Selection and Preparation
After initial experiments to select a suitable growth medium, that of Murashige & Skoog (1962) was selected for routine use. Stocks of the inorganic macro and micro nutrients were individually prepared at 100-fold concentration and stored in bulk at 4°C. Organic supplements at 100-fold concentration were stored at -17°C in 10 ml aliquots until required.
Auxins (IAA and 2,4-D) were dissolved in warn ethanol to give a 5% aqueous solution; whilst the cytokinins (BAP and FAP) were dissolved in 5 ml of warm 0.1 N HC1 and the volume then made up to 100 ml with distilled water. Plant growth regulators were pre-batched at a concentration of 500 ug ml ^ and were deep frozen at -17°C until required.
When required for use, stock solutions were allowed to equilibriate to room temperature and dispensed into approximately half of the final volume of distilled water in a pyrex volumetric flask. - 79-
Aliquots of heat-stable organic additives such as vitamins, amino acids, 2,4-D, FAP, BAP and sucrose were then added, and the solution made up to volume. M & S medium had a pH of approximately 4.7 units, when freshly prepared. This value did not change significantly after autoclaving and therefore the medium was routinely adjusted to pH 5.6 by the addition of 0.2 N KOH at the time of preparation.
Note Although some hydrolysis of sucrose occurs when sugar is autoclaved in bulk with mineral salts (Ball, 1953) there was no detectable difference in the growth of Mentha cultures maintained on media in which sucrose had been autoclaved en-mass as opposed to being added aseptically after the bulk of the media had been autoclaved.
Suspension Cultures
Suspension cultures were established by transferring approximately 1 g of actively growing callus material into 100 ml of pre-sterilized M & S liquid medium within a 250 ml erhlenmeyer flask. Preconditioning of the medium was not a necessary prerequisite for good growth of suspension cultures, even though relatively small explants were used, particularly during the subculture of established cell lines. Environmental conditions were selected so as to closely reflect those experienced by the intact plants grown as part of this study. The photoperiod was —2 set at 14 hours with an intensity of 6 Wm (PAR) and cultures were incubated at 25°C + 2°C within an oribital shaker operating at 150 rpm (Gallenkamp & Co Ltd).
« Because of the gradual release of phenolic substances; which may be phytotoxic; the 'starter callus' was aseptically removed from new cultures after the first 14 days growth. The new cell suspension cultures were grown for a further 14 days prior to subculture into fresh medium. In established suspension cultures, cells were subcultured every 14th day by transferring 10 ml of the cell suspension into 90 ml of fresh medium. This was equivalent to an innoculun of 70— 5 mg dry weight of cell material per 100 ml of culture medium. Although cell cultures were once thought to be uniform and homogenous, it is now appreciated that suspension cultures comprise a heterogeous collection of cell types. For this reason the growth Kinetics of M piperita suspension cultures were defined in terms of cell density (dry weight) (Street, 1973) and medium conductivity, (Hahlbrock & Kuhlen, 1972, Hahlbrock et^ al, 1974) prior to their use in biosynthetic studies. Material for experimental purposes was obtained from subcultures 2-5 only, in order to limit the possible variability of material.
In an attempt to improve the growth and extend the viability of cultures, cells were also grown in M & S medium with the inclusion of 2% (w/v) soluble polyvinylpyrrolidine (PVP; rmm 40K). The latter is reported to absorb and detoxify plant polyphenols, which may be partly responsible for the premature senescence of cell cultures. (Siegel & Enns, 1978).
Results are presented in Section 4.3.1.
3.6.2 BASAL TERPENOID METABOLISM
Callus cultures
Tissue samples of approximately one gram in weight were taken from 3rd generation callus cultures after 5 weeks growth on M&S media containing a range of plant growth regulators. - 81-
Surplus agar was rinsed from the tissue surface using sterile distilled water and the samples subjected to steam and steam-acid distillation for the recovery of terpenes and terpene-glycosides respectively. Direct-volatilisation GLC was also used to screen fragments of tissues, which frequently exhibited diverse forms of growth within a single culture.
Suspension cultures
At various points during the growth cycle, representative suspension cultures were screened for the presence of monoterpene compounds. Cells were harvested during the lag phase (day 2), exponential growth (day 10) at maturity (day 18) and senescence (day 24). They were filtered through a 0.4 mm mesh nylon sieve, rinsed with 100 ml of sterile distilled water and one gram of the fresh cell material subjected to steam and steam-acid distillation for the recovery of terpenes and terpene glycosides respectively. Direct-volatilisation GLC was also used to screen variously pigmented cell clusters which arose spontaneously during the routine culture of M piperita cell suspensions.
3.6.3 ENVIRONMENTAL EFFECTS
Environmental conditions are known to affect essential oil yield and quality in plants of Mentha spp. (Biggs & Leopold 1955, Burbott & Loomis 1967, Virmani & Datta 1970, Clark & Menary 1979).
i) Effect of cell culture conditions
To establish whether the conditions employed in this study would support monoterpene biosynthesis per se, plantlets were grown in cell culture flasks under identical conditions to those routinely employed for the growth of cell cultures. In order to minimise the carry-over of preformed metabolites from the parent plant, plantlets were regenerated from small, surface-sterilized nodal sections of chamber-grown plants. The plantlets were grown in 100 ml erhlenmeyer flasks containing 20 ml of agarified M&S medium lacking plant growth regulators. Explants were maintained under the conditions described previously for the growth of callus cultures (25°C — 2°C 1.6 Wm ^ PAR) and intact plants (25°C - 3°C, 20 Wm~2 PAR).
A control set of explants were regenerated in loam and remained in the plant growth chamber until required for analysis. After 7 weeks growth all plantlets were examined, weighed and the tissues subjected to steam and steam-acid distillation for the extraction of monoterpenes and glycosides respectively. The oils produced by the plantlets were isolated and analysed quantitatively and qualitatively by the standard methods.
Requirement for light
In order to resolve whether light or photosynthesis are direct pre-requisites for monoterpene biosynthesis, plantlets were regenerated from nodal explants and maintained in darkness. After 7 weeks growth the plantlets were examined, weighed and the tissues subjected to steam and steam-acid distillation for the extraction of monoterpenes and glycosides respectively. The oils produced by the plantlets were isolated and analysed quantitatively and qualitatively by the standard methods. 3.6.4 INHIBITION OF PRIMARY GROWTH
Primary growth was restricted as a possible means of increasing the yield of secondary metabolites from M piperita cell suspension cultures.
Initial experiments were carried out simply by rinsing a cell sample from 14-day old cultures with regulator-free medium and then transferring 10 g (fresh weight) into fresh M&S medium from which the growth regulators; 2,4-D and BAP had been withdrawn. Cultures were then maintained as for a routine subculture for a further 5 days before investigation.
An alternative approach was to use chemical means to override the effect of endogenous growth regulators. Reference to the literature aided in the selection of colchicine, chlorocholine chloride and gibberellic acid as possible inhibitors. In this instance the cell sample was rinsed with regulator-free M&S medium and then 10 g (fresh weight) transferred into the same medium . . -3 -4 -5 . . -1 containing 10 , 10 or 10 molar colchicine, 5 mg 1 _3 gibberellic acid (GA^) or 10 molar chlorocholine chloride
(CCC).
After 5 days subculture, experimental materials were harvested, a sample taken for dry weight determination and the remaining tissues examined for the presence of free and bound monoterpenes using standard techniques. A comparison was made with the growth and basal metabolism of a similar innoculum transferred into standard M&S medium. All cultures were maintained under the normal conditions described in section 3.6.1. 3.6.5 CHEMICAL AND PHYSICAL "STRESS"
3.6.5.1 The biosynthesis of secondary products is frequently associated with conditions of environmental or metabolic "stress" (Vickery & Vickery 1981). The application of stress-related plant growth regulators was therefore investigated as a possible means of inducing the accumulation of essential oil in M piperita cell cultures.
10 g (fw) of cells were collected by filtering 14 day old suspension cultures through a 0.4 mm mesh, nylon sieve. The cells were rinsed with 100 ml pre-sterilized M&S medium, transferred to a 50 ml distillation flask and resuspended in 10 ml of fresh medium.
Paraquat, abscisic acid (ABA) and ethephon (2-CEPA) were prepared as 1 millimolar solutions in ethanol, and at zero time, a 10 ml aliquot of the chosen regulator added to the cell suspension to give a one micromolar effective concentration. The flasks were closed "air-tight" with silicone-greased ground glass stoppers; to retrict the loss of volatiles; and were incubated on an orbital shaker (150 rpm) with continuous illumination (6 Wm ^ PAR) at 25°C as for the normal growth of suspension cultures.
After 6, 12, 18, 24 or 48 hours, flasks were unstoppered and transferred directly to the Likens-Nickerson distillation apparatus. The internal standard (1 mg n-tetradecane) was added and distillation for the recovery of monoterpenes commenced. The post distillation residue was subsequently steam-acid distilled for the recovery of monoterpene glycosides. Comparison was made with cells resuspended in standard M&S medium, and maintained under identical conditions. -85 -
3.6.5.2 14-day old M piperita suspension cultures were subjected to 24, 48, and 72 hours of high temperature stress at 35°C, as this • had been reported to induce essential oil accumulation in cell cultures of Jasminum grandiflorum (Bush Boake Allen research report 1980).
After exposure, the cells were returned to normal growth • conditions for a further 24 hours, prior to being filtered, rinsed and steam/steam-acid distilled.
The experiment was repeated using a subculture of the jasmin cell line previously studied at BBA, but maintained under the • conditions selected for the growth of M piperita cultures.
3.6.6 SUBSTRATE AVAILABILITY
It was considered that the availability of specific substrates • maybe one of the factors limiting essential oil accumulation in Mentha cell cultures.
Dimethylallyl alcohol (DMA-OH) and beta-methyl crotonic acid (beta-MCA) were prepared as 1 molar solutions in ethanol and ^ supplied to cells as potential substrates at 1 millimolar concentration. DL-mevalonic acid (MVA) was supplied as its lactone ester. The acid was released by alkaline hydrolysis for 30 mins at 40°C in 0.02 molar KOH (Gray & Kekwick, 1973) prior to its use as a substrate. MVA was applied at a 2 millimolar concentration since only the naturally occuring D-isomer; accounting for approximately half of the mass; enters physiological processes.
Cells were collected by filtering 14 day old suspension cultures through a 0.4 mm mesh nylon sieve. The material was rinsed with 100 ml pre-sterilised M&S medium (including regulators), and 10 g (fw) transferred to a 50 ml round bottomed distillation flask containing 10 ml of fresh medium. At zero time a 10 ul aliquot of the selected substrate was added to the resuspended cells, giving an effective substrate concentration of 1 mM.
Flasks containing experimental material, and control samples without substrate additions were sealed and incubated for 2-24 hours as described in section 3.6.5, prior to in-situ steam and steam-acid distillation.
CHEMICAL REGULATION
Compounds based upon a trimethyl cyclohexyl ring structure have previously been shown to promote the biosynthesis of higher terpenoids such as carotenoids and sterols. (Gooday (1978), Dandekar et al (1980)). Such compounds are believed to act by deregulating an early, enzymically-controlled stage in the terpenoid pathway - probably pyrophosphomevalonate decarboxylase, which catalyses the conversion of 5-pyrophosphomevalonate into isopentenyl pyrophosphate. Although this process is common to the biosynthesis of all terpenoids, their use as a means of enhancing monoterpene biosynthesis was considered for the first time in this study.
Beta-Ionone ( C ^ ^gO) an<* Ret:*-no^ ^ 2 0 ^ 2 8 ^ were prepared in ethanol for use at ImM final concentration in M & S medium.
Fresh cells (10 g) from 14-day old suspension cultures were filtered, rinsed and incubated for 12-48 hours with the reagents as described in section 3.6.5. The cells and medium were then steam/steam-acid distilled for the recovery of monoterpenes and their glycosides respectively. -87-
3.6.8 ENZYMATIC POTENTIAL
Cell cultures of M piperita were exposed to a number of dilute, purified monoterpenes, in order to investigate their ability to produce the full range of monoterpenes found in a natural essential oil. The metabolism of monoterpenes under these conditions was monitored.
L-isomers of monoterpenes were prepared in ethanol, overlain with nitrogen gas, and stored within septum-sealed vials, whilst the r-isomers of pulegone and neomenthol; because of their low alcohol solubility; were added directly to the culture medium when required.
Log phase cells were filtered and rinsed, then 10 grams, transferred to a distillation flask and resuspended in 10 ml, M&S medium. Monoterpenes were added to give a final concentration of 2.5 mM since higher concentrations were found to cause relatively rapid browning of the cell material. The flasks were then sealed and the cells incubated for 2-24 hours on an orbital shaker (150 rpm) with continuous illumination (6 Wm ^ PAR) at 25°C. At various time intervals the contents of individual flasks were distilled in order to retrieve volatile products, and subsequently steam-acid distilled to release the glycosides of terpene alcohols.
Control incubations were examined to identify endogenous production of monoterpenes (cells suspended alone) and to monitor the "non-enzymatic" changes of monoterpenes in aqueous solution (monoterpene and M & S medium alone). In addition, cells from log phase suspension cultures of Nicotiana tabacum were also assessed for their ability to carry out the biotransformation of monoterpenes, and hence determine the degree of culture specificity required for this process. 3.6.9 CELLULAR COMPARTMENTATION
Whilst cell cultures appear organisationally simple, both intra and intercellular compartmentation of precursors, intermediates, enzymes and reaction products may restrict their ability to perform certain metabolic activites. It has also been shown that the addition of surfactants to the media of microbial (Udagawa et: al 1962) and plant cell cultures (Tanaka e_t al 1974) can result in the "leakage" of metabolites, and lead to an overall increase in product yield.
Dimethyl sulphoxide (DMSO) was selected as an agent with which the permeability of cell membranes and hence, compartmentation could be reversibly altered. (Rammler & Zaffaroni 1967, Delraer 1979).
Prefiltered and washed log-phase cells from 14-day old cultures were incubated in M&S medium containing 5% (v/v) DMSO for periods of up to 24 hours under the conditions described in Section 3.6.5, and subsequently assayed for the presence of free and bound monoterpenes. After various exposure periods cell samples were suspended in a 0.25% (w/v) solution of Evan's Blue prepared in M & S medium, and an assessment of integrity made using light microscopy (Gaff et: al 1971).
3.6.10 MORPHOLOGICAL DIFFERENTIATION
There is considerable evidence to suggest that secondary metabolite production is intrinsically linked to some degree of differentiation in many plant cell cultures (Staba 1980, Yeoman £t al 1980). It was concluded that controlled morphogenesis may therefore substantially increase the yield of essential oil in M piperita cultures. Furthermore, if plantlets were regenerated which accumulated essential oil in detectable amounts, their examination might also provide a means of quickly assessing the genetic uniformity and stability of M piperita cell cultures by effectively providing a chemical fingerprint. Aliquots of 1 ml were taken from 14 days old suspension cultures and plated onto agarified M & S medium for proliferation as described by Earle (1965). Five days later cell clumps which had shown signs of new growth were individually transferred onto a range of media formulated to promote organogenesis. Such "regeneration media" were based upon M&S salts and vitamins, 1% (w/v) agar, 3% (w/v) sucrose and combinations of the following additives:-
activated charcoal (1% w/v) naphthalene-l-acetic acid (NAA, 0.5-5mgl ^) 2,3,5-triiodobenzoic acid (TIBA, 10 ^-10 ^M), gibberellic acid A (GA , 0.05-5 mgl ^), . ^ - 1 6-benzylaminopurine (BAP, 1-10 mgl ) -3 chlorocholine chloride (CCC, 10 M) 2,4 -Dichlorophenoxyacetic acid (2,4-D, 1 mgl ^).
10 replicate cultures were initiated on each medium and were visually examined for signs of differentiation after 14, 28, 42 and 56 days using a xlO binocular microscope. The cultures were initially placed in darkness for 24 hours and then maintained under a 14 hour photoperiod (PAR 20 Wm ^) at 25°C. The essential oil yield of various cultures was determined using direct-volatilisation GLC or steam distillation. 4 RESULTS DEVELOPMENT OF PLANT MATERIAL
To provide a supply of uniform material for experimental examination and the initiation of cell cultures, M piperita plants were grown from cuttings of a single clone and maintained under controlled environmental conditions.
After approximately 7 weeks growth at 25 — 3°C with a 14 hour photoperiod, the plants were approximately 30 cm tall and had developed 15 pairs of leaves. They exhibited strong, erect vegetative growth. Those leaves initially present on cuttings (i.e. Nos 1 and 2) had by this stage started to senesce and in some instances had fallen from the plants. Since these leaves were preformed when cuttings were taken, and were therefore liable to have experienced some degree of metabolic perturbation during their early growth, they were not included in any investigations of monoterpene metabolism.
ISOLATION OF TERPENOID COMPOUNDS
Essential oils comprise a heterogeneous collection of organic molecules covering a great variety of chemical types. It is obvious therefore, that any extraction procedure will be a compromise between maximum total recovery and the optimum recovery of individual constituents. In this study, maximum recovery of total monoterpenes, at their in-vivo concentrations was the primary determinant in the selection and development of suitable techniques. A. 2.1 STEAM DISTILLATION
Steam distillation; using a Likens-Nickerson apparatus modified for microscale work; proved a very effective means of isolating essential oil from samples of M piperita tissue between 0.1 and 10 g in weight.
The time course for recovery of essential oil from an authentic sample of natural peppermint oil and fragmented midstem leaves of M piperita was determined experimentally in order to develop a working method for routine use. Results have been presented graphically (figure 10).
In the early stages of distillation, monoterpene recovery from the plant material was somewhat slower than from free solution. Since both oils were ultimately shown to be qualitatively similar, this was assumed to be due to the structural components of the plant tissue partly restricting the release of volatiles. Additionally, a brief temperature lag, due to the slower heat transfer through plant material may also have contributed.
During the course of distillation, individual monoterpenes were recovered at different rates. Monoterpene hydrocarbons appeared most rapidly in the distillate, whilst those derivatives with higher boiling points and lower vapour pressures such as ketones, alcohols and esters were respectively slower.
A standard distillation time of 60 minutes was selected for the optimum recovery of total monoterpenes from M piperita tissues. Under these conditions recovery of available monoterpenes was shown to be approximately 95% complete (figure 10) and the profile of individual components identical to that of the original oil. Figure 10. The effect of distillation period upon recovery of monoterpenes
Total terpenes recovered from:- x piperita oil in aqueous solution o natural oil from fresh M piperita leaf material (1 g) 4.2.2 DIRECT VOLATILISATION GAS LIQUID CHROMATOGRAPHY
In order to study the distribution of monoterpenes within samples of plant material as small as 1-5 mg, direct- volatilisation GLC was employed.
The most suitable volatilisation period was determined by heating M piperita leaf discs (2 mm diameter) at 200°C within the chromatogram injector unit for periods of between 15 and 180 seconds, followed by immediate withdrawal. The GLC analysis was completed and then the same sample reinjected for a further 120 seconds volatilisation at 200°C. The recovery of monoterpenes obtained by the initial volatilisation was then expressed as a percentage of the combined total for each sample. The results have been presented graphically (figure 11).
Figure 11. THE EFFECT OF DIRECT-VOLATILISATION PERIOD UPON RECOVERY OF MONOTERPENES -95-
Using direct-volatilisation GLC the relative proportions of lower boiling volatiles in plant tissues was always higher than when determining essential oil content by other means. This was believed to be a genuine effect; indicating that the process of steam distillation; even in an efficient Likens-Nickerson apparatus; can cause some degradation and losses of such materials. The essential oil profile obtained m by the direct-volatilisation of midstem leaf discs from M piperita grown under laboratory conditions is presented in figure 12.
The technique of direct-volatilisation GLC was shown to be particularly suitable for the detection of small quantities of volatile substances in situ. Volatilisation for 60 seconds at 200°C gave quantitative release of monoterpenes - approximately 90% of the maximum recovery - whilst still maintaining good resolution of the individual components. It was estimated that the procedure was capable of detecting approximately 1 x 10 ^ g of any individually resolved component. Figure 12. Essential oil profile of midstem leaves, by direct volatilisation GLC
*
TIME(minutes)
The early elution of 2-methyl butan-l-al followed by 2-raethyl butan-l-ol is a feature of the terpene profile of M piperita leaves when examined by direct-volatilisation GLC. These components were only detected in trace amounts when a steam distillate of similar material was examined by GLC. -97-
4.2.3 QUANTIFICATION OF TERPENE GLYCOSIDES
Glycosidic derivatives of monoterpenes are not steam volatile, and hence usually remain "tissue-bound" following the recovery of an essential oil from plant material by steam distillation. The easiest method of quantification for this class of compound is by the preliminary removal of free terpenes by distillation, followed by acid hydrolysis of the plant residue to yield the volatile aglycones of the terpene glycosides. The latter are usually monoterpene alcohols and can be simply quantified following a secondary distillation.
A crude sample of 1-menthyl alpha and beta glucopyranosides was prepared in the laboratory and subjected to acid hydrolysis (3N HC1 pH 0.1) followed by steam-acid distillation in order to assess the stability of menthol under these rigorous conditions. The stability of menthol under acid conditions was verified and results showed that menthol was quantitatively released from its glycoside by acid hydrolysis for 60 minutes at 60°C, followed by a 60 minute steam-acid distillation period.
The effect of various timed periods of acid pretreatment on the release and recovery of bound menthol from fragmented midstem leaves of M piperita was also studied. The results have been presented in figure 13, where it can be seen that an acid pretreatment for 60 minutes at 60°C improved the recovery of bound-menthol from leaf material; over that obtained by direct steam-acid distillation; by almost thirty percent.
Steam distillation of plant material followed by incubation at 60°C for 60 minutes in 3N HC1 and subsequent steam-acid distillation has proven to be an effective means of isolating terpene-glycosides. — 8ure I3*— Ihe effect of acid pretreatment on release and recovery of bound-menthol from midsteam leaves of M piperita 4.3 ANALYSIS OF TERPENOID COMPOUNDS
Gas liquid chromatography was found to be an excellent tool for the separation, characterisation and quantitative analysis of the main constituents of essential oils. However, when investigating complex mixtures using packed column GLC there is always a possibility that some constituents will co-chromatograph on the selected GLC stationary phase. Therefore, prior to the investigation of experimentally derived materials, two commercially distilled peppermint oils were examined in great detail using a combination of more sophisticated techniques.
4.3.1 REFERENCE MATERIALS
Combined gas chromatography - mass spectrometry was used to fractionate and identify the constituents of Yakima and Willamette peppermint oils, whilst high resolution GLC was used to calculate accurate retention indices for each component relative to n-tetradecane, the internal standard used throughout this study. Provisional identification of components was made by electronic comparison with relative retention times obtained previously for known standards. The integrated peak areas were then corrected by relevant FID response factors in order to perform a quantitative analysis.
The detection limit of the GC-MS analysis was calculated to be approximately 1 x 10 ^ g for the identification of a single compound, whilst the high resolution GLC system with Pye DP 101 integrator was able to quantify down to 5 x 10 ^ g of a singly resolved compound. A typical trace obtained for Yakima Peppermint oil using the high resolution GLC system is illustrated in figure 14. O 5 6 A 2 0 5 6 A 2 0 5 6 A 2 0 6 2 A 6 8 10 12 1A 16 15 20 22 2A 26 25 30 32 3A 36 35 AO Analysis fused50silica performedusingcolumnWCOT theSP2100containing stationarym, as phase, temperature60-200°C fromprogrammed at 3°Cper minute. (See for table 3 %composition) FID RESPONSE iue 14.piperita profile (Yakima)ofFigure oil EssentialM resolution byhigh GLC* ME ( nut ) s te u in (m E IM T _ _ to Jus iUU AJ IA J-J j UL
101
Although originating from the same plant material, oils from M piperita, grown in Willamette (Oregon) and Yakima (Washington State) were found to be chemically different. Environmental conditions were considered to be responsible for the main differences, which lay in the relative proportions of 1-menthone, menthofuran and pulegone. The major constituents of the two oil samples are presented in table 3.
Table 3. Characterisation and identification of essential oil components in Willamette and Yakima Peppermint Oils
CONSTITUENT Response Relative Mass Concentration Factor Retention (parts per thousand) Time Willamette Yakima 41 2-methy1-butan-l-al 0.93 0.130 0.2 0.3 2-ethyl furan 0.86 0.144 trace trace 2-methyl butan-J-ol 0.95 0.160 0.1 trace cis-3-hexen-l-ol 0.90 0.238 0.2 trace hexanal 0.92 0.247 trace trace alpha thujene 0.60 0.311 trace trace alpha pinene 0.60 0.321 4.9 5.5 camphene 0.60 0.335 0.1 trace sabinene 0.60 0.363 3.3 4.0 beta pinene 0.60 0.370 6.7 8.1 oct-l-en-3-ol 0.87 0.372 1.6 trace nyrcene 0.60 0.382 1.8 1.6 octan-3-ol 0.88 0.387 2.7 3.0 car-3-ene 0.60 0.401 0.4 trace alpha terpinene 0.60 0.418 3.6 1.9 para-cymene 0.60 0.422 0.7 0.5 limonene 0.60 0.432 10.3 15.2 1-8, cineole 0.83 0.438 51.4 52.6 cis-ocimene 0.60 0.441 1.9 1.9 trans-ocimene 0.60 0.458 0.8 0.7 gamma-terpinene 0.60 0.474 6.3 3.9 sabinene hydrate 0.74 0.483 3.9 9.6 terpinolene 0.60 0.514 1.6 1.0 amy 1-2-methyl butyrate 0.91 0.528 ) linalool 0.91 0.531 ) 4.2 5.4 2-methy1-butyl-isovalerate 0.91 0.536 1.8 0.5 1-menthone 0.83 0.599 276.1 143.7 iso-menthone 0.83 0.612 34.4 20.4 menthofuran 0.81 0.618 4.3 110.0 neo-menthol 0.84 0.627 33.0 20.6 1-menthol 0.84 0.646 422.4 445.1 neo-isomenthol 0.84 0.658 6.4 3.8 isomenthol 0.83 0.666 4.4 1.9 pulegone 0.82 0.719 2.9 23.3 piperitone 0.82 0.742 6.5 4.0 neo-menthyl acetate 0.89 0.789 2.1 2.9 1-menthyl acetate 0.89 0.814 35.2 42.9 isomenthyl acetate 0.89 0.835 1.9 2.3 alpha copaene 0.74 0.950 1.4 trace beta-bourbenene 0.74 0.963 5.4 6.4 caryophyllene 0.74 1.008 19.0 20.7 alpha-humulene 0.74 1.040 0.2 0.3 trans beta farnesene 0.74 1.053 3.0 4.4 germacrene D 0.74 1.090 23.8 23.3 bicyclo germacrene 0.74 1.110 3.9 3.5 delta-cadinene 0.74 1.146 0.7 trace sesquiterpene alcohol 0.8 1.236 1.6 1.9 delta-cadinol 0.8 1.255 2.1 3.0 TOTAL 1000.0 1000.0 -102-
4.3.2 GAS LIQUID CHROMATOGRAPHY - WORKING METHOD
Although offering a less refined means of fractionation than was possible using high resolution capillary chromatography, packed column GLC was routinely employed for the analysis of terpenes isolated from experimental material. The typical resolution obtained is illustrated in figure 15, which for comparison # purposes refers to a sample of the same Yakima peppermint oil as examined by the high performance system (figure 14).
What appear as singly resolved peaks when using packed column GLC, have been shown to be comprised of several closely related molecular types using the more sensitive procedures.
In table 4 the main components of each peak has been listed first, and trace materials indicated in order of decreasing magnitude. In subsequent discussions only the major components have been referred to by name, though this should not be taken as to imply that other materials were not also present in trace amount s.
Although the resolution and hence detection limit was slightly poorer than that obtained using high resolution capillary systems, it was nevertheless possible to detect approximately -9 1 x 10 g of a singly resolved component. This was equivalent to approximately 1 microgram of monoterpene present within the original sample of biological material. FID RESPONSE Chrorasorb (w/w) temperature 10% SP2100, programmed from70-200°Cand W 1.5° perat minute. in from numberedelutionPrimary2.75peaksorderof ra, (id) 3 GLCcolumns packedmm with iue 15. (Yakima) profilepiperita EssentialFigureoil of M byworking method \u fl 13 Q ■w Q U 3
2 TABLE 4
COMPONENTS ISOLATED FROM M PIPERITA ESSENTIAL OIL BY GLC WORKING METHOD
PEAK RELATIVE FID IDENTIFICATION NUMBER RETENTION RESPONSE (GC/MS) INDEX FACTOR
1 0.132 0.93 2-methyl butan-l-al, amyl alcohol 2 0.235 0.60 alpha-pinene 3 0.294 0.60 beta-pinene, sabinene 4 0.315 0.74 octan-3-ol, myrcene 5 0.325 0.60 alpha-terpinene, para-cymene 6 0.373 0.68 1,8-cineole, cis-ocimene, limonene 7 0.405 0.60 trans-o c imene 8 0.429 0.67 sabinene hydrate, gamma-terpinene 9 0.472 0.60 terpinolene 10 0.488 0.91 amy1-2-methyl butyrate, linalool 11 0.525 0.91 2-methyl-butyl-isovalerate 12 0.561 0.83 menthone, isomenthone 13 0.586 0.83 menthofuran, neomenthol 14 0.604 0.84 1-menthol, isomenthol 15 0.704 0.82 pulegone 16 0.729 0.82 piperitone 17 0.777 0.89 neomenthyl acetate 18 0.803 0.89 1-menthyl acetate 19 0.829 0.89 Isomenthyl acetate 20 0.969 0.74 beta-Bourbenene ★ 1.000 0.76 n-tetradecane (internal standard) 21 1.020 0.74 caryophy1lene 22 1.071 0.74 alpha-humulene 23 1.107 0.74 trans beta-farnesene 24 1.129 0.74 germacrene D 25 1.162 0.74 bicyclogerraacrene 26 1.239 0.80 sesquiterpene Alcohol 27 1.257 0.80 delta cadinol 4.4 INTACT PLANT STUDIES
Vegetative plants of M piperita were grown under controlled conditions in order to investigate the distribution, localisation and development of monoterpenes during ontogenesis. Aerial tissues of chamber grown plants were found to contain approximately 37 micromoles of free monoterpenes after 7 weeks growth, corresponding to an average of 1.5 percent of their dry weight. The leaves appeared to be the primary site of monoterpene accumulation, containing over 90% of the total free monoterpenes. Stem tissues contained a lower proportion of monoterpenes; 5-7% of the total; though the oil profile was qualitatively similar to that observed for the leaf fraction. Roots did not appear to contain an essential oil.
Steam-acid distillation revealed the presence of small amounts of menthyl-glycoside in the leaf (2.3 micromoles) and stem (2.7 micromoles) fractions, but not root tissues. Menthyl-glycoside
was found to account for approximately 20% of the total menthol present in leaf tissues, and almost 85% of the total menthol of the stem fraction, lending further support to the hypothesis that it might be a transport/storage metabolite particularly in the latter case.
4.4.1 DEVELOPMENT OF ESSENTIAL OIL DURING ONTOGENESIS
The examination of leaves of different ages revealed a number of changes in monoterpene content which have been related to changes in leaf morphology and organisation. For ease of reference, leaves have been numbered in chronological order of emergence, and their growth monitored in terms of dry weight. Results are presented in table 5, where micromoles of various monoterpenes have been calculated per leaf as the average obtained by the duplicate analysis of 2 leaf pairs. TABLE 5 - DEVELOPMENT OF MAJOR MONOTERPENES IN M PIPERITA LEAVES* (micromoles per leaf **)
Leaf pair No f 15 14 13 12 11 10 9 8 7 6 5 4 3
Dry weight (mg) 4.2 10 25 42.5 50 57.5 65 72 75 71 60 51 36
Pulegone 0.07 0.09 0.23 0.30 0.16 0.17 0.14 0.05 - - - --
Menthofuran 0.07 0.08 0.19 0.24 0.19 0.39 0.26 0.36 0.39 0.22 0.21 0.14 0.08
Menthones 0.13 0.30 0.66 0.99 0.81 1.40 0.95 0.99 0.92 0.53 0.29 0.14 0.04
Menthols - - 0.01 0.07 0.13 0.47 0.46 0.74 1.02 0.74 0.59 0.38 0.12 -106- Menthy1 Acetate —— — — 0.01 0.02 0.02 0.03 0.04 0.03 0.03 0.02 0.01
Others 0.04 0.08 0.18 0.28 0.21 0.41 0.34 0.39 0.42 0.26 0.19 0.12 0.03
Total Monoterpenes 0.31 0.55 1.27 1.88 1.51 2.86 2.17 2.68 2.84 1.78 1.31 0.80 0.28
Bound Menthol - - - 0.01 0.035 0.125 0.115 0.155 0.16 0.155 0.145 0.125 0.115
* Plants grown at 25°C i 3°C, 14 hr photoperiod, 20 Wm“^ PAR ** Data presented are uMoles per leaf, calculated as average obtained by duplicate analysis of 2 leaf pairs f Leaves numbered in chronological order of appearance The accumulation of "free" monoterpenes during leaf development has been documented in figure 16. An initial period of monoterpene accumulation was observed coincident with early leaf growth. With the activation of secondary meristems and laminal expansion (leaves 11-8), the level of monoterpenes was observed to fluctuate, rising gradually to a plateau of some 2.5 micromoles per leaf.
15 14 13 12 11 X) 9 8 7 6 5 4 3
(young) Leaf pair No. (old)
Once the leaf dry weight started to fall so too did the level of monoterpenes. Interestingly the level of menthyl-glycoside was found to fall less rapidly over the same period. This data is presented in figure 19. It was therefore concluded that monoterpenes are not inert secondary metabolites, but undergo biosynthesis and degradation during leaf development. -1 0 8 -
The concentration of monoterpenes in leaf tissue was investigated on a dry weight basis. Young leaves were shown to contain the highest concentration of monoterpenes, since as they developed, overall dry weight gain exceeded the rate of oil accumulation, leading to an effective fall in monoterpene concentration. With the activation of secondary meristems there was a temporary increase in monoterpene concentration which subsequently declined concomitant with the overall decrease in leaf dry weight. The data is summarised in figure 17.
Figure 17. Concentration of monoterpenes during leaf development
The high concentration of monoterpenes in young leaf tissues was taken to indicate that terpenoid metabolism was particularly active prior to leaf expansion. Unlike most secondary products therefore, monoterpene biosynthesis did not appear to be restricted to mature tissues which had completed their primary growth. Mole 7. major monoterpenes uigla eeomn i ouetd infigure 18. isleafdocumented duringdevelopment individualthetheinof proportionsVariationmain monoterpenes ohyugadod tissues.andoldboth young the levelsimilarofatimescale decreasedover whilst menthofuran proportion of menthol as the leaves matured. The pulegone content Thepulegone leaves as theofproportionmatured. menthol Drama ticoccurredchanges inthepiperita ofprofile M monoterpene iue 8 Vraini ao ooepnsdrn leafin 18.duringdevelopment VariationFigure monoterpenes major hc i unrlt t aiu hss f leaf ofdevelopment.relate in tophasesturn various which hsooia rdcino etoeadacnoiatrs i the in reductionconcomitantphysiologicalrisea andof menthone eandcntn i ise evs rsn t ihrlvl in to risinglevels inleaves,remainedhigher constantmidstem isnhss itrovrinaddgaain adta thesethat and interconversionanddegradation, biosynthesis, evsdrn hi eeomn. ot infcn o hs a a was significantofthese Most leavestheirduringdevelopment. changes can be related to gross variations in monoterpene content, in togrossvariations relatedmonoterpene canchanges be t ws ocue ta individual thatundergo,Itmonoterpenes concluded was yu e (old) (youne) Leaf pairNo. 109- 9 0 -1
r
-110-
Menthyl - Glycosides
Tissue-bound menthy1-glycosides were isolated from leaf pairs by steam-acid distillation, and quantified by GLC. Accounting for approximately 5% of the combined total of free and bound monoterpenes, the level was observed to increase from young leaves to a plateau of some 0.16 micromoles per midstem leaf. There was a slight decrease observed in older leaves, though this was not so rapid as the overall decrease in free menthol or the fall in total terpenes of these leaves.
Figure 19. Variation in "Free" and "Bound" menthol during leaf development
15 14 13 12 11 10 9 8 7 6 5 4 3 (young) (old) Leaf pair No. -111-
Menthyl glycoside was considered to represent a minor constituent of M piperita leaf tissues, though its relatively high concentration in stem material did suggest a possible role for this compound within the wider metabolism of the intact plant. The formation of menthy1-glycoside is hypothesised as being partly responsible for the decrease in free essential oil observed in older leaf tissue. However, closer investigation of the temporal accumulation of the glycoside in stem sections and its subsequent metabolism would be required to reveal what proportion of total menthol is actually translocated or metabolised via this route.
4.4.2 LEAF SURFACE TOPOGRAPHY - SCANNING ELECTRON MICROSCOPY
The surface topography of M piperita leaves at various stages of development were examined by scanning electron microscopy. Plate 3 illustrates the typical appearance of a young leaf. Three distinct types of epidermal appendages were identified and their distribution documented. Similar glandular structures were present on shoot tissues but in reduced numbers.
They have been termed
1. Papillate hairs These simple hairs were primarily found overlying veinal tissue. Constituent cells were initially vacuolate, but were frequently collapsed in mature tissues.
2. Glandular Hairs These structures comprised a single secretory head cell borne upon a single-celled stalk. They were widely distributed but were particularly common overlying veinal tissue. -112-
3. Glandular Trichomes These most complex epidermal structures comprised eight secretory cells surmounted upon a unicellular stalk. They appeared to be evenly distributed over the interveinal leaf lamina, strongly suggesting their origin from endogenous meristemoids during leaf development.
The density of glandular appendages on young leaves was similar for both upper and lower epidermis. However, reference to more mature leaves revealled that gland initials do not all develop at the same rate. Whilst many glandular trichomes reached 80-90 micrometers diameter on midstem leaves, some had not expanded above 25 micrometers. This is consistent with the observations of Lemli (1955a) who also reported a gradual "filling" of glandular trichomes during leaf development. -113-
PLATE 3. LOWER EPIDERMIS OF M PIPERITA LEAF ILLUSTRATING VARIETY OF EPIDERMAL APPENDAGES (Scale xl20)
#
Sample viewed by scanning electron microscopy. Glandular trichomes appear as globose appendages comprising eight secretory cells. Glandular hairs ; with a single , terminal secretory cell; are particularly numerous overlying veinal tissues. 4.4.3 LOCALISATION OF ESSENTIAL OIL
I) Leaves of M piperita plants have been shown to bear epidermal glands from an early stage. The contents of large glandular trichomes were isolated using glass capillaries and then examined for the presence of monoterpenes by direct- volatilisation GLC. Comparison was made with the terpene profile of complete leaf discs from the same material.
The analysis revealed that glandular trichomes contained an essential oil which was qualitatively quite similar to that of the intact leaf at the same developmental stage (leaf number 7). Leaf discs, however, exhibited relatively high levels of 2-methyl butan-l-al, 2-methyl butan-l-ol and the lower terpene hydrocarbons alpha pinene, beta pinene and alpha terpinene. In addition they contained menthyl acetate, which was only detected in trace amounts in the isolated glands.
TABLE 6. Main Terpenoids of isolated glands and leaf tissue identified by direct-volatilisation GLC
Constituent Mass Concentration (parts per thousand)
Leaf Discs Isolated Glandular Trichomes
2-methyl butan-l-al 81.6 trace 2-methyl butan-l-ol 43.5 trace alpha-pinene 10.7 8.4 beta-pinene 16.3 14.9 alpha-1e rpinene 7.2 2.1 1,8-cineole 54.3 68.0 sabinene hydrate 22.5 25.8 menthone 207.9 235.1 menthofuran 148.0 197.5 menthol 316.2 362.7 pulegone 45.6 52.3 menthyl acetate 15.3 trace caryophyllene 14.9 15. 1 trans beta farnesene 7.4 8.5 B icyclogermacrene 8.6 9.6
Total Constituents 1000.0 1000.0 It was estimated that individual glandular trichomes contained 3 x 10 ^ moles, or 0.05 micrograms; of essential oil. However, there was considerable variation between individual glands, and the problem of sample variability proved to limit the usefulness of the technique for the preparation of quantitative data. For that reason, a more effective means was required for the collection and analysis of a large number of glands.
II) The isolation of intact epidermis from leaves of M piperita proved very difficult. Using a scalpel and forceps only small fragments were obtained, and these frequently included patches of photosynthetic tissue adhering to the underside. However, the direct volatilisation of M piperita epidermis isolated from young stem tissue revealed the presence of a large number of the monoterpene compounds known to be constituents of the essential oil of the intact plant (figure 20). Using this technique the epidermal oil yield was estimated at 0.25% on a dry weight basis.
Analysis of cells from the underlying mesophyll and parenchyma revealed a very different profile (see table 7) with the overall yield of fewer monoterpene components, amounting to only 0.01% on a dry weight basis. Mesophyll tissues also contained high levels of 2-methyl butan-l-al and 2-methyl butan-l-ol, both structurally related to the terpene "primer" compound; dimethylally1 alcohol (3-methyl but-2-ene-l-ol). Of the monoterpenes; only alpha terpinene, 1-menthol and menthyl acetate were detected in significant quantities in mesophyll tissues. #
FIGURE 20. Comparison of volatiles recovered from M piperita stem epidermis and internal tissues by direct-volatilisation GLC -116- Table 7. Main terpenoids of stem epidermis and internal tissues identified by direct-volatilisation GLC.
Peak No Constituent micromoles per gram (dry weight)
Epidermis Internal
5-Carbon Compounds 1 2-methyl butan-l-al 0.10 1.94 2 2-methyl butan-l-ol 0.17 0.97
Monoterpenes 3 alpha-pinene 0.15 - 4 beta-pinene 0.27 - 5 alpha-terpinene 0.04 0.10 6 1,8-cineole 1.10 - 7 sabinene hydrate 0.42 - 8 menthone 3.75 - 9 menthofuran 3.27 - 10 menthol 5.78 2.37 11 pulegone 0.86 - 12 menthyl acetate 0.01 1.16 13 caryophyllene trace - 14 trans beta farnesene trace - 15 Bicyclogermacrene trace 0.10
TOTAL MONOTERPENES 0.25 0.01 (% dry weight)
Analysis of epidermal and internal tissues revealed that monoterpenes were primarily accumulated in the epidermis, where the profile of components quite closely reflected the composition of the whole-plant essential oil. However, the relatively high concentrations of alpha-terpinene and menthyl acetate in the internal tissues suggested that these may be the primary site for the accumulation of these particular components. The latter time also contains relatively high levels of the 5-carbon compounds 2-methyl butan-l-al and 2-methyl butan-l-ol, both structurally related to known terpenoid precursors. Ill) Scanning electron microscopy showed that epidermal oil glands were successfully removed from M piperita leaves by the use of PVA films (see plates 4&5). Large numbers of the smaller glandular hairs, however, remained attached to the leaf after the PVA treatment. With the removal of the PVA film, glandular trichomes were usually severed at the junction of the stalk and secretory cells, the latter remaining within the PVA film. The essential oil content of each PVA films and the control (untreated) leaf of the specified leaf pairs was subsequently determined by steam distillation.
Recovery of essential oil by direct distillation of the PVA films did not prove to be a practical means of study. At elevated temperatures the PVA caused foaming within the distillation apparatus, breaking down the physical compartmentation of liquid and vapour phases required for its effective use- However, distillation of treated leaves and untreated controls of the same developmental age provided the following results (table 8).
Table 8. The effect of PVA treatment on recovery of essential oil from leaf tissues of M piperita*
Untreated PVA Treated % Essential Control Leaf Oil in PVA Film
Essential Oil 2.85 0.37 87 (micrograms)
* average determination from duplicate analysis of 6 leaf pairs -119-
Plate 4. Upper epidermis of M piperita leaf showing distribution of oil glands (Scale x60)
Plate 5. Upper epidermis of equivalent leaf after PVA treatment (Scale x60) -120-
Plate 6. Lower epidermis of M piperita leaf showing distribution of oil glands (Scale x 60)
Plate 7. Lower epidermis of equivalent leaf after PVA treatment (Scale x 60) -121-
Almost 90% of the essential oil normally associated with M piperita leaves was removed with PVA films.
The preparation of PVA films from the surface of M piperita leaves facilitated the isolation of large numbers of glandular trichomes. Analysis revealed that almost 90% of the total leaf essential oil was removed with PVA films and that the components present were similar to those obtained by the direct-volatilisation of epidermal tissues and individual oil glands.
All characteristic components of the essential oil were detected in epidermal tissues and glandular trichomes, though the levels of 2-methyl butan-l-ol, 2-methyl butan-l-al, alpha and beta pinene, alpha-terpinene and menthyl acetate were proportionately lower than observed for samples of the intact leaf.
IV) PVA solution was applied to a number of young leaves of M piperita, allowed to dry and then peeled off to remove the glandular trichomes. As in previous cases, the initial recovery of leaf essential oil was approximately 90% complete. Leaves were then left attached to the parent plant for a further 18 days growth, prior to steam and steam-acid distillation for the recovery of free and bound monoterpenes respectively. The removal of glandular trichomes from M piperita leaves by PVA treatment appeared to cause a perturbation of normal leaf metabolism, such that laminal expansion and dry weight gain were both restricted.
Analysis showed that during this period there was no further increase in the essential oil content of the PVA treated leaves, whilst the untreated controls continued to accumulate monoterpenes. It was also noted that although PVA-treated leaves; remained green and apparently healthy; they did not follow a normal course of growth and expansion. Leaves which had been only partly treated with PVA and the film left in situ did not appear to suffer any of these effects.
The following explanations are proposed to account for the observations
1. The removal of glandular trichomes might cause water stress as a result of excessive transpiration via the broken stalk cells. Under such conditions leaves would not grow and expand normally.
2. Monoterpenes are largely accumulated in glandular trichomes at a rate closely reflecting the dry weight gain of young leaves. Therefore, the lack of monoterpene accumulation observed in the absence of glandular trichomes may be due to either
i) intimate coupling of monoterpene biosynthesis with the pathways responsible for dry weight gain or leaf expansion (i.e. no growth then no monoterpenes). -123-
ii) monoterpene biosynthesis being physically restricted to glandular tissues within the epidermis (i.e. no glands then no monoterpene biosynthesis).
iii) the requirement for an isolated compartment for monoterpene accumulation in order to avoid feedback repression of the enzymes of monoterpene biosynthesis.
In order to investigate the possible role of glandular trichomes in monoterpene metabolism, specimens were examined by light and electron microscopy. The development of glands and their relationship with adjacent tissues was documented.
4.4.4 OIL GLAND ULTRASTRUCTURE & DEVELOPMENT
Light microscopy
Glandular trichomes were visible arising from the epidermis of very young leaves once they had reached an axial length of approximately 0.5 mm. Even at this early stage a highly organised leaf anatoiny was associated with glandular trichomes.
Observations of young tissues by light microscopy suggested that the development of glandular trichomes commenced with the vertical enlargement of an epidermal initial. This would then divide periclinally into a basal cell; which would remain within the epidermal plane; and a daughter cell; erect from the plant surface. A subsequent periclinal division of the latter would lead to the formation of a head and stalk cell. The head cell would then divide anticlinally into first two, then four and finally eight secretory cells in the mature glandular trichome. Plate 8 illustrates the arrangement of a glandular trichome in relation to the surrounding tissues of a young leaf.
Plate 8. Section of young leaf of M piperita illustrating glandular trichome and surrounding tissues (Scale x350)
Glandular trichomes appeared to be tightly appressed to the leaf, lying within small depressions on the surface. The connecting stalk cell was usually compact and firmly bound to a large basal cell, which was itself embedded within the epidermal plane. Mesophyll cells in the region of the basal cell were densely packed, with few visible intercellular air spaces. These features suggested the existence of close metabolic coupling between the underlying photosynthetic cells and the aerial gland complex. Vital Staining
Cells and tissues were characterised by the use of toluidine blue, although the stain was unable to penetrate the cuticle of M piperita leaf sections. The most useful material proved to be small fragments of leaf epidermis and mesophyll, excised and stained for 20 minutes immediately prior to examination. Observations have been summarised in table 9.
Table 9. Differential staining of M piperita tissues by toluidine blue
Tissue Colour Reaction
epidermal cells blue-green guard cells unstained subsiduary cells unstained glandular hairs blue-green glandular trichomes purple-violet leaf mesophyll purple-violet
These results indicate that; on histochemical grounds; glandular hairs appeared to be closely related to other epidermal cell types, whilst trichomes tend to reflect the status of mesophyll tissues. The relationships existing between glandular structures and adjacent tissues was investigated in more detail using transmission electron microscopy.
Transmission electron microscopy
The ultrastructural organisation of glandular hairs and trichomes was examined using transmission electron microscopy -126-
All component cells of the glands were found to contain a prominent nucleus and a complement of darkly staining plastids which appeared to lack well-developed thylakoids. During their early development these plastids frequently contained paracrystalline inclusions - a feature common to many epidermal cell plastids. The external surface of glandular hairs and trichomes was usually covered by an extensively thickened cuticle from an early stage in their development. In fully developed glands the cuticle of trichomes reached a thickness of some 350 nanometers and for hairs approximately 250 nanometers.
The following characteristics were observed as common features of both glandular hairs and trichomes in M piperita.
Stalk Cell
This cell mediates the supply of metabolites into the secretory cells and as such appeared classically modified to effect short range metabolic transport. The cross-walls adjacent to the secretory and basal cell were perforated by numerous plasmodesmata; considered capable of the relatively specific transport of salts, sugars, amino acids and organic acids (Carr 1976); whilst the external wall was heavily adcrusted with a complex mixture of cutin and suberin. The latter, coupled with the intimate association of cuticle and cell wall at this point, would effectively restrict the apoplastic flow of water-soluble materials via the cell wall free space of hydrated cells. Within the peripheral cytoplasm groups of microtubules were negatively stained, and appeared to be aligned concentrically around the cell. In addition to performing a structural role by determining the orientation of cellulose microfibrils within the cell wall it is proposed that such a shield of microtubules would inhibit the fusion of vesicles with the plasma membrane at the outer cell faces, and thereby impose some degree of polarity to the cellular transport processes. Examination of a large number of glandular hairs and trichomes suggested that a viable stalk cell was required for the extensive accumulation of terpenoids in the secretory cells, and these may therefore play an important role in determining overall oil yield.
Secretory Cells
The secretory cells appeared to be the primary site of monoterpene accumulation in the plant. The secretory cells of young hairs and trichomes contained a dense cytoplasm, including closely appressed sheets of membranes and numerous ribosomes; both free and associated with long strands of endoplasmic reticulum. The cells contained large prominent nuclei whilst small fragmented vacuoles were observed in the peripheral cytoplasm. During their development the plasma membrane of secretory cells was seen to invaginate, either as a consequence of physiological activity, reduced cell turgor, or as an artifact arising during sample preparation. Since it was observed in several separately prepared samples and had previously been reported, it was considered to be a genuine feature of the tissue.
Diffuse globules of darkly staining, osmiophilic material were frequently observed around the extracellular face of the plasma membrane, usually at points where the endoplasmic reticulum and plasma membrane appeared to be intimately associated. The subsequent development of an extraplasmatic space; between the cuticle and the cell wall of secretory cells was frequently observed in both glandular hairs and trichomes. Closer examination revealed that the split usually occurred between the pectic and cellulosic layers of the secretory cell wall (see plate 11). Such extracellular compartments were generally seen to contain a homogenous osmiophilic material, presumed to be the essential oil which is known to be associated with these cells. It was concluded that the membranes within and delimiting the cells of oil glands were likely to play an important role in the biosynthesis,transport and accumulation of terpenoid metabolites.
Glandular Hairs
Plate 9 represents a typical glandular hair at an early stage of development, illustrating the prominent nuclei and well- developed cuticle. The accumulation of essential oil within the secretory cell and the extraplasmatic space, along with an abundance of membranes and ribosomes can be clearly seen. It was considered that the presence of essential oil droplets within the cell may have been responsible for the compaction of the remaining cytoplasm, since examination of similar tissues prior to oil formation showed that the secretory cell volume was largely occupied by a tubular network of smooth endoplasmic reticulum and prominent dictyosomes. The latter was interspersed with infrequent sheets of rough endoplasmic reticulum and free ribosomes. (see plate 10)
Whilst plasmodesmata were seen in large numbers between the stalk and adjacent cells, these were not detected between the basal cell and adjacent underlying parenchyma. This supported the results of vital staining which showed that glandular hairs exhibited similar physiological staining to other epidermal cells, but differed from mesophyll tissues. -129-
Plate 9. Longditudinal section of young glandular hair viewed by transmision electron microscopy (Scale x8,845)
extraplasmatic space containing secreted materials
osmiophilic materials prior to secretion
cross-walls of stalk impregnated with suberin
prominent nucleus
plasmodesmata
basal cell within epidermal plane
/ Plate 10. Detail of secretory cell and cell wall organisation in glandular hair of M piperita (Scale x22,020)
The cytoplasm of the terminal secretory cell is based upon an elaborate network of endoplasmic reticulum and Golgi cisternae. The darkly staining plastid appears to be characteristic of glandular structures in M piperita , as does extensive membrane invagination at the cell wall region. -1 3 1 -
Glandular Trichomes
The general features of this type of gland have been summarised in a schematic diagram, (figure 21) itself compiled from a large number of electron micrographs. Particular features to note include the adcrustation of the stalk cell which extends deep within the tangential cross-walls, and the additional presence of plasmodesmata linking the basal cell with underlying mesophyll tissues. This symplasmic continuity has physiological implications which may affect monoterpene biosynthesis, and is a fundamental difference between glandular trichomes and hairs. In addition, vital staining has indicated that glandular trichomes exhibit a physiological status similar to mesophyll, rather than epidermal tissues.
The intimate association between glandular trichomes and underlying mesophyll; particularly the strengthening of the shared tangential cross wall; is also thought to be responsible for the difficulties in isolating intact epidermis from leaf tissues (section 4.4.3).
Plate 11 illustrates the fine structure of a secretory cell, and shows quite clearly, the formation of an extraplasmatic cavity, the split occuring between the darker staining pectic layer and the fibrous cellulosic layers of the cell wall. In general, the secretory cells of trichomes were less densely packed with sheets of membrane than was observed for glandular hairs. -132-
Figure 21. Schematic Representation of M piperita glandular trichome*
* based upon various observations by light and electron microscopy. -133-
Plate 11. Detail of secretory cell, cell wall organisation and formation of extraplasmatic cavity in glandular trichome of M piperita (Scale x30,580)
The formation of an extraplasmatic cavity is ascribed to the separation of the cellulose layersof the cell wall, from the outer , wax-based cuticle. Cutin and pectins are restricted to the darkly staining separation zone, and together with the wax , may reach a thickness of 350 nm. Note the presence ofthe characteristic plastid-type, and prominent nucleus. -134-
4.4.5 ULTRASTRUCTURAL LOCALISATION OF BETA-D-GLUCOSIDASE
Small quantities of menthyl glucoside were detected in mature, vegetative tissues of M piperita. Its role in monoterpene metabolism is not fully understood, though similar derivatives have been reported by various authors (Croteau & Martinkus 1979, Sakata & Mitsui 1975, Sakata & Koshimizu 1978) and frequently implicated as transport metabolites (Francis & O'Connell 1969, Croteau & Martinkus 1979, Pogorel's Kaya et al 1979). If this were to be so, then a high glucosidase activity might be expected at the site where free menthol is accumulated - viz glandular trichomes.
Sections of young shoots and leaves were simultaneously incubated with naphthol ASBI beta-D-glucopyranoside; as a cytochemical substrate for beta-glucosidase and freshly diazotised para-acetoxymercuric aniline as the coupling agent. In order to assess the specificity of the technique, controls were also examined in which:-
I) Endogenous enzyme activity was inactivated by heating. II) No cytochemical substrate was supplied. III) The substrate was chemically hydrolysed prior to incubation.
No staining was apparent in the experimental sections, or any controls other than those including previously hydrolysed substrate. In the latter case, diffuse vacuolar staining was observed in mesophyll and parenchyma tissues, whilst palisade, epidermis and epidermal glands remained relatively unstained.
Penetration of the incubation medium into samples appeared to be non-uniform, and limited to those tissues with a relatively "open" structure. In experimental sections, the release of phenolics from damaged cells may also have reduced endogenous enzyme activity. Therefore, whilst the technical validity of the procedure was established in terms of substrate and stain coupling; its practical use with M piperita tissues failed to convincingly localise enzyme activity because of unreliable penetration of certain tissues. The technique therefore failed to clarify in any more detail the possible role of menthyl-glucoside as a transport metabolite or precursor of "free" menthol.
4.3 PLANT TISSUE AND CELL CULTURE STUDIES
4.3.1 GROWTH KINETICS
In order to select a suitable medium for the growth of M piperita cells in culture, several defined media were prepared including various plant growth hormones. Their ability to support the initiation and development of M piperita calli was quantified. Suspension cultures were subsequently initiated in a liquid version of the medium selected for routine callus growth. Their growth cycle was documented in order to provide uniform cellular material with defined qualities for experimental use (figures 22 & 24).
4.3.1.1 Callus Cultures
Although originally devised as a defined medium for the culture of tobacco cells, Murashige & Skoog (M&S) medium, supplmented with 3% sucrose, 1 mg 1 ^2,4-D and 0.1 rag 1 ^BAP proved to be a most suitable medium for M piperita callus growth, as judged by dry weight gain. There were only marginal differences in growth observed after changing the inorganic fraction of the medium to B5 formulation, though qualitative and quantitative hormone changes had a more pronounced effect. 2,4-D and BAP sustained good growth, whilst both IAA and FAP failed to stimulate lasting cell divisions in cultures. Data pertaining to the callus growth obtained from shoot explants on M&S medium with various combinations of 2,4-D and BAP have been presented graphically. Figure 22 illustrates the observed dry weight after 40 days of primary culture, whilst representative samples of the experimental material are illustrated in plate 12. Callus growth rate was slow, reaching approximately 1.5 mg dry weight gain per day on the medium selected for growth.
Plate 12. The effect of 2,4-D and BAP upon callus growth from M piperita shoot explants -1 3 7 -
Figure 22. The effect of 2,4-D and BAP upon initiation and dry weight gain of M piperita callus -138-
Suspension cultures
Actively growing suspension cultures were maintained successfully on the same medium as selected for callus cultures. The cell growth rate was however considerably improved; with dry weight gain averaging approximately 45 mg day ^ during the 21-day culture period.
The growth cycle of cells used for experimental purposes was determined as a function of cell density and medium conductivity. After an initial lag period of approximately 70 hours, growth was rapid; with the cell density doubling approximately every 80 hrs. Dry weight gain reached a plateau after 18 days of culture, with senescence and the concomitant release of vacuolar phenolics considered responsible for the gradual fall observed after 22 days. The results have been presented graphically in figures 23 and 24 respectively.
Monitoring the conductivity of the growth medium provided an alternative index of culture growth, (see fig 24). During the growth cycle the conductivity fell to approximately 50% of its starting value as inorganic nutrients were taken up. Over the same period, the pH of the medium fell from 5.7 to 4.6, suggesting the preferential utilisation of ammonium as opposed to nitrate, as the primary nitrogen source. The gradual increase in medium conductivity which occurred after 20 days was taken to indicate the leakage of ionic metabolites from cells as they underwent lysis.
During the culture period the material was comprised of unpigmented free cells and clusters, mixed with chlorophyllous cell nodules. However, the proportion of compact, chlorophyllous nodules appeared to gradually decrease after prolonged culture (i.e. 8 or more subcultures). Figure 2i. Growth cycle (cell density, mg ml dry weight) of M piperita suspension cultures
confidence limits established for p 53 0.05 #
Figure 2k, Growth cycle (conduct:ivity, tnftlhos) of M piperita suspension cultures -on-
confidence limits established for p30.05 CELL DENSITY (mg dry weight mf^) ofcell (w/v)tothegrowth 2%promoteappear at not did medium xeietlcniin eecrflyslce t nue that toselectedensureconditionscarefully were Experimental Figure 25. The effect of PVP upon dry weight gain ofpiperita gain 25.dryM PVPweighteffect ofupon The Figure h nlso fplvnlproioe (PVP)the ingrowthinclusionof pyrrolidonepolyvinylThe purposes.experimental for 2-6subcultures from ofonly,thecellsanduse both the morphology and metabolic status of cell tissues statusofwerethecellandboth metabolic morphology bevtoso h rwhcceo ieiacl suspension thecellonof piperitaobservations growthcycle M utrs tog tdd pert rln theirtoit appearviability thoughprolong cultures, did autocatalytic senescence, and might be a major factor limiting factora senescence,and be major autocatalyticmight cultures led to the selection of a 14-day subculture period,14-day subculture selectiona ledoftheto cultures the viability of M piperita cell cultures under commercialculturescell theunder piperitaof viability M ossetdrn h oreo h suy Preliminary study.thethecourseduringofconsistent 12-| indirectly, perhaps as a result of causing a slightdecreaseofresultcausinga asa indirectly, perhaps leaching of phenolics from a few cause fromagedcellsleachinga of phenolics might ngot ae fgr 2) Hwvr i a cnldd that it the concluded However, was (figure 25).inrategrowth LU LH O Q3 ''O O fermentationconditions. T 2 suspensioncultures i 10-12 2 1 - 0 1 8 CULTURE PERIOD PERIOD CULTURE --- 1 --- [ -141- --- % 1 --- 6 8 0 22 20 18 16 (days) 1 --- 1 --- 1 --- 1 --- 2k 1 --- 6 28 26 1 ---
r
r ■ -r
30
Control PVP
-142-
4.3.2 BASAL TERPENOID METABOLISM
Callus and suspension cultures did not produce the essential oil characteristic of the parent plant. However, a number of volatile compounds were isolated by direct-volatilisation GLC and steam distillation. These were identified by GLC as; alpha terpinene, 1-menthol and trace amounts of menthyl acetate. In addition, large quantities of the 5-carbon compounds 2-methyl butan-l-ol, 2-methyl butan-l-al; which are closely related to the terpene precursors DMAPP and IPP; were routinely detected in cell cultures.
Callus Cultures
There was no obvious peak period of monoterpene accumulation during the growth of callus cultures. The typical monoterpene concentration for cells throughout their viable growth period was estimated to be 0.03% of dry weight.
Suspension Cultures
Volatile compounds were present at all stages in the growth of suspension cultures, but were accumulated at the highest levels when growth was most rapid (day 10 sample). At this point monoterpenes accounted for approximately 0.15% of cell dry weight.
Results are presented in table 10. Table 10. Basal terpenoid metabolism of M piperita suspension cultures during the growth cycle
Days of Subculture Monoterpene Content * (% of dry weight)
2 0.10 - 0.01 10 0.15 - 0.01 18 0.12 - 0.01 24 0.08 - 0.02 * confidence limits calculated for p = 0.05
Callus and suspension cultures of M piperita were capable of producing and accumulating low levels of monoterpenes, though not in the same quantity or variety as observed in fully developed plants. Relatively large quantities of 2-methyl butan-l-ol and 2-methyl butan-l-al were produced, though it was not determined whether their presence was indicative of substrate degradation or as a result of poor metabolic coupling of such precursors into the monoterpene pathway.
On the basis of these results and those previously recorded for young leaves (Sect 4.4.1) it is hypothesised that monoterpene precursors are produced as a result of growth-related processes. If these materials are the same as; and accessible to; those functioning within the pathways of higher terpenoid biosynthesis then the precursor pool will be regulated by the demands for intermediates at various levels. However, if as seems to be the case; monoterpene biosynthesis is compartmentalised and regulated by its own negative feedback systems, the concentration of precursors may be regulated by degradation and/or release of volatile derivatives from plant tissues.
This is proposed as a simple mechanism by which plants; and particularly undifferentiated cell cultures; might regulate their terpenoid metabolism. This hypothesis is also supported by the fact that lower levels of the volatile precursors were found in cells from the later stages of the growth cycle. -144-
4.3.3 ENVIRONMENTAL EFFECTS
i) Effect of cell culture conditions
To establish whether the conditions employed for plant cell culture growth would support monoterpene biosynthesis per se, plantlets were regenerated from nodal sections of chamber-grown M piperita plants. Regeneration from sections placed on a medium lacking sucrose was infrequent and therefore the latter was routinely included in the medium at 3% (w/v). Regenerated plantlets produced an essential oil yield only slightly less than that determined for chamber-grown plants on a dry weight basis.
% However, plantlets developing within sealed culture flasks exhibited weak growth and bore small leaves, characteristic of immature foliage. Another feature of flask grown plantlets was the early necrosis and browning of the stalk cell in glandular trichomes. Collapsed secretory cells were frequently observed, containing only small quantities of oil even after several weeks growth in culture.
Free and bound terpenes were recovered from the plant tissues by steam and steam-acid distillation respectively. Quantitative results have been presented in table 11.
Table 11. The effect of plant cell culture conditions on the essential oil yield of regenerated M piperita plantlets*
EXPLANT LIGHT INTENSITY MONOTERPENES MOLE PERCENTAGE LOCATION (Win”2 PAR) (% DRY WEIGHT) PULEGONE
Flask 1.6 1.4 17 Flask 20.0 1.2 18
Chamber Control 20.0 1.5 8
* Average of triplicate analysis for combined sample from 10 plantlets The highest oil yield was obtained from plantlets regenerated in the open growth chamber used for the maintenance of stock plants of M piperita. These plantlets exhibited sturdy growth and bore well-developed, dark green leaves. ii) Requirement for Light
Light intensity was shown to affect both quantitative and qualitative aspects of the monoterpene profile of cultured M piperita explants (table 12). Light did not appear to be an absolute prerequisite for monoterpene biosynthesis, though even at relatively low levels it did appear to favourably affect the yield.
Table 12. The effect of light intensity upon the essential oil yield of M piperita plantlets regenerated under plant cell culture conditions*
EXPLANT LIGHT INTENSITY MONOTERPENES MOLE PERCENTAGE LOCATION (Wm-2 PAR) (% DRY WEIGHT) PULEGONE
Flask Darkness 0.6 30
Flask 1.6 1.4 17
Flask 20.0 1.2 18
Chamber (control) 20.0 1.5 8
* Average of triplicate analysis for combined sample from 10 plantlets.
Plantlets maintained in darkness were found to contain only 42% of the essential oil observed for plantlets grown under low _2 intensity light (1.6 Wm PAR). Increasing the light intensity -2 . . . further to 20 Wm however, resulted m a slight decrease in oil content of some 15%. -146-
The oils obtained from all sets of light-grown plants were qualitatively similar, but dark grown material exhibited a markedly different profile, containing high levels of pulegone and only a trace of alpha terpinene (table 13).
Table 13. Monoterpene composition of essential oils from light and dark grown M piperita plantlets*
Essential Oil % Composition Components Dark Light
alpha pinene 1.0 1.0 beta pinene 1.3 1.2 alpha terpinene trace 0.3 1,8 cineole 3.3 2.2 1-menthone 19.8 12.2 menthofuran 31.2 38.0 1-menthol 9.6 25.8 pulegone 33.0 18.0 1-menthyl acetate 0.8 0.6 caryophyllene trace 0.4 trans-beta-farnesene trace 0.3
100.0 100.0
* Average of triplicate analysis of combined sample from 10 plantlets
During the earlier examination of variously aged leaves, (Sect 4.4.1) it was noted that the mole percentage composition of certain essential oil components was a useful guide as to the origin of the oil within the plant. Using pulegone as an indicator compound, the essential oil quality of plantlets grown in culture flasks was found to correspond to the juvenile foliage of chamber-grown plants. In dark grown plantlets, pulegone appeared to be accumulated as the end-product of monoterpene biosynthesis. -147-
The predominance of essential oil accumulation in leaf tissue has been illustrated (section 4.4), and therefore, it was appreciated that the leaf-to-shoot ratio has a marked effect on the oil yield of a complete plant. Flask-grown plantlets tended to have a relatively low ratio, (i.e. small, unexpanded leaves) accounting at least in part for the observed oil yield. It is also hypothesised that the micro environment within closed culture systems - particularly high humidity and balance may have contributed to the slight shortfall in oil yield of flask-grown plantlets when compared with similar chamber grown material.
It was concluded that the conditions employed for the growth of cell cultures supported monoterpene biosynthesis in plantlets regenerated from nodal explants. However, the conditions promoted the formation of slightly reduced quantities of an "immature" type of oil.
Light did not appear to be an absolute prerequisite for monoterpene synthesis, though its presence appeared to have both direct and indirect effects on essential oil accumulation. Biosynthesis appeared to be increased per se, and there was an increase in the leaf to shoot ratio, which would also contribute to the overall increase in essential oil content.
It is significant to note that monoterpene biosynthesis is not a light-dependent process, since the provision of lighting and additional cooling for large scale fermenters could otherwise represent a significant barrier to achieving a commercially viable means of supporting monoterpene biosynthesis in plant cell cultures. -148-
4.3.4 INHIBITION OF PRIMARY GROWTH
Many secondary products occur in nature only after an organisms primary growth has ceased. Whilst the evidence presented earlier in this study suggests that monoterpenes are produced by young, growing tissues; it has also been shown that individual oil glands are fully developed in advance of other leaf tissues. It is possible, therefore, that the primary growth of oil gland cells may well have ceased prior to monoterpene accumulation.
The growth of M piperita cells was partially reduced when resuspended in regulator-free M&S medium. The average dry weight gain of five replicate cultures was documented and is illustrated graphically in figure 26.
The primary growth of M piperita suspension cultures was also -3 -4 -5 variously reduced by the inclusion of 10 , 10 and 10 molar colchicine or 5 mg 1 ^ gibberellic acid (GA-) in the J -3 culture medium. Chlorocholine chloride (CCC) at 10 molar concentration was used to specifically inhibit the latter stages of higher terpenoid biosynthesis thereby hopefully freeing substrates for monoterpene biosynthesis. The primary growth of cultures was decreased by CCC, presumably as a result of the shortage of higher terpenoids affecting membrane biosynthesis and functionality.
The effect of colchicine, CCC and GA^ on the dry weight gain of cell suspension cultures is illustrated graphically in figure 26.
It was considered that in the presence of an adequate carbohydrate supply, limited primary growth might lead to the initiation of enhanced monoterpene biosynthesis. However, none of the reagents promoted an increase in the amount, or variety of volatile constituents detected in cell cultures after periods of exposure ranging up to 5 days. Figure 26. The effect of CCC, GA colchicine and regulator removal on the growth of M piperita suspension cultures
H O H untreated control 0 regulators withdrawn □ 5 mgl * GA- _3 J * 10 H colchicine _ 3 A 10 M CCC
control; confidence limits established at p 8 0.05 experimental; average of five replicates 4.3.5 CHEMICAL AND PHYSICAL STRESS
Environmental and metabolic "stress" has often been considered responsible for initiating the accumulation of secondary products in plants and microorganisms.
4.3.5.1 The application of stress-related plant growth regulators was therefore investigated as a possible means of inducing the accumulation of monoterpenes in M piperita cell cultures. Cell cultures were individually exposed to paraquat, abscisic acid and ethephon, each at one micromolar active concentration.
Contact with paraquat and ABA for greater than 24 hours resulted in a visible darkening of the cultures, and rapidly led to cell death. Whilst ethephon did not appear to cause the same degree of cellular disruption over the time course studied, there was no evidence to suggest that monoterpene biosynthesis was enhanced.
The essential oil profiles obtained by steam and steam-acid distillation of experimental materials were similar to those of control cultures maintained under standard conditions, and did not reveal an increase in the yield or variety of monoterpene compounds. The yield obtained from both control and experimental cultures fell slightly after 24 hours within the sealed flask.
4.3.5.2 It has been reported that cell suspension cultures of Jasminum grandiflorum produce an authentic aroma after exposure to high temperatures (35°c) for a short period of time. (Bush Boake Allen Research Report R4/80). -151-
14 day old M piperita suspension cultures were subjected to 24, 48 or 72 hours of high temperature stress at 35°C, allowed to equilibriate for 24 hours under normal conditions at 25°C, and then assayed for monoterpenes and monoterpene-glycosides. However, the treatment at elevated temperatures did not appear to promote the accumulation of monoterpenes in this case. Similarly, jasmin cultures (ex BBA) failed to generate an aroma when the experiment was repeated under the conditions selected for the growth of M piperita cultures.
Reference back to the earlier experiments carried out at Bush Boake Allen, revealed that the culture medium contained indol-3y1-acetic acid (IAA) as a plant growth regulator rather than 2,4 D. Since indole and a number of related products are known constituents of Jasmin odour, it seems possible that the metabolic transformation of IAA could have partly accounted for the fragrant aroma associated with such cultures. The true effect of the high temperature treatment was also rather confused by the absence of comparative data for control samples. Since cultures grown on an IAA - containing medium without the heat shock failed to develop a strong aroma, it seems unlikely that IAA induced morphological changes within cultures which would thereby facilitate the accumulation of essential oils, but that high temperatures played some role in catalysing the formation of fragrant compounds derived from or regulated by IAA. The results obtained with M piperita and J grandiflorum grown on other media however, suggests that high temperature treatment cannot be regarded as a universal "trigger" for secondary metabolite production. -152-
4.3.6 SUBSTRATE AVAILABILITY
Enhancing the supply of potential precursors was considered as a means of promoting the accumulation of monoterpenes in M piperita cell cultures. Cells from 14 day old cultures were therefore resuspended in M&S medium containing mevalonic acid (MVA), beta-methyl crotonic acid (beta-MCA) or dimethylallyl alcohol (DMA-OH) at one millimolar active concentration.
The utilisation of beta-MCA and DMA-OH was quantified by GLC during the routine screening for monoterpenes. The results have been summarised in figure 27. Both beta-MCA and DMA-OH were rapidly metabolised by M piperita cells; disappearing from the growth medium within 12 hours of addition. However, there was no detectable rise in the level of monoterpenes or monoterpene-glycosides during this period.
Figure 27. The metabolism of beta-methyl crotonic acid and dimethylallyl alcohol by M piperita suspension cell cultures.*
* Average of duplicate analysis from 3 separate cultures -153-
Since it is relatively non-volatile, the fate of MVA was not routinely monitored; once it had been added to cell cultures. However, examination of the steam and steam-acid distillates, failed to reveal the presence of additional quantitities of monoterpenes or their glycosides.
It was concluded that increasing the supply of these exogenous substrates was not an effective means of enhancing monoterpene biosynthesis in M piperita cell cultures. Since monoterpenes are formed, albeit in small amounts, it is hypothesised that terpenoid precursors are probably adequately available in cell cultures of M piperita, but that competitive metabolic and physico-chemical processes quickly reduce the level of exogenous substrates to a minimum equilibrium level. The failure of cells to utilise beta-MCA and DMA-OH may have been due to:-
(i) An inability of substrates to penetrate the functional sites of monoterpenes biosynthesis. (ii) The enzymes of monoterpene biosynthesis may not have been active at the point in time when the substrates were applied. (iii) Monoterpenes biosynthesis may be self-regulating, the addition of precursors failing to stimulate further biosynthesis in the absence of other changes (eg morphologic). (iv) Exogenous substrates may have been incorporated into higher terpenoids with more clearly defined physiological roles. 4.3.7 CHEMICAL REGULATION
Beta-Ionone and retinol are known to promote the biosynthesis of carotenoids and other higher terpenoids in plant and microbial tissues. In this study their ability to promote the accumulation of monoterpenes in M piperita cell cultures was examined for the first time.
Since retinol is non-steam volatile, its fate once added to cell cultures was not ascertained. However, no new monoterpene products were detected following exposure of the cells to the reagent.
Beta-Ionone at one millimolar concentration was actively metabolised by cell cultures, but did not lead to a detectable increase in the yield or diversity of terpenoid products over a 48 hour period. The results obtained by analysis of the incubation have been summarised graphically in figure 28.
Figure 28. The metabolism of beta-Ionone by M piperita cell cultures*
* Average of duplicate analyses from 3 separate cultures -155-
4.3.8 ENZYMATIC POTENTIAL
The possibility that cell cultures do not possess the full complement of enzymes necessary for the biosynthesis of a complete, multicomponent essential oil may limit their commercial usefulness. However, if cultures can biotransform relatively simple terpenoid compounds into more valuable derivatives or mixtures thereof, this may represent a significant commercial opportunity. Such studies, if carried out taking into account the known interrelationships between various essential oil components, may also assist in the identification of the particular link in the pathway which normally appears to limit essential oil accumulation in many cell cultures including M piperita.
M piperita cells were suspended in M&S medium containing purified monoterpenes at 2.5 millimolar concentration, and incubated under standard conditions. Higher concentrations of monoterpenes were found to be unsuitable for use, causing visible browning within the cultures relatively quickly (within 2-6 hours).
Controls were examined in which:-
I) No cells were added to the dilute monoterpenes. II) No monoterpenes were added to the cells and suspending medium.
After periods of between 2 and 24 hours, combined cells and medium were steam/steam-acid distilled to recover monoterpenes and their glycosides. In all cases the level of endogenous monoterpenes in the cell innoculum was insignificant when compared to the material supplied for experimental purposes. Chemical losses were small during the typical procedure, varying between 5 and 10% depending upon the monoterpene under evaluation. It has been assumed that these losses are primarily incurred during the initial transfer of substrate into the cell suspension, and their subsequent transfer to the distillation unit at the end of the incubation period. -156- •
The exogenously supplied monoterpenes; geraniol, alpha-terpineol, r-pulegone, 1-menthone, 1-menthol and r-neomenthol were all metabolised by M piperita cell cultures over the course of 24 hours. These simple terpenes were transformed, leading to a temporary accumulation of the structurally related compounds, summarised in table 14. The net result of such processes was an overall decrease in the total monoterpene concentration, over the incubation period. Glycosidic derivatives; when formed were also metabolised, but relatively slowly. The biotransformation of each monoterpene has been considered separately in the following sections.
Table 14. Biotransformation of monoterpenes by M piperita cell cultures
SUBSTRATE PRODUCT REACTION 4.3.8.1 Geraniol biotransformation
Geraniol was rapidly metabolised by cultures, reflected by a temporary accumulation of citral. Both reactant and product were concurrently catabolised; each falling to a low concentration after 24 hours incubation. Neither geraniol nor citral were detected in control cultures, and "chemical" losses were virtually constant at some 10% of initial geraniol concentration. The latter was presumed largely lost during the initial transfer of geraniol into the suspended cells, and their subsequent transfer for distillation at the end of the incubation period.
The oxidation of geraniol appeared to be a relatively unspecific reaction; since cultures of Nicotiana tabacum were also found to be capable of effecting this transformation, though the reaction rate was not quantified.
Figure 29. Biotransformation of’Geraniol by M piperita cell cultures
TIME ( hours)
Exposure period (hours) o = GERANIOL □ = CITRAL * Average of duplicate analyses from 3 separate cultures. -158-
4.3.8.2 Pulegone biotransformation
The reduction of pulegone to isomenthone was relatively slow in M piperita cell cultures, with chemical losses small and effectively constant at 5% of the initial pulegone concentration.
Cultures of N. tabacum were unable to effect the reaction within a 24 hour period, indicating that some degree of culture specificity may be associated with this transformation.
Figure 30. Biotransformation of pulegone by M piperita cell cultures
TIME (hours)
Exposure period (hours) o = PULEGONE D = ISOMENTHONE
* Average of duplicate analyses from 3 separate cultures 5.3.8.3 Menthone biotransformation
L-menthone was rapidly reduced to a mixture of isomeric alcohols, of which 1-menthol and r-neomenthol were identified as the main components. These alcohols were subsequently glycosylated; effectively driving the equilibrium of the reduction reaction quicky to completion. The level of menthone, menthols and glycosides fell steadily over the course of time, but no further products were detected. Chemical losses were small and estimated to be approximately 5% of the initial menthone level.
Cultures of N. tabacum were capable of performing the same reaction sequence though somewhat more slowly.
Figure 31. Biotransformation of menthone by M piperita cell cultures
TI ME" (hours)
Exposure period (hours) o = MENTHONE □ = MENTHOLS x = GLYCOSIDES * Average of duplicate analyses from 3 separate cultures 4.3.8.4 Menthol biotransformation
Both 1-menthol and r-neomenthol were apparently glycosylated by cell cultures of M piperita at a similar rate. The level of menthy1-glycoside produced by the reaction rapidly reached a plateau, only to subsequently fall slowly. However, volatile terpenes or related compounds were not detected arising from the further metabolism of the glycosides.
N . tabacum cultures performed a similar transformation, leading to the temporary accumulation of menthy1-glycoside.
Figure 32. Biotransformation of menthol by M piperita cell cultures
TIME(hours)
Exposure period (hours) o = MENTHOL □ = GLYCOSIDE * Average of duplicate analyses from 3 separate cultures -161-
From the work carried out on monoterpene biotransformation it has been possible to make the following general statements as regards the potential use of plant cell cultures for the production or modification of essential oils.
1) Monoterpenes are potentially cytotoxic to plant cells. Concentrations of 10 millimolar or greater caused visible browning of cellular material relatively rapidly (^ 2 hours).
2) The majority of monoterpenes are metabolically labile under fermentation conditions. They are metabolised to non-volatile derivatives relatively quickly.
3) Cell cultures from M piperita were unable to recreate the complete oil profile of the parent plant, even when supplied with purified, individual para-menthane monoterpenes.
4) Most of the monoterpenes were biotransformed by relatively unspecific reactions (oxidation, glycosylation) in which cultures of N.tabacum appeared similarly as effective as M piperita on a comparative dry weight basis.
5) Glycosylation of menthols appeared to be a general detoxification process. - 162 -
A.3.9 CELLULAR COMPARTMENTATION
It has been hypothesised that the compartmentat ion of precursors and relevant enzyme systems may restrict the metabolic activity of cell cultures. Likewise, the presence of low concentrations of simple terpenes or related products at their site of synthesis, may exert an inhibitory effect, restricting the further biosynthesis of monoterpenes.
Cells from M piperita cultures were resuspended in M&S medium containing 5% (v/v) dimethyl sulphoxide (DMSO) and assayed for the production of terpene compounds at intervals over 24 hours. However, the presence of DMSO did not appear to influence the accumulation of terpene compounds in M piperita cultures or the suspending medium.
The use of Evan's blue (Gaff et aJL 1971) suggested that cell viability and integrity was retained after the DMSO treatment and there was no apparent cytoplasmic disruption visible at the light microscope level. This was consistent with the experience of Delmer (1979) who described the ability of DMSO to render the plasmamembrane permeable to small molecules, facilitating rapid cytoplasmic leaching; whilst vacuolar contents were largely unaffected. The viability of experimental material was also supported by the fact that, when plated onto agared medium, there was regular regrowth of callus from most cell clumps.
It was tentatively concluded that increasing the permeability of the plasmamembrane did not enhance the accumulation or secretion of monoterpenes in M piperita cell cultures. Nevertheless, the localisation of essential oil within glandular trichomes of the intact plant did lend support to the hypothesis that some degree of morphological compartmentation; for the deposition of monoterpenes away from the mainstream of metabolism; might warrant study. -163-
4.3.10 MORPHOLOGICAL DIFFERENTIATION
Morphological differentiation provides the opportunity for secondary products; which are often phytotoxic; to be localised and segregated away from normal physiological processes; thereby facilitating their accumulation or excretion. In the intact plant this is frequently seen as an important feature of secondary metabolism.
Combinations of various additives, which had previously been reported to stimulate shoot regeneration in plant cell cultures were assessed for their ability to induce morphological differentiation and promote essential oil accumulation in M piperita cultures.
Microscopic examination of cell nodules from suspension cultures revealed that cultures grown under normal conditions contained groups of cells whose walls had undergone secondary thickening and lignification (see plate 13). There was thus, some degree of cellular differentiation frequently associated with such cultures. Indeed the deposition of lignin; is itself classically regarded as a phenomenon of secondary metabolism.
Plate 13. Groups of lignified cells from M piperita suspension cultures (Scale x350) When such nodules were plated out onto "regeneration" media, rootlet formation was relatively easy to control by variation in the auxin: cytokinin ratio. A high ratio (e.g. 2,4-D 0.1 mgl \ BAP 0.01 mgl ^), and in particular a low 2,4-D concentration proved effective. Embryogenesis or shootlet formation however, was less reliably controlled with well known regulators, though a small number of plantlets were obtained from cultures transferred to M & S medium containing 0.1 mgl ^ 2,4-D and 1 mgl ^ FAP. The plantlets developing within a culture were seen to produce a morphologically juvenile foliage, characteristic of plantlets regenerated from nodal explants in culture, and similar to that of M piperita seedlings.
The essential oil of regenerated plantlets was isolated by microscale steam distillation, and the overall yield determined at approximately 0.9% dry weight. This compared with an average of 1.2% for plantlets regenerated from nodal explants and maintained under identical conditions. The reason for this difference was not fully established through it was suspected that uptake of nutrients (including carbon) from the medium was less effective in the case of regenerated shoots on callus, possibly also accounting for their slightly weaker growth. Although the residual callus material appeared to cease growing once shoots were established, its presence would also contribute to the slight reduction in essential oil content, calculated on a dry weight basis.
Whilst the shoots arising from within cultures; bore secretory appendages; relatively few glandular trichomes appeared to contain essential oil. In many instances the stalk cell was brown and necrotic, with the secretory cells collapsed, non-viable and lacking essential oil. -165-
The other reagents examined - IAA, NAA, GA^, CCC, TIBA and active charcoal caused a variety of morphogenetic effects when applied to M piperita cell nodules. Their morphogenetic effects on cell nodules after 8 weeks culture growth have been compared with growth of nodules on the control medium (M&S).
IAA Callus growth much reduced, (0.001 lOmgl"1) but no regeneration
NAA callus growth was much reduced, (0.5-5 m g r 1) cultures were compact and dark green
ga3 no observed effect at 0.05 mgl 1 but at (0.05-5 mg!"1) 5 mgl 1 culture growth was dark green and compact
CCC only slight culture growth with some
(io"3m ) browning of older cells
TIBA callus growth moderate, dark green and
(i o "7-io"3m ) compact at all concentrations.
Activated rootlet regeneration encouraged, especially Carbon on low auxin media. Callus growth slightly (10 gl_1) reduced.
It was concluded that morphological differentiation in the form of shootlet regeneration facilitated the accumulation of raonoterpenes, in M piperita cell cultures. However, regeneration was unpredictable, and none of the reagents specifically selected for this purpose proved to be particularly effective. Where shootlets had formed, the oil yield was 30% lower than that obtained with similar material regenerated from nodal explants. Nevertheless, morphological differentiation did lead to a considerable increase in monoterpene concentration over that observed for undifferentiated callus or suspension cultures. Further work to determine accurate control over the degree of differentiation would appear to be well worthwhile. -166-
5 DISCUSSION -167-
DISCUSSION
The objective of this study was to investigate the theoretical and practical feasibility of producing para-menthane type monoterpenes from cultured plant cells. Based upon the results of initial studies using intact plants, and reference to the literature, original approaches to the production and • modification of monoterpenes have been made using cell cultures.
Monoterpenes are commonly regarded as secondary products, arising through the activities of a non-essential biochemical pathway. They are a commercially significant class of compound • amongst the isoprenoids and are found as the principal constituents of many essential oils. (Nicholas 1973, 1973a, Lawler 1977). Widely used as flavourings and fragrances, the natural sources of supply have largely failed to keep pace with the demand created by the growth of modern consumer products. • (Arnadau 1981). Although many constituents can be chemically synthesised, (Erickson 1976) only a few are individually consumed in such quantities as to warrant economic manufacture. Since the olfactory qualities of synthetic materials are usually inferior to the natural alternative, and their use in food products also frequently incurs undesirable labelling, the application of biological systems (BIOPROCESSING) for the preparation of natural products has many attractions.
Some of the implications of bioprocessing have been discussed by ^ various authors (Lee & Scott 1979, Tabata 1977, Yeoman et^ a_l 1980, Fowler 1981, Crocomo et^ al^ 1981, Ruttloff 1982), the key points of which are summarised below.
+ ADVANTAGES OF BIO-PROCESSING
1. Production can be controlled and located near to consuming centres. 2. Uses cheap, renewable raw materials. 3. Biomass is free from microbial and insect contamination. 4. Product yield and quality should be consistent and predictable subject to genetic stability of cultures. 5. Supply of strategic biochemicals should be free from political interference (Stout & Schultes, 1973). 6. Transport and storage costs should be reduced 7. Pricing structure should be stabilised. 8. Labour costs reduced for semi-automated biological process 9. Products will be "natural" for legislative purposes. 10. Bioprocesses should only produce physiologically active isomers, therefore yield and purity will frequently be better than competing chemical routes. 11. Proceeds under mild reaction conditions (low temperature, low pressures, solvent-free) 12. Flexibility - basic system applicable to many products. 13. Effluents are usually harmless, sometimes potentially useful. 14. Cell lines are self-propagating 15. Production capacity per unit area increased over field-grown crops.
However, because it involves considerable capital investment, biotechnology is most likely to succeed where it can offer innovative products and processes or dramatic cost reductions, rather than substitutions for, or marginal improvements to, existing materials. Its potential application to the flavour industry appears to be highly attractive, given the identification of suitable commercial targets - where the flavour is comprised of a small range of chemically similar compounds, preferably commanding a high value. -169-
Because the success of these investigations with plant material and derived cell cultures was dependent upon the development of effective techniques for the extraction and analysis of trace amounts of terpenoid metabolites, the first part of the investigation concentrated on this aspect.
The techniques employed for the isolation of essential oils were developed to give an accurate reflection of those materials present in vivo. Enzymatically induced changes; which may occur prior to the extraction of fresh plant material; were minimised by immersing tissues in liquid nitrogen during preparation. (Loomis 1974). The latter also restricted the formation of n-hexenal; an artifact often produced by the autoxidation of plant tissue in air (Major ej: al^ 1963) whilst losses attributable to evaporation (von Rudloff 1967); appeared to be minimal, both during sample preparation and short storage periods employed at -17°C. The use of relatively small or fragmented samples facilitated rapid heat transfer during distillation, leading to the inactivation of endogenous enzymes and hence to improved recovery within relatively short distillation periods.
Direct-volatilisation GLC and Likens-Nickerson distillation caused minimal degradation and losses since the volatile constituents received limited exposure to high temperatures within the apparatus (Pickett e£ al_ 1975). Although the Likens-Nickerson distillation did not appear to fully recover the most volatile 5-carbon compounds such as 2-methyl butan-l-al and 2-methyl butan-l-ol; recovery of monoterpenes from plant tissue was better than 90% for both techniques when optimised for work with M piperita. -170-
In recent years the role that monoterpene glycosides might play in monoterpene metabolism has been the subject of experimental investigation and much conjecture. Monoterpene glycosides have been implicated as intermediates in monoterpene biosynthesis in Tanacetum vulgare (Banthorpe & Mann 1971), Rosa dilecta (Francis & Allcock 1969, 1969a, Francis & O'Connell 1969, Pogorel 'Skaya 1979,) Camellia sinensis (Takeo 1981, 1981a) and cell cultures of Ocimum basilicum (Lang & Horster, 1977), whilst Croteau & Martinkus (1979) have shown that neomenthyl B-D-glucoside is produced in significant quantities from (-)-menthone in flowering Peppermint. Since monoterpene glycosides are not steam-volatile, they usually remain associated with the residual plant material following distillation (Francis & Allcock 1969). For this reason the most effective means of isolation proved to be by the initial removal of free terpenes by steam distillation, followed by hot acid hydrolysis (3N HC1, at 60°C) and subsequent steam-acid distillation (similar to the method of Croteau & Martinkus 1979). The monoterpenes then released were presumed to have originally been present as glycosidic derivatives. In order to validate this procedure a crude sample of 1-menthyl alpha/beta-D- glucopyranosides was synthesised according to the method of Sakata & Iwamura (1979). The controlled release of 1-menthol under the conditions routinely employed, gave a recovery approaching 90% of the theoretical yield.
The composition of essential oils isolated from M piperita was initially investigated in detail by gas chromatography and mass-spectrometry (Duffield 1969). The efficiency of GLC fractionation is a function of column length, column diameter, stationary phase, phase loading, temperature and carrier gas flow rate. Hiltunen & Raisanen (1981) have reviewed the selection of stationary phases for the GLC examination of plant constituents, and have also drawn attention to the compromise required between the high resolution of individual constituents achievable using long columns, at the simultaneous risk of increased thermal, catalytic and absorptive losses as a result of the longer residence times on-column. Similarly, von Rudloff (1968) warned that tertiary alcohols, such as linalool, may suffer dehydration and rearrangement at injector and column temperatures higher than 120°C.
The most advanced procedures employed in this study revealed forty-eight different constituents in the essential oil of Peppermint. Retention indices for the various components were expressed relative to n-tetradecane; the internal standard selected for use in this study; in order to reduce errors which might arise as a result of variation between separate analyses (Lawrence 1971, Jennings & Shibamoto 1980). Quantitative analysis was performed by correcting the integrated peak areas using predetermined response factors for each known constituent (McNair & Bonelli 1969). All chromatographic data were processed using a Hewlett Packard 3390A reporting integrator, either recording directly from the GLC, or computing pre-recorded traces from the data storage system. The latter was constructed during the course of this investigation to provide a means of storing basic chromatographic data pertaining to a destructive analysis. With the packed column GLC system routinely employed, the detection limit for single components -9 was approximately 1x10 g, though the fundamental studies using a high resolution capillary GLC revealed levels down to 5xl0”n g.
Intact Plant Studies
Equipped with reliable and accurate techniques for the assay of monoterpenes, the first area of experimental study was to determine the level of monoterpenes and their glycosides in various tissues of intact M piperita plants. It is well known that monoterpene metabolism in M piperita is influenced by environmental factors (Langston & Leopold 1954, Biggs & Leopold 1955, Ahlgrimm 1965, Hefendehl 1962, Clark & Menary 1980, 1980a and Steward 1962), hence plants were grown under conditions known to promote vegetative growth and monoterpene biosynthesis (Burbott & Loomis 1967). -172-
Plants which had been grown from a single clone for 7 weeks at 25°C under a 14 hr photoperiod were harvested and divided into leaf, stem and root fractions* The aerial parts were found to contain an essential oil comprising of 37 micromoles of free monoterpenes and additionally approximately 5 micromoles of menthyl glycoside. The former was largely associated with leaf tissue (90%) whilst the latter was more equally distributed between leaves (46%) and stems (54%). Analysis of root material failed to reveal the presence of free or bound monoterpenes, in agreement with the earlier work of Dimitrova et^ al^ (1961).
Since they appeared to be the primary site of monoterpene accumulation, a more detailed investigation was carried out on leaves at various stages of development. It has been assumed that; for plants growing under a constant environmental regime; the sequence from young to old tissues represents a time course of monoterpene biosynthesis and interconversion. Similar % studies have been carried out with citrus spp (Attaway e t aJL 1967) M suaveolens (Hendriks & van Os 1972) and M piperita
(Lemli 1955, Reitsema et^ a_l 1957, Loomis 1967, Croteau 1980). During leaf ontogenesis the level and types of monoterpenes in M piperita were found to change considerably. In the first instance the accumulation of monoterpenes was found to closely reflect the rate of gain in dry weight of leaves themselves, reaching a maximum of some 2.5 micronoles per leaf. As the leaf dry weight subsequently decreased, however, then so did the level of monoterpenes. These observations led to the belief that unlike many secondary products; which are produced in small quantities over a limited period of an organism's life; monoterpenes undergo active metabolism throughout the plant's development, and are far from inert, waste products. If the concentration of monoterpenes was considered as a function of dry weight, then young leaf tissues were found to contain the highest levels, though there was a transient rise observed in midstem leaves, possibly associated with the activation of secondary meristems. Together with these gross changes in monoterpene content there was considerable underlying variation in the proportions of the individual constituents. Most noticeable of these was a reduction of menthone and concomitant rise in the menthol content of midstem leaves, whilst the level of pulegone was seen to progressively decrease with the age of leaf tissue. It was concluded that individual monoterpenes undergo biosynthesis, interconversion and degradation, leading to major changes in the profile observed in leaves of different ages.
Menthyl glucoside was detected as a minor constituent in vegetative plants of M piperita; accounting for some 5% of total leaf monoterpenes and reaching a peak level of approximately 0.16 micromoles in midstem leaves. However, its formation; and additional presence at relatively high levels in stem tissues; suggested that it may perform a role in the translocation or metabolism of menthol. Croteau & Martinkus (1979) proposed that monoterpene glycosides may represent an energy-rich storage reserve in such cases, though this was not investigated further.
The next stage in the investigation of M piperita leaf material was to locate and characterise the sites of monoterpene accumulation. Three types of epidermal appendage were observed by scanning electron microscopy - papillate hairs, glandular hairs and ten-celled glandular trichomes. The former were primarily found overlying veinal tissue on the ventral leaf surface, whilst glandular hairs and trichomes were more uniformly distributed at approximately equal density on ventral and dorsal surfaces. Similar structures were present on stem tissues but in reduced numbers. On midstem leaves there was considerable variation in the dimensions of glandular trichomes, ranging from 25 to 90 micrometers diameter. The larger examples appeared as distended vesicles with refractile contents - later shown to be essential oil. -174-
Similar structures have been shown to be associated with the accumulation of complex resins in Newcastelia viscida (Dell & McComb 1977), monoterpenes and sesquiterpenes in Salvia spp (Croteau et al 1981b), Majorana hortensis (Croteau 1977a), Pogostemon cab1in (Henderson et al 1970), Nicotiana tabacum (Michie & Reid 1968), M piperita (Lemli 1955a, Amelunxen et al 1969), Lophanthus anisatus (van Horne & Zopf 1948).
By using glass capillaries drawn to a fine point, the contents of a number of trichomes were collected and examined by direct- volatilisation GLC (Malingre et al^ 1969). Analysis revealed that the trichomes contained an essential oil with less pinenes, alpha-terpinene and menthyl acetate than present in whole leaf samples together with trace amounts of 2-methyl butan-l-al and 2-methyl butan-l-ol . Using this technique it was possible to determine that fully developed glandular trichomes contained approximately 3x10 ^ moles of monoterpenes, equivalent to _8 approximately 5x10 g. However, in order to overcome the problem of sample variability, a more effective means of collecting large numbers of oil glands was developed. First attempts to isolate the epidermis from leaves proved very difficult but material was obtained from young stems. The latter proved advantageous, since it was then also relatively easy to make a comparative examination of the underlying internal tissue. The yield of monoterpenes in epidermis and mesophyll was determined experimentally as being some 0.25 and 0.01 percent of dry weight respectively. Whilst the composition of the epidermal sample was similar to that previously determined for isolated glandular trichomes, the mesophyll tissue only contained alpha terpinene, 1-menthol and menthyl acetate in detectable quantities. However, high levels of 2-methyl butan-l-al and 2-methyl butan-l-ol; both structurally related to DMAPP (3-methyl but-2-ene-l-ol), the fundamental terpenoid primer unit; were detected in mesophyll tissues. It was concluded that whilst monoterpenes were present in both epidermal and mesophyll cells, the latter appeared to be the primary site of synthesis for key precursors, and to a lesser extent the accumulation of certain monoterpenes, such as alpha terpinene and menthyl acetate. However, most essential oil constituents were preferentially accumulated in the epidermis, principally associated with glandular trichomes.
In order to quantify the distibution of monoterpenes between epidermal glands and other tissues a number of leaves were treated with a solution of Polyvinyl alcohol (PVA) which was then dried to a film. When the film was removed, scanning electron microscopy revealed that the glandular trichomes were virtually completely removed from leaves, though many of the smaller glandular hairs still remained intact. Examination of both untreated and treated leaves revealed that almost 90% of the total leaf monoterpenes were recovered by the use of PVA films, and that with the exception of menthyl acetate and alpha terpinene, all essential oil components appeared to be preferentially accumulated in the glandular appendages. This technique was first employed by Hefendehl (1967, 1968) who claimed to have isolated 98% of the glandular trichomes of peppermint leaves and achieved a highly selective recovery of essential oil compounds.
This approach was then employed to investigate further the biosynthesis and accumulation of monoterpenes, which appears to take place in a sequential fashion from the mesophyll to the epidermal trichomes. Trichomes belong to a class of structures called "meristemoids", the cells of which not only undergo their own brief pseudomeristematic activity, but also exert some sort of inhibitory influence over the initiation of other meristemoids within their vicinity (Bunning 1952). In order to investigate whether monoterpenes would be accumulated in the absence of glandular trichomes or indeed whether new trichomes will differentiate on treated leaves, PVA solution was applied to a number of young leaves, allowed to dry and then peeled off to remove the glandular trichomes. The leaves were then left attached to the plant and after a further 18 days assayed for free and bound monoterpenes.
Unfortunately, the removal of glandular trichomes appeared to cause a perturbation of leaf metabolism such that further laminal expansion, dry weight gain and monoterpene accumulation were all restricted. Furthermore, initiation of new trichomes over the experimental period was not apparent. It is hypothesised that the removal of glandular trichomes caused injury to the leaf tissues, leading to various physiological aberrations and restricting laminal expansion. Since monoterpene biosynthesis appears to be at least partially growth-related; these changes would undoubtedly tend to limit monoterpene biosynthesis.
Although leaf turgidity was retained, transpirational water losses may also have restricted leaf growth. In practice Ahlgrim (1956), Franz & Wunsch (1972), Hefendehl (1964a) and Kalitzki (1954), have all shown that enforced wilting will actually increase the essential oil content of M piperita leaves. This increase has been shown to occur partly as a consequence of the hydrolysis of endogeneous proteins; leading to the additional incorporation of leucine into monoterpenes (Franz & Wunsch 1972, 1973); and also as a result of the depolymerisation of triterpenoid acids such as oleanolic and ursolic acids, into monoterpene units (Brieskorn 1953). However, these observations were made on mature tissues where primary growth was already complete and monoterpene metabolism had been previously established for some time. It was therefore difficult to draw any further conclusions from this particular area of study and a complementary approach, investigating the structural relationship between glandular trichomes and adjacent tissues was initiated. The ultrastructural features of glandular tissues in plants have recently been reviewed by Fahn (1979) and Heinrich (1979).
Glandular trichomes were visible on young leaf primordia once they had reached approximately 0.5 mm axial length. Examination of sections from slightly more mature tissues by light microscopy, revealed that sub-glandular mesophyll cells were densely packed and the tissue characterised by a noticeable lack of intercellular air spaces. This is similar to the situation in Patchouli (Pogostemon cablin) where the glandular cells; which accumulate a sesquiterpene-based essential oil; appear to be closely associated with photosynthetic cells in the leaf, or actually develop within the plant tissue where they are located within the phloem of roots and shoots (Henderson nt a \ 1970, Jones & Krishadethan 1973, Jones 1982).
The implication of metabolic coupling between the glandular trichomes and underlying photosynthetic cells in M piperita was strong; lending support to the results of earlier biochemical analyses in which relatively high concentrations of potential precursors were detected in the mesophyll tissues. This relationship was investigated further by the use of "vital" physiological stains.
Toluidine Blue was used to characterise individual cell and tissue types based upon their physiological status (O'Brien e t al 1964, Stadelmann & Kinzel 1972). It was discovered that glandular hairs and trichomes were differentially stained, the former identical to epidermal and the latter similar to mesophyll tissues. A more detailed examination of the glandular tissues was carried out using transmission electron microscopy. -178-
The latter technique revealed that plasmodesmata provided symplasmic continuity (Carr 1976) between the cells of individual oil glands, but that they also provided a link to the underlying mesophyll in the case of glandular trichomes. This was not found to be the case for glandular hairs. In many other respects the two types of glands were similar in organisation, being comprised of a basal cell within the epidermal plane, an adjoining stalk cell and terminal secretory cell(s).
The ultrastructural observations presented in this study were largely consistent with those made previously by Amelunxen (1964, 1965, 1967, et al 1969a) though several additional features have been identified.
Both glandular hairs and trichomes were encompassed by an extensively thickened cuticle from an early stage of development. The latter was intimately fused with the stalk cell; cutin and suberin penetrating the entire depth of the cell walls at this point, and forming a barrier to the apoplastic flow of water-soluble materials. It can be assumed therefore that any metabolites reaching the secretory cells from mesophyll tissue would be necessarily transported via the cytoplasm of the stalk cell. Numerous plasmodesmata were visible linking the stalk with secretory and basal cells and a collar of concentrically aligned microtubules; negatively stained against the general cytoplasm; are envisaged as imparting polarity to cellular processes by inhibiting the fusion of membrane-bound vesicles with the plasma membrane at the vertical outer faces of the stalk cell. Stalk cells thus appeared to be classically modified for short distance, active transfer.
Microscopic examination and biochemical analysis of a large number of glands indicated that in cases where the stalk cell was necrotic, the essential oil content of secretory cells was minimal implying that a living stalk cell may be necessary to transport necessary precursors to the secretory cells. -179-
The secretory cells; which have been shown to be the primary site of monoterpene accumulation; contained prominent nuclei, an extensive network of endoplasmic reticulum and darkly staining plastids which appeared to lack well developed thylakoid lamellae. The latter were similar in appearance to those described in the epithelial cells of Pinus resin ducts by Wooding & Northcote (1965, 1965a); the secretory cells of Solanum tuberosum (Lyshede 1980) and to the leucoplasts isolated from Citrofortunella mitis which have been shown to be capable of the biosynthesis of the monoterpene hydrocarbons; limonene, alpha and beta-pinene; from 1-^C-isopentenyl pyrophosphate (Gleizes et al 1982).
Chloroplasts have also been shown to play a key role in the biosynthesis of certain higher terpenoids such as carotenoids, chlorophylls, alpha tocopherol, alpha tocoquinone, phylloquinone
K, and plastoquinone 9 (Rogers et al^ 1966, Treharne et a l 1966, Charlton et al 1967, Buggy et al 1974, Lichtenthaler £t al 1982). As chloroplasts develop, their ability to utilise exogenous mevalonate generally decreases (Treharne £t a_l 1966) due to changes in membrane permability. Chloroplastic terpenoids are then derived endogenously via the fixation of C0~. Croteau (1972, 1972a) showed that the monoterpenes of z . 14 young M piperita leaves were rapidly labelled by CC^; suggesting a plastidic route; whilst sesquiterpenes were preferentially labelled by exogenous MVA (Croteau 1972b). Similar results have recently been obtained by Carde et al (1980) and Gleizes et al (1980a) for the biosynthesis of sesquiterpenes by an endoplasmic reticulum fraction obtained from pine needles. These authors also indicated that there is frequently a high degree of compartmentation even between the biosynthesis of what appear to be closely related groups of lower terpenes. In 1982 Carde nt a l carried out an extensive survey of essential oil containing plants; and related the ultrastructural features of glandular tissues to their terpenoid composition. They have proposed that the formation of monoterpene hydrocarbons is associated with the presence of leucoplasts in secretory cells, whilst a well developed endoplasmic reticulum is a feature of cells containing oxygenated monoterpenes or sesquiterpenes. The relative proportions of these organelles observed in the secretory cells of M piperita in this study was consistent with their hypothesis, though similar plastids; frequently containing paracrystalline inclusions; were also observed during the early development of many epidermal cells which did not appear themselves to accumulate monoterpenes.
Another characteristic of the secretory cells in M piperita was the regular invagination of the plasma membrane at points of association with the extensive intracellular network of endoplasmic reticulum. The extracellular space which developed was frequently seen to contain darkly staining osmiophilic materials, representing metabolites secreted from the cytoplasm. In certain areas the space became more extensive as the glands matured, ultimately splitting the cell wall between the pectic and cellulosic layers and generating a large extraplasmatic cavity. The latter was frequently found to contain an electron opaque, homogeneous material, previously shown by Amelunxen (1965, et aT 1967, 1969a) to be essential oil. The excretion of essential oil appears to be a widespread phenomenon in higher plants and is frequently associated with some degree of morphological differentiation. The reason for localised excretion is not clear, though the fact that many essential oil constituents are cytotoxic at relatively low concentrations (Jones 1974) suggests that it may be an obligate metabolic requirement. e
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Since trace amounts of menthyl glycoside were detected in vegetative tissues of M piperita, it was hypothesised that the sites of free menthol accumulation might exhibit high glycosidase activity. A technique for the cytochemical localisation of beta-glucosidase was developed based upon earlier work by Smith & Fishman (1969), Ashford & McCully (1970,a,b) Bowen (1973), and Stewart & Pitt (1977). Naphthol AS-BI beta D-glucopyranoside was employed as the substrate, and freshly diazotized para-acetoxymercuric aniline as the coupling agent. However, penetration of the reagents into the tissues was poor, and only a control sample containing previously hydrolysed substrate showed a staining reaction which was itself confined to areas of relatively open cell structure. Because of such technical problems this avenue of study was not pursued further.
It has been concluded from the preliminary work on M piperita plants, that anatomical and ultrastructural features of leaf organisation indicate metabolic coupling between glandular trichomes and the underlying mesophyll tissues. Vital-staining also supported this possibility. Analysis of the two tissues revealed that the mesophyll specifically contained high levels of volatile five-carbon compounds such as 2-methyl butan-l-al and 2-methyl butan-l-ol; which are potential terpenoid precursors or derivatives thereof; although monoterpenes were barely detected. In epidermal tissues the situation was effectively reversed, monoterpenes being accumulated, whilst putative volatile precursors were present in only trace amounts.
It is proposed that monoterpene precursors are primarily produced in mesophyll tissues during their primary growth. Transport of these compounds through the basal and stalk cells of the glandular trichome would then provide the terminal secretory cells with a source of readily available carbon skeleta for monoterpene biosynthesis. 9 -182-
Once within the secretory cells, the specialised plastids together with the endoplasmic reticulum might be responsible for * effecting the final conversion of precursors into monoterpenes. Since monoterpenes are frequently cytotoxic, their degradation, compartmentation or secretion would be expected* A network of endoplasmic reticulum within the secretory cells appeared to provide secretory channels, with their contents being discharged • into a subcuticular extraplasmatic cavity, physically isolated from all other physiological processes.
Plant tissue and cell culture studies
• An experimental approach to the study of monoterpene metabolism in Mentha cell cultures was developed from the knowledge obtained using intact plants. Since cell cultures increase in dry weight and undergo multiple cell divisions it has been assumed that they are capable of producing higher terpenoids 0 such as steroids. Green cultures probably also possess the ability to produce phytol and carotenoids amongst their terpenoid profile.
Firstly, callus cultures were established from leaf and stem explants of M piperita on a variety of defined media. Initial cell divisions occurred at cut surfaces in the vicinity of vascular elements, and led to the formation of callus material in a number of cases. Jablonski & Skoog (1954) made similar observations during the initiation of tobacco pith cultures and showed that the cambium was a rich source of natural cytokinins. e
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Although not necessarily reflecting the optimum conditions for monoterpene accumulation; cultures were initially grown for maximum dry weight gain in order to provide healthy, viable material for biosynthetic studies. The medium selected for routine use was similar to that developed by Lin & Staba, (1961) based upon the inorganic formulation of Murashige & Skoog (1962), supplemented with 3% (w/v) sucrose, 1 mg 1 ^2,4-D, 0.1 mg 1 ^ BAP and 1% (w/v) Difco Bacto agar . At a temperature of 25 — 2°C and a 14 hour photoperiod (1.6 Wm PAR) this medium supported callus growth of approximately 1.5 mg dry weight gain per day.
Because of their relatively slow growth rate, callus cultures were not considered as a particularly attractive material for the commercial production or modification of monoterpenes. Since they are grown with only one surface regularly in contact with the solidified medium, the transfer of exogenous precursors, substrates and reaction products is also limited, and results in metabolic gradients within the tissues.
Suspension cultures; initiated from an actively dividing callus cell line; were used for the majority of biosynthetic studies. The growth of cultures was considerably improved when cells were grown in a liquid version of the same medium as employed for callus initiation. For established cultures, an average daily dry weight gain of 45 milligrams was determined over a 2 1-day culture period. Such batch cultures of M piperita cells comprised a heterogeneous collection of chlorophyllous cell clusters and unpigmented free cells.
Suspension cultures were subcultured every 14 days, at approximately 70% of their peak cell density. Cultures were sustained over several periods prior to experimental use, though there was a noticeable decrease in the proportion of chlorophyllous tissues after 9 -1 0 subcultures. ©
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It has been proposed that the microenvironment within cell clusters promotes greening by a combination of low endogenous sugar and high CO2 concentration, also related to their relatively slow growth rate* (Staba 1969, Thomas & Stobart 1970). It was, therefore, inevitable that during the course of successive subcultures, green cell nodules were gradually 'diluted' to a lower proportion of the total cell mass. These observations are consistent with those of Dobberstein (1966) who documented an inverse correlation between growth rate and chlorophyll content once cell aggregates of M arvensis (var piperascens) had reached approximately 2mm diameter.
At this point it is pertinent to draw attention to the fact that though there are many reports in the literature concerning the monoterpene metabolism of plant cell cultures; very few authors have properly defined the origins and physiological status of their experimental material. Indeed, it has almost invariably been assumed that cultures are homogenous in their cell-type composition, though this is known not to be the case (Blakely & Steward 1962,a,b). In order to ensure comparability and consistency of experimental material, cells for experimental use were only obtained from subcultures 2-6. The growth cycle of cultures over this period was predictable and defined for the purposes of this study (see figures 23 & 24).
M piperita cultures typically became darkened, and growth slowed after only 18-20 days in culture. This short growth cycle of M piperita suspension cultures was regarded as a potential drawback, imposing constraints upon both their commercial and experimental use. The cost of the medium is frequently a high proportion of the total cost of producing plant cell material, (Bush Boake Allen 1980, Goldstein et^ aJL 1980), whilst regular resterilisation is time-consuming, expensive and introduces the risk of microbial contamination. In laboratory experiments, the implication of short growth cycles is that cells are in a rapidly changing enviroment, and are likely to be altering their own metabolism at least as rapidly. Several other workers have reported a similar brown discolouration with suspension cultures of Acer pseudoplatanus L. when the exogenous auxin concentration was decreased. They also recorded a concomitant rise in the level of phenolic compounds (Gathercole & Street 1967, King 1976, Westcott & Henshaw 1976, Withers 1976). Ellis (1971) has studied the formation and metabolism of phenolics in Mentha cell cultures in detail, and in a closely related species; Ocimum basilicum; Lang et^ £l^ (1979) have shown that during the log phase of batch cultures there was a rapid increase in the vacuolar tannins, which were then transferred into the cytosol co-incident with the culture's arrival at stationary phase. The accumulation of such materials in the cytosol of 25-day old cells was considered to be one of the main reasons for their premature death.
As a possible means of extending the viability of M piperita suspension cultures; polyvinyl pyrrolidone (PVP) was included in the growth medium at a level of 2% (w/v). PVP has been widely used to adsorb the phenolics and their polymerised derivatives which are released during the maceration of plant tissue for enzyme isolation. (Loomis & Battaile 1966, Andersen & Sowers 1968, Loomis 1974, Loomis e_t al^ 1979). PVP was also considered by Siegel & Enns (1979) as a means of adsorbing excess polyphenols from the medium of soybean (Glycine max L) suspension cultures. They postulated that PVP is excluded from the cells themselves because of its molecular dimensions
(RMM •£: 40K), but that it acts by reversibily altering the permeability of the plasmamembrane in such a fashion as to allow phenolic materials to escape. Once within the extracellular medium, the potentially cytotoxic phenolics are rapidly adsorbed by the PVP in solution. In this study, the growth of M piperita cultures was somewhat decreased in the presence of PVP, thereby shifting and extending the growth period slightly. However, since the same final cell density was eventually reached, any metabolic benefits arising from its use were not obvious. ©
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The basal monoterpene metabolism of both callus and suspension cultures was investigated experimentally in order to determine the background level of activity. Undifferentiated callus and suspension cultures were found to contain monoterpenes at 0.03% and 0.15% of dry weight respectively. In addition to being much lower than the intact plant (1.5% dw) the range of components was also much simpler. The principal monoterpene constituents; alpha terpinene, 1 -menthol and menthyl acetate together with large quantities of the 5-carbon compounds 2-methyl butan-l-ol and 2-methyl butan-l-al actually gave rise to a profile similar to that of internal shoot tissue. The profiles of callus and suspension cultures were qualitatively similar, although the concentration within suspension cultured cells; at 0.15% of dry weight; was approximately five-fold higher than callus material. This observation; together with the earlier identification of large quantities of monoterpenes in young leaves, strongly supports the hypothesis that monoterpene biosynthesis in Mentha is a growth-related phenomenon.
It has been proposed in this report that in the intact plant, monoterpene precursors; 5-carbon compounds such as DMAPP and IPP; are primarily produced in internal, chlorophyll-containing tissues. Their translocation into glandular appendages borne on the epidermis would facilitate the maintenance of a low endogenous concentration within the inner tissues and subsequent condensation into monoterpenes would then be localised within the epidermis. In contrast, it is proposed that undifferentiated tissue cultures produce monoterpene precursors during their growth, which may then be released as volatile 5-carbon compounds, thereby similarly maintaining the endogenous tissue concentration of monoterpenes at a minimal level. ©
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It is suggested that the "simple volatiles" reported by Becker (1970) in undifferentiated callus cultures of Pimpinella, Foeniculum, Levisticum, Origanum, Salvia, Rosemarinus and Mentha spp and those in Matricaria chamomilla (Reichling & Becker 1976) which disappeared with the formation of differentiated leaves and shoots; were also 2-methyl butan-l-ol and 2-methyl butan-l-al or closely related compounds. Becker noted the disappearance of such "simple volatiles" in cultures where morphogensis had been initiated, and at the same time recorded the development of an essential oil within the tissues. It is proposed that the decrease in the level of "volatiles" following culture morphogenesis was the result of their more effective conversion into monoterpenes within the secretory sturctures of newly differentiated plantlets.
Various authors have documented the basal metabolism of plant cell cultures derived from flavour and fragrance crops. Their broad conclusion (Kuzovkina 1975) that cultures do not retain the ability to synthesise the characteristic odour of the intact plant remains undisputed, though it could be more accurately stated such that "when monoterpenes are produced by plant tissue cultures, their composition often differs from that observed in the intact plant and they are present in much reduced quantities". This is supported by the present results and those of many other authors - Kireeva e^t aJ 1977, (Rosa damascena), 1978 (M piperita), Reznickova et^ al 1978 (R damascena, M piperita), Sardesai & Tipnis 1969 (Coriandrum sativum), Sugisawa & Ohnishi 1976 (Perilla frutescens), Tomoda et^ al^ 1976 (Jasminum grandiflorum, Ruta graveolens), Downing & Mitchell 1975 (Nepetaria cataria), Lang & Horster 1977 (Ocimum basilicum), Justice 1967, Banthorpe & Justice 1972c (Tanacetum vulgare) who have all independently reported the production of trace amounts of monoterpenes and derivatives in undifferentiated cultures of various aromatic plants. In all cases the essential oil was found to contain relatively few constituents, each of which would normally be found in the intact plant, but in different ratios. However, many of the early studies frequently suffered from a lack of relevant control materials, the use of relatively crude analytical procedures and in many cases the suitability of the culture material itself appears questionable. For example, experiments were often carried out using colourless, heterotrophic cells from stationary phase cultures, which had been grown under constant temperature and continual illumination. In contrast, the conditions employed in the present study reflected more closely those experienced by intact plants which were known to produce an essential oil of acceptable yield and quality. The variation in both the light and temperature regime employed in the present study gave rise to mixed, partially chlorophyllous cultures. Metabolically active cells from a consistently defined point in the log phase were used as experimental material.
The remainder of this thesis discusses the results obtained from a series of experiments whose purpose was to investigate the biosynthesis and accumulation of monoterpenes in M piperita cell cultures. The study was structured into three general areas and sought to answer a number of key questions. Firstly the influence of various environmental conditions were studied, secondly, biochemical means of enhancing monoterpene biosynthesis were investigated and finally, the control of culture morphogenesis and its influence upon monoterpene accumulation was assessed.
Is the microenvironment within culture vessels suitable for sustaining monoterpene biosynthesis?
Environmental conditions such as temperature (Clark & Menary 1980a, Burbott & Loomis 1967), light intensity (Cantoria 1974, Paupardin 1976) relative humidity (Croteau 1977) and post harvest conditions (Franz & Wunsch 1972, 1973, Hefendehl 1964a, Kalitzki 1954) have been shown to exert a marked influence over the content and quality of essential oil in intact Mentha spp (Baslas 1970,a, Virmani & Datta 1970). -189-
For the purposes of this investigation cell cultures were grown under an environmental regime previously shown to support monoterpene accumulation in chamber-grown plants. In adopting this approach it was considered noteworthy that whilst Jones & Krishnadethan (1973) succeeded in regenerating plantlets of Patchouli (Pogostemon cablin) under constant environmental conditions, biosynthesis of the sesquiterpene-based essential oil was only initiated following their exposure to a cyclical regime of cool nights and long days. However, it should also be mentioned that undifferentiated Pogostemon cablin suspension cultures failed to accumulate sesquiterpenes even when maintained under this apparently desirable regime.
The suitability of the specific microenvironment created within sealed culture flasks under the conditions selected for this study was investigated by regenerating M piperita plantlets from pre-sterilised nodal explants. The essential oil concentration was depressed by approximately 20% for plants grown within culture flasks, when compared with the equivalent dry weight of chamber-grown plant material. Plantlets developing within culture vessels exhibited weak growth, bore small leaves and generated an oil qualitatively typical of immature foliage. Pulegone comprised approximately 18% of the total essential oil.
These results are similar to those obtained by Bricout & Paupardin (1975), who reported that culture-grown plantlets derived from nodal explants of M piperita exhibited an oil profile similar to young tissues and infloresences. In a more extensive study (Bricout et al, 1978a) plantlets were regenerated from M piperita, M rotundifolia, M aquatica, M citrata, M viridis and M pulegium. Analyses of their monoterpene profiles revealed the presence of the same constituents as would normally be found in intact plants, but with a predominance of the more oxidised derivatives. -190-
For example they reported a pulegone concentration approaching 30% in cultured M piperita plantlets, though it should be stressed that with continous low level illumination at 600 lux; -2 equivalent to 1.7Wm over a 400-780nm spectrum; the culture conditions which they employed would not be experienced by plants grown normally. This was similarly the case with the work of Paupardin (1976), where cultured plantlets maintained _2 under 400 lux (1.2Wm ) produced many shoots yielding an essential oil which was comprised of 80% combined menthofuran and pulegone.
From analysis of the intact plant it has been concluded that the concentration of essential oil is usually highest in young tissues. It was surprising therefore that the determination for cultured material was less than the whole-plant-average for plantlets grown under open chamber conditions. However, since cultured plantlets exhibited a juvenile foliage, with a particularly low leaf-to-shoot ratio and since leaves are the primary site of monoterpene accumulation this could account for the reduction in the overall concentration.
One of the parameters which might differ significantly between the culture flask and open chamber enviroment would be relative humidity. High humidity; such as might be experienced within a sealed culture flask; has previously been shown to decrease the essential oil content of field- grown M piperita by effecting changes in the cell wall hydration and cuticle permeability of glandular trichomes (Croteau 1977). The latter phenomenon is therefore also hypothesised as contributing to the slightly decreased activity observed in flask-grown plantlets.
It was concluded that the conditions employed for the growth of cell cultures would support monoterpene biosynthesis in plantlets regenerated from nodal explants, although they promoted the formation of slightly reduced quantities of an "immature" type of oil. Is light a prerequisite for monoterpene biosynthesis?
Wide ranging evidence suggests that monoterpene biosynthesis may be related to the presence of light and/or photosynthetic activity in higher plants. Whether there is an obligate requirement for light or, whether photosynthates merely represent accessible carbon sources has wide implications for cell culture studies, and is a fundamental issue which has never been fully resolved even in the intact plant.
The presence of light has also been shown to affect qualitative aspects of the essential oil profile of Matricaria chamomilla (Szoke 1978, 1979) and Ruta graveolens cultures. (Reinhard et^ al 1968, 1971, Corduan & Reinhard 1972 and Nagel & Reinhard 1975).
Several authors; including Rogers et 14 Croteau (1972, a, b, 1973) discovered that whilst CO^ at physiological concentrations was the most efficient precursor of monoterpenes in M piperita; acetate and mevalonate were preferentially incorporated into the sesqui- and triterpenes. Similar results were obtained by Banthorpe et al (1975) and also by Gliezes et al (1980,a), who recorded the uptake of ^^CO^ into the mono- and sesquiterpenes of Pinus pinaster needles in the light. 14 Interestingly, they reported that the incorporation of 1- C- 14 and 2- C-acetate into monoterpenes was also strongly enhanced by light, though only achieving a level of some 10% of that 14 observed with CO^* The equal incorporation of 1- and 2-labelled acetate suggested that the substrate was probably initially degraded by the tricarboxylic acid cycle, and both C *2- - and C reincorporated as (X^ into terpenoids via photosynthetic carbon fixation. They also showed that high _2 intensity illumination (160 Wm ) had a strongly stimulating effect on monoterpene biosynthesis. Spectral maxima for the 14 incorporation of CO^ into monoterpenes were observed at 480 nm and 685 nm, though since no comparable action spectrum for photosynthesis was prepared it was not possible to determine whether monoterpene synthesis was regulated by simple availability of photosynthetic metabolites or the direct activation of another photodependent process. In 1972 Francis reported a light induced stimulation in the uptake of labelled glucose and sucrose into the sesquiterpenes of Pogostemon cablin leaf discs. It is suggested that light-induced stomatal opening may influence precursor incorporation by facilitating access of exogenous substrates into the metabolically active leaf tissues. However, it is also likely that ATP availability from photosynthesis may stimulate the active uptake of metabolites by plant cells, the subsequent utilisation of precursors depending upon competing and constructive metabolic pathways. Photosynthesis may generate competing substrates or divert exogenous substrates into primary growth, though in balance it appears to facilitate increased biosynthesis of monoterpenes and improved utilisation of exogenous precursors. Supplementary evidence has been provided by several authors who have recorded the presence of characteristic plastids; frequently enveloped with sheaths of endoplasmic reticulum; in glandular cells associated with the secretion of monoterpenes. (Wooding & Northcote 1965, a, Gliezes et_ al_ 1980, Araelunxen 1964, 1965, et_ al 1969, a, b, Arnott & Harris 1973, Akers £t al 1978, Hammond & Mahlberg 1978). -193- The role that plastids play in terpene biosynthesis has been discussed with particular reference to monoterpenes; by Gleizes et al (1982) and Carde e£ al (1980, 1982). In 1973 Rasmussen & Jones reported the natural occurrences and of liberation from illuminated leaf discs of Hamamelis sp; the five-carbon, terpenoid "Isoprene". Isoprene had been conceptually identified as an intermediate in terpenoid biosynthesis many years earlier, though only in its "active" forms as IPP and DMAPP. More recently Tingey et al (1980, 1981) examined the effect of environmental conditions on isoprene emission from oak and pine, and concluded that its production is linked to the biosynthesis of common intermediates of photorespiration such as glycolate. It has been shown (Jones & Rasmussen 1975) that the primary influence of light is to drive isoprene biosynthesis, though by affecting stomatal aperture and altering the resistance of the diffusion pathway, volatilisation and hence biosynthesis may be regulated to some extent. In fact, the vapor pressure of isoprene is such that variation in stomatal aperture only plays a slight role in regulating volatilisation. Isoprene emissions rapidly decrease in the dark as the endogenous pool becomes depleted - maintaining a low concentration within the leaf tissues at all times. Isoprene biosynthesis, its possible incorporation into other terpenoids, and the regulation of its metabolism via the volatilisation process has not been widely studied. Certainly, there is reason to believe that its metabolism and biogenetic relationship with other terpenoids in fragrant crops such as M piperita would warrant closer study. Light appears to have both direct and indirect effects on the accumulation of monoterpenes in plant material. Morphological changes affect the essential oil yield, whilst the photocontrol of physiological processes such as photosynthesis, photorespiration, plastid development and enzyme induction all undoubtedly contribute to observed changes in monoterpene metabolism and accumulation. For example it has been shown (Thomas & Stobart 1970) that during greening, the mevalonate kinase activity of Kalanchoe crenata callus cultures increased markedly, reflecting an increase in the numbers and development of photosynthetically active chloroplasts. If the chloroplastic isozyme of MVA kinase is capable of coupling into monoterpene biosynthesis then this could contribute to the higher levels of monoterpenes observed in light-grown, green tissues of M piperita. In the only study of its kind, Scora (1973) examined the essential oil content and composition of green tissues and the adjacent albino sections of leaves from a variegated cultivar of Myrtus communis. Although it is recognised that metabolic transfer within sections of the leaf lamina was likely to occur, his results showed that both albino and green foliage yielded essential oils which were qualitatively similar. Though all components were present at highest absolute levels in green tissues (average 4.5 fold concentration), by transforming his data it is possible to discover that alpha and beta pinene, 2-methyl butan-l-ol, 1,8-cineole and caryophyllene were all preferentially localised in the green tissues. Interestingly, this is similar to the situation reported in this study, where samples from internal tissues have also been shown to contain relatively high proportions of pinenes and 2-methyl butan-l-ol/al, when compared with epidermal samples. Since an exogenous carbon source (sucrose) was required for the maintenance of all plant material grown in culture, it was not possible to examine monoterpene biosynthesis in tissues solely dependent upon photosynthesis as a carbon source. However, the presence of light was shown to positively affect quantitative aspects of the monoterpene profile of cultured M piperita explants; though it did not appear to be an absolute prerequisite for monoterpene biosynthesis per se since dark-grown plantlets also produced trace amounts of monoterpenes. In 1976, Paupardin showed that the monoterpene content of cultured M piperita shoots was increased five-fold by increasing _2 the light intensity from 400 lux (1.2Wm ) to 4000 lux —2 (12Wm ). However, in the present study, increasing the light -2 -2 intensity from 1.6 Wm to 20 Wm actually resulted in a slight decrease in monoterpene content. It is suggested that here the rise in flask temperature, associated with the higher intensity lighting may have negated any increase in monoterpene biosynthesis per se. Plantlets regenerated in darkness produced small quantities of an essential oil which contained a high proportion of pulegone (approx 30%). It is presumed that the sucrose present in the medium was sufficient to sustain both plant growth and monoterpene biosynthesis. This would be consistent with the results of Bricout & Paupardin (1975) who demonstrated that glucose from the medium was the preferred substrate for monoterpene biosynthesis even when M piperita plantlets were cultured in the light. In a similar experiment, Benayoun & Ikan (1980) grew Pinus halepensis seedlings in darkness, nourished only by their own endosperm reserves. They concluded that resin biosynthesis can proceed both in darkness and in the light. In this case the resin; which contained various monoterpenes such as alpha and beta pinene, myrcene and limonene; was qualitatively similar from light and dark-grown plantlets, though the yield was some 2.4 fold higher in light-grown material. It is concluded that monoterpene biosynthesis occurs in plant tissues even under conditions of carbon shortage such as might be experienced in darkness. Whilst CC^ may be an effective monoterpene precursor in illuminated intact plants, it has been shown that other accessible carbon sources; such as sucrose, glucose or endosperm reserves can prove at least partially effective with cultured material. Although non-photosynthetic microorganisms such as Ceratocystis moniliformis (Lanza et al^ 1976, Lanza & Palmer 1977) and others (Collins 1976, 1979 Collins & Halim 1970, Sprecher & Haussen 1982) also produce monoterpenes, the variety of evidence discussed above has been taken to support the involvement of a plastidic pathway during the early stages of monoterpene biosynthesis in higher plants, or cells thereof. In non-photosynthetic plant tissues such as rose petals, there is also evidence that plastidic starch as a direct precursor of monoterpenes (Pogorel'Skaya e£ al 1980). The effect of light upon monoterpene biosynthesis is therefore seen as a complex interaction between morphogenetic changes; such as leaf size and structure; together with the stimulation of biosynthesis per se. However, it is significant to note that monoterpene biosynthesis is not exclusively a light-dependent process, since the cost of lighting and cooling large-scale ferraentors could represent a significant hurdle to achieving an economically viable means of supporting monoterpene biosynthesis in plant cell cultures. Can monoterpene yield be increased by restricting primary growth and freeing substrates for secondary metabolism? Monoterpenes are commonly regarded as secondary products, arising through the activities of a non-essential biochemical pathway (Vickery & Vickery 1981). It has been hypothesised therefore, that in common with many other secondary products; their biosynthesis may be restricted to periods in which the primary growth of the plant has ceased. It has also been proposed that metabolic resources will generally only be allocated to the production of secondary metabolites if the requirements of primary metabolism have already been satisfied (Luckner 1979). The accumulation of secondary products in plant cell cultures is, therefore, frequently associated with cell vacuolation and maturation at the end of the growth phase (Forrest 1969). At this stage the cells start to lyse, their vacuolar and cytosolic contents react and flavouring principles are often leached into the medium (Ogutuga 1970). The accumulation of various flavour components in cultures of Coffea arabica (Townsley 1974a, Frischknecht et al^ 1977) Theobroma cacao (Townsley 1974, Jalal & Collin 1979) and Camellia sinensis (Forrest 1969, Ogutuga 1970) is strongly linked to the stationary phase of growth. Similarly, Yeoman ej: al (1980) have shown that in order to increase the yield of the pungent principle capsaicin from cell cultures of Capsicum frutescens it is necessary to divert common precursors such as phenylalanine, away from the competing primary pathways of protein biosynthesis and lignin formation. For the purposes of this study the primary growth (dry weight gain) of M piperita cell suspensions was variously restricted by chemical means, whilst maintaining an adequate carbohydrate supply for possible utilisation in secondary pathways. At its simplest, removal of 2,4-D and BAP from the culture medium partially restricted cell growth, whilst reference to the literature aided in the selection of colchicine, gibberellic acid and chlorocholine chloride as potential inhibitors. These compounds were variously successful as growth inhibitors, each acting in a different fashion. Colchicine ^ 22^25^ 6 ^ was selected for a number of reasons, not least being its previously reported ability to treble the essential oil content of cultured M piperita shootlets (Bricout et al 1978). Closer examination revealed that in this case there were changes induced in the leaf form and leaf:shoot ratio which were probably responsible for the major changes in oil yield. However, colchicine also exhibits antimitotic properties, restricting growth and divisions in plant cell cultures by disorganisation and depolymerisation of the microtubular cytoskeleton. (Nooden 1971). Umetsu et: al_ 1975 studied the effect of colchicine on cell suspension cultures of Glycine max, and discovered that it induced a drastic reduction in cell aggregation, whilst promoting the enlargement of the resulting individual cells. Similar observations were made on Daucus carota cultures by Okamura (1979) and Lloyd et al (1980), who described three levels of ultrastructural aberration starting with irregular cell plate formation at 10 "*M, the formation of large spherical cells at -4 . . . 10 H and the extensive disorganisation of microtubular systems -3 at 10 M colchine. In the present study, colchine failed to significantly reduce dry weight gain beyond that effected by the withdrawal of 2,4-D and BAP, nor did it promote the accumulation of monoterpenes in undifferentiated M piperita cultures. Inhibition of the final stages in gibberellin synthesis was investigated as a means of enhancing the biosynthesis of lower terpenes. Whilst acting directly upon the terpenoid pathway, it is proposed that the reduced demand for higher terpenoids; because of restricted primary growth; may also support the biosynthesis of monoterpenes. Chlorocholine chloride (CCC) was prepared in ethanol for use at a final concentration of 1 millimolar - previously shown to inhibit the formation of Kaurene from geranylgeranyl pyrophosphate in Gibbberella fujikuroi (Shechter & West 1969, Echols et al 1981). CCC caused a noticeable reduction in dry weight gain of M piperita cultures though failed to generate any noticeable change in the monoterpene concentration. The application of CCC to intact peppermint plants is reported to cause the development of compact sturdy plants with small, dark green leaves - though the concentration of essential oil (as % of dry weight) was also found to remain constant (Hook et: a_l 1973). Interestingly, gibberellic acid itself has also been shown to inhibit the growth of M arvensis and M spicata cultures by Dobberstein (1966) and Lamba & Staba (1963) respectively. GA„ - 1 J was applied exogenously at a concentration of 5 mgl and led to a reduction in culture dry weight. -199- The dry weight gain of intact mint plants is also reported to be reduced (Gjerstad I960, Hefendehl 1964), though in the latter case, essential oil concentration (as % dry weight) was also quite significantly reduced as a result of a major shift in the leaf:shoot ratio. In summary, it appears that the simple restriction of primary • growth does not promote the accumulation of monoterpenes in undifferentiated M piperita cultures. Monoterpene concentration as a percentage of dry weight remains effectively constant over the experimental period. The effect of regulators such as colchine, CCC and GA^ on the intact plant or cultured • plantlets is further complicated by morphogenetic changes affecting the leaf:shoot ratio and laminal expansidn, which have significant effects on essential oil yield. Will the imposition of stress induce secondary metabolism and • the accumulation of monoterpenes? Various forms of stress are known to induce the formation of secondary products. For example, Yeoman et al (1980) successfully employed "nutrient stress" (low nitrogen and low sucrose medium) to effect a considerable increase in the capsaicin content of Capsicum frutescens callus cultures. Examples in which terpenoid metabolism has been affected by stress include the promotion of latex flow in Hevea brasiliensis by 2-chloroethyl phosphonic acid (2-CEPA) (Audley 1979), induced resinosis in Pinus spp following the application of paraquat (Brown & Nix 1975, Birchem ejt al 1979) and the production of sesquiterpenoid stress metabolites such as solavetivone, lubimin, rishitin and phytuberin by Solanum tuberosum when challenged by cultures of Phytophthora infestans (Alves e£ jil 1974). Furthermore, peppermint plants grown under moisture stress have been shown to produce high levels of soluble leaf sugars, which are subsequently utilised as both an energy and carbon source for monoterpene biosynthesis (Berry 1981). In all cases other than moisture stress, ethylene has been implicated as a common mediator of the changes in metabolism. -200- In the present study cells from log phase cultures of M piperita were exposed to paraquat, ABA and ethephon (2-CEPA) for periods • of up to 48 hours. Paraquat, being a highly polar compound; would be expected to be strongly absorbed onto the cellulose of cell walls relatively quickly (Brown & Nix 1975) though in this study, over exposure ( ^ 2 4 hours contact) resulted in rapid cell death with no enhancement of monoterpene biosynthesis. • These results are similar to those obtained by Birchem et: a_l (1979) who noted that whilst paraquat induces oleoresin synthesis in cells throughout the stems of Pinus elliottii seedlings; there was no initiation of monoterpene biosynthesis when cell cultures were treated. Cell cultures suffered • membrane damage and eventual death as a result of either prolonged exposure or increased concentrations of paraquat. The latter authors proposed that paraquat and related materials stimulate oleoresin biosynthesis via indirect means. Those f cells directly exposed to the reagent are disrupted and upon approaching senescence liberate increased levels of ethylene, which then diffuses within adjacent tissues. It is in fact this compound which is believed to stimulate oleoresin biosynthesis in unspecialised cells. The effects of ABA on M piperita cell cultures were similar to paraquat, though somewhat less severe. It has previously been shown by Ammirato (1974) that low concentrations of ABA tends to slow down cell divisions, improve embryogenesis and reduce the frequency of genetic aberrations in cultures of Carum carvi. However, no evidence to suggest that it enhances monoterpene biosynthesis was obtained during the course of the present study. - 201 It is well known that cell suspension cultures produce ethylene (Mackenzie & Street 1970, Gamborg & La Rue 1971) and that maximum production occurs at the approach to stationary phase (La Rue & Gamborg 1971). Ethephon (2-CEPA) is also known to generate ethylene jjl situ when applied to plant tissues (Yang 1969) and has previously been employed with Acer cell cultures to suppress differentiation and inhibit the rise in tannin production in the later stages of growth (Westcott 1976). In the light of the suspected involvement of ethylene in the regulation of certain areas of secondary metabolism, M piperita cultures were exposed to 2-CEPA. However, following short term treatment there was no detectable change in the qualitative or quantitative composition of volatile terpenes associated with M piperita cultures. High temperature stress was not validated as a means of enhancing monoterpene biosynthesis in M piperita cultures, although it had previously been reported to be effective with Jasmin cultures (Bush Boake Allen 1980). Such cultures were assessed by professional perfumers after various priods of 35°C temperature stress, and particularly those grown on media containing IAA were described as exhibiting a floral, jasmin-like odour. Jasmin cultures grown on IAA-containing media at normal temperatures, or in which IAA was omitted developed only a weak, vaguely floral aroma. Although not evident at the light microscope level (x 400) it is possible that IAA promoted some degree of morphogenesis or exerted a regulatory effect in Jasmin cultures. This might have facilitated the accumulation and/or excretion of various unidentified odouriferous compounds whilst elevated temperatures may have exaggerated this effect. Indeed it is even possible that certain products derived from the metabolism of IAA; provided at a 2 millimolar concentration; may also have contributed to the floral odour at high dilution. The ability of various forms of stress to induce monoterpene accumulation in M piperita suspension cultures was not validated in this study. It is proposed that in general, monoterpene biosynthesis is associated with metabolically active tissues, though accumulation may be restricted to cells dedicated for this purpose. Will the addition of precursors promote the biosynthesis and accumulation of monoterpenes? Shortage of precursors has frequently been cited as a reason for the low yield of secondary metabolites in plant tissue cultures. For example, undifferentiated onion cultures (Allium cepa) fail to generate an authentic flavour and aroma due partly to a shortage of precursors (Davey £t al 1974, Freeman et al 1974). In this case the precursor pool is only some 10% of that available within a comparable weight of tissue from the intact plant (Selby & Collin 1976, et^ al^ 1979). Similarly, Buckland (1975) has shown that suspension cultures of coffee (Coffea arabica) contain low levels of flavour precursors by comparison to the green bean (caffeine 0.038% vs 1.15%, chlorogenic acid 1.3% vs 6.5%). For the purposes of the present study, M piperita cells were supplied with a 1 millimolar solution of "available" 14 substrates. Earlier experiments with C-labelled substrates have shown that in many instances, biosynthetically produced terpenoids are non-uniformly labelled (Allen e_t aJL 1976). These observations have been interpreted as indicating that terpenoids are based upon an initial 5-carbon "primer" - Dimethylallyl pyrophosphate (DMAPP) to which successive molecules of isopentenyl pyrophosphate (IPP) are condensed to yield the higher homologues. (eg 10, 15, 20, etc carbon atoms). - 203 - Dimethylallyl alcohol was therefore selected for use with M piperita cells since it is reasonable to assume that if the above model is correct, there will be a high demand for DMAPP to facilitate monoterpene biosynthesis. However, although the substrate disappeared from the culture medium it did not appear to lead to an increase in the yield or variety of terpenoid compounds present in M piperita cell cultures. One of the earliest studies concerned with the production of secondary metabolites by plant tissue culture was performed by Arreguin & Bonner in 1950. They showed that cultured stem sections of Guayule (Parthenium argentatum) which had formed peripheral callus tissue would synthesise rubber; a polyterpene; when a leaf extract from the parent plant was added to the culture medium. They later showed that acetate, acetoacetate, acetone, glycerol and beta methyl crotonic acid (beta-MCA) could duplicate this effect (Bonner 1965). Since it possesses the same branched carbon skeleton as isoprene, the latter compound was selected as a potential terpenoid precursor. It was rapidly metabolised by M piperita cultures, though failed to generate any new terpenoid metabolites. The third potential substrate studied was mevalonic acid (MVA). First shown to be a key intermediate in terpenoid biosynthesis by Tavormina e_t al (1956), MVA has been shown to enhance the synthesis of a number of isoprenoid compounds in tobacco callus (Chen & Hall 1969) and to promote callus growth though direct incorporation into a cytokinin - active ribonucleoside (isopentenyl adenosine) (McChesney 1970, Murai et al 1975). Labelled MVA has also previously been supplied as a terpenoid precursor to callus cultures of Andrographis paniculata (Overton 1977) and Nepeta cataria (Downing & Mitchell 1975). 204 - In the former case, label was eventually transferred into paniculides A, B and C (sesquiterpene lactones) though there was * no net increase in overall pool size. With the latter case, even though the enzymes for MVA activation were able to generate MVA-P, MVA-PP and IPP, there was no production of the characteristic methyl cyclopentanone monoterpenes such as nepetalactone (Regnier et al 1968). • It is interesting to note that in plant tissues which accumulate significant amounts of terpenoids the level of free MVA is always very low (Wills & Scurr 1975). # In the present study none of the substrates led to an increase in the accumulation of free or bound monoterpenes associated with M piperita cells. However, both DMA-OH and beta-MCA were actively metabolised by cultures, disappearing from the incubation medium within 12 hours. The most likely explanation ^ for this would appear to be their utilisation in the biosynthesis of non-volatile, higher terpenes or their oxidative degradation as a result of endogenous epoxidase and epoxyhydrase activity associated with the cultures (Banthorpe et^ al 1972b, Banthorpe & Charlwood 1972d, Banthorpe et^ al 1977). Can chemical regulators enhance monoterpene biosynthesis and accumulation? The biosynthesis of higher terpenoids has been extensively studied and as a result, a number of pure chemicals have been identified which exert regulatory control over specific aspects of terpenoid metabolism. One of the most interesting groups of terpenoid "activators" is a family of compounds sharing a common structural form, based upon a trimethyl cyclohexyl ring structure substituted with a side chain containing unsaturated * double bonds (Dandekar et al 1980). In 1964 Reyes et al reported that the application of low levels of beta-ionone to cultures of Phycomyces blakesleanus led to an overall stimulation of isoprenoid biosynthesis, particularly carotenoids and sterols. They showed that beta-ionone deregulated higher terpenoid biosynthesis by influencing the activity of the rate-limiting enzyme; beta-hydroxy-beta- methylglutaric acid CoA reductase. The effect was validated by Lowry & Chichester (1967), who recorded increased incorporation 14 of (2- C)-MVA into the carotenoids and sterols of carrot root slices when exposed to beta-ionone. At the same time, Thomas et al (1967) reported that carotogenesis could be induced in the (-) strain of Blakeslea trispora by 18-carbon terpenoid derivatives; the trisporic acids. Trisporic acids (TSA) are found in nature as the diffusible chemicals responsible for the switch from asexual to sexual forms in Mucorales fungi. They also lead to a general increase in the accumulation of terpenoids within such tissues (Gooday 1978). More recently Dandekar et al (1980) studied the activation of carotenoid biosynthesis in Blakeslea trispora by TSA and structurally related compounds such as alpha and beta-ionones, vitamin A and ABA. Based upon such studies, beta-ionone and vitamin A (retinol) were selected as a possible means of enhancing monoterpene biosynthesis in M piperita cell cultures. Their influence on lower terpene metabolism has not previously been recorded. Retinol Beta-ionone Neither reagent led to a detectable increase in the level of free or bound monoterpenes during the course of a 48 hour incubation with log phase peppermint cells. However, beta-ionone was actively metabolised, falling to a low concentration by the end of the incubation period. It is possible that this reagent was directly incorporated into the higher terpenoids of the experimental material. It was concluded that monoterpene accumulation is regulated by means other than the normal point of control for higher terpenoid biosynthesis - the activity of beta-hydroxy-beta-methylglutaric acid CoA reductase. Are cell cultures capable of producing the variety of monoterpenes found in an intact plant? In general, most studies to date have shown that cell cultures of aromatic plants fail to produce the same spectrum of volatiles as is associated with tissues of the intact plant. For example, nepetalactone was not synthesised by cells of Nepeta cataria (Downing & Mitchell 1975) and isothujone was noticeably absent from Tanacetum vulgare cultures (Banthorpe & Wirz-Justice 1972c, Justice A.M. 1972); each of which would normally be a major constituent of their respective essential However, cultures frequently seem to possess traces of an aroma, which is comprised of some of the normal plant constituents and a variety of "novel" or unexpected compounds. Such has been the case with Matricaria chamomilla (Reichling & Becker 1976, e£ al 1978), Pimpine11a anisum (Becker 1970), Ocimum basilicum (Lang & Horster 1977) and Andrographis paniculata (Overton 1974). Therefore, although cultures may have been described as sweet smelling or with a mild aroma (Staba 1969, 1980) this is no guarantee that they are capable of producing a commercially acceptable replacement for the original plant product. Several authors have studied Peppermint and Spearmint cultures, failing to detect an essential oil similar to that of the parent plants. (Krikorian & Steward 1969, Becker 1970, Wang & Staba 1963). However, the work presented in this thesis has employed more refined and sensitive analytical techniques than available to earlier authors, and trace amounts of several monoterpenoid compounds have been detected within cultured material. It is postulated that the presence of low levels of an incomplete essential oil profile in such tissues may be due to an inability to produce or subsequently utilise one or more key intermediates. Since the addition of 5-carbon substrates did not enhance monoterpene biosynthesis, it was considered that cultured cells may be deficient in one or more of the enzymes of monoterpene interconversion itself. Cells were therefore exposed to various, purified monoterpenes in an attempt to identify any biosynthetic break. The earliest studies of this type were carried out by Staba (1965, 1969). However, working with Mentha cell cultures and applying monoterpenes at relatively high concentrations (6.5 millimolar) he failed to record any biotransformation of menthol, menthone piperitenone or pulegone. By comparison, individual monoterpenes at 2.5 millimolar concentration were relatively rapidly utilised by cells from the log phase of M piperita cultures in the present study. The monoterpenes - geraniol, r-pulegone, 1-menthone, 1-menthol and r-neomenthol appeared to be metabolically labile under fermentation conditions, rapidly disappearing from the medium at the same time as the temporary accumulation of related compounds. Recent studies by Galun & Aviv (1978), Aviv & Galun (1978) and Aviv et al (1981) have also confirmed that Mentha cell lines can perform single-stage reactions such as the conversion of pulegone to isomenthone and menthone to neomenthol respectively. In the present study menthyl glycosides were produced by cultured cells from menthone, menthol and r-neomenthol, though these too rapidly disappeared during the incubation period. Similar observations were also made by How (1970), who reported that lower terpenes were not accumulated in Rosa damascena cultures because such materials were either assimilated or degraded. When cultures were incubated with geraniol or nerol; geranial, neral, citronellol and their respective glycosides were subsequently detected as transient constituents of the culture medium (Jones 1974). It is proposed that the formation of monoterpene glycosides may be part of general detoxification process, such as is known to exist in the case of phenolic glucosylation by cultures of Datura innoxia (Tabata et al 1976). Jones (1974) also suggested that since geraniol; but not geranyl pyrophosphate (GPP); was utilised by the cultures to generate cyclic monoterpenes, the enzymes necessary to "decouple" GPP from an endogenous pool destined for higher terpenoid biosynthesis, might be absent or inactive, thereby limiting the biosynthesis of monoterpenes in cultures. However, since the uptake of GPP into cells was not documented it is questioned as to whether this is a valid statement or whether incorporation was limited simply as a result of differential membrane permeability towards the two components. There is certainly evidence to suggest that terpenoid metabolism is tightly regulated in vivo, such that even the addition of elaborate precursors such as monoterpenes themselves, may not lead to an increase in overall yield. For example, cannabinoids are produced by the condensation of an acyclic monoterpene (geraniol or nerol) with olivetol or olivetolic acid in the intact plant; and are then accumulated in the cannabis resin. Since derived callus cultures failed to produce the characteristic resin, they were exposed to an exogenous supply of geraniol (Itokawa et al 1976, 1977). Although the latter was rapidly utilised, it was simply transformed and appeared in the culture medium as citral, there being no indication of further cannaboid biosynthesis. A wide variety of species have now been studied by various authors and increasing evidence supports the view that living plant cells can effect biochemical transformations (Suga et_ al 1976, Reinhard & Alfermann 1980, Alfemnann & Reinhard 1980). However, in many cases the reaction products; such as monoterpenes; are unstable under culture conditions and are degraded rather than accumulated (Wood 1969). There also frequently appears to be relatively little culture specificity associated with certain reactions though there may be a marked stereo specificity for certain substrates (Itokawa et al 1976, 1977, Takeya & Itokawa 1977). For example, although tobacco does not normally accumulate monoterpenes, tobacco cell cultures have been shown to be capable of effecting monoterpene biotransformation in the current study, and also previously recorded by several authors. (Suga et_ al 1976, Suga & Hirata 1977) and summarised by Reinhard (1980). Figure 33. Products of geraniol biotransformation* 10-hydroxygeraniol Citronellal citral a citral b *Nicotiana tabacum (Reinhard & Alfermann 1980) 210 From the present study it became clear that even if M piperita cell cultures were supplied with simple monoterpenes, they were • unable to recreate the complete oil profile of the parent plant. Those biotransformations effected were; in the majority of cases; relatively unspecific and equally amenable to transformation by quicker growing tobacco cultures. In all cases studied to date, the products of monoterpene • biotransformation have failed to accumulate, but are rapidly remetabolised into non-volatile derivatives. Until this latter problem can be overcome the potential application of the technique will inevitably be limited. In • order to achieve effective biotransformation with minimal secondary losses it appears that the reaction products must be rapidly removed from the cell environment. Since the products of monoterpene biotransformation are frequently found in the suspending medium there may be several means of achieving this. 4 For example, cells could be grown and subsequently packed into a column through which culture medium containing the precursor is continously fed and then collected for extraction of the product(s). The contact time between precursor and cells will notionally determine the degree of biotransformation and the quantities of product available for recovery. Alternatively, the "exposed" medium may be recirculated via a solvent trap containing an inert hydrophobic solvent such as a natural triglyceride (Bisson et^ «il 1983). The latter would concentrate terpenoids and other lipophilic products, effectively removing them from the culture environment ready for purification and concentration. The regular reintroduction of fresh substrate would of course be required in both cases. If; as seems to be the case; monoterpenes are unstable under plant and microbial culture conditions (Wood 1969) it is hardly * surprising that undifferentiated cultures fail to accumulate such products. - 211 - The basal level detected in plant tissue cultures is therefore presumed to reflect an equilibium between biosynthesis and • subsequent utilisation. It is hypothesised that physical or metabolic compartmentation would be required if further accumulation of monoterpenes was desired since in this way, monoterpenes might be continually removed, localised and accumulated away from general metabolism. There is also • evidence supporting this hypothesis from the work of Vassiliev & Carde (1976); who showed that by "tapping" the resin canals of P sylvestis and P abies and thereby continuously removing the resin; the overall yield was increased considerably. • Can changes in intracellcular compartmentation enhance monoterpene accumulation? There is evidence to suggest that a variety of intracellular, metabolic pools may participate in terpenoid metabolism % (Banthorpe et al 1972a, Wu & Baisted 1973, Suga e_t a_l 1974, Allen et^ al 1976, Banthorpe & Ekundayo 1976) and also that intercellular compartmentation plays a role in the metabolism of 1-menthone in peppermint (Croteau & Winters 1982). It is therefore also reasonable to assume that in order for terpenoid metabolism to proceed, various enzymes and relevant precursors must be accessible to interact at certain points in time. Should the compartmentation of cultured cells be different from tissues of the intact plant, it may interfere with their ability to produce and accumulate monoterpenes. One of the best known cases where cellular compartmentation is responsible for restricting flavour generation is that of onion. Both in the intact plant and derived cell cultures the flavour is only developed when separately compartmentalised precursors (alkyl-l-cysteine sulphoxides) and the enzyme * "alliinase" are brought together by maceration or disruption of the tissues (Freeman 1974). 212 - In the present study 5% (v/v) DMSO was used to reversibly alter the compartmentation of cultured M piperita cells by increasing the permeability of cell membranes (MacGregor 1967, Rammler & Zaffaroni 1967, Delmer 1979). In light of the results obtained from biotransformation studies with M piperita cells, and the perceived need to continually remove monoterpenes from the culture environment if they are to be stabilised, it would seem that if DMSO were to facilitate the release of monoterpenes from cell material it may prove a valuable adjunct to any process of this type. However under normal incubation conditions DMSO alone failed to effect any detectable changes in the terpenoid profile over a 24 hour contact period. The compartmentation of monoterpene metabolism has been studied in Pine needles by Gleizes et al (1980a). They found that when fragments of needles; rather than intact tissues; were employed, monoterpenes were not labelled by ^CO , ^C-acetate, 14 14 . C-MVA or C-IPP though their uptake into sesquiterpenes was invariably increased. By fractionating the fragmented tissues after a period of 1-^C-acetate uptake, the same authors were able to show that the label was predominantly associated with sesquiterpenes in the endoplasmic reticulum. However, since lipophilic terpenoids would automatically tend to migrate into the membrane fraction, these results can only be taken as being indicative of the differential compartmentation of mono- and sesquiterpene biosynthesis. Indeed, recent evidence strongly suggests that anatomical and morphological features of the intact pine needle may be more important factors in determining monoterpene accumulation (Bernard-Dagan et al 1980), such that lone tissue fragments would not carry out effective monoterpene biosynthesis. Is monoterpene accumulation linked to morphological differentiation? There is an increasing body of evidence to support a correlation between the "differentiation" of plant cell cultures and secondary metabolite production (Yeoman et al 1980, 1981). In general, cultures which undergo organogenesis appear to produce secondary compounds in amounts and proportions approaching those of the intact plant (Reinhard et al 1968, Becker 1970, Corduan et al 1972, Tabata £t al^ 1972) whilst undifferentiated material only produce trace amounts (Goodwin & Williams 1962, Williams & Goodwin 1965, Reinhard et al 1971) or novel compounds (Butcher & Connolly 1971, Overton & Picken 1977, Overton 1977). Since plant cells are totipotent, theoretically at least, all metabolites which are found in the intact plant can be expected to be found in their derived cell cultures, provided that these can be induced to follow the same biochemical and structural modifications as do cells of the intact plant (Constabel et al 1974). Cell cultures may therefore be suitable for the production of secondary metabolites provided that their growth can be regulated in a relevant fashion. However, cells which divide with short interphases do not usually accumulate secondary products, even though their daughter cells may quickly become differentiated, facilitating the expression of a more specialised metabolism. "Differentiation" does not exclusively imply a morphogenetic response, since many aspects of cellular specialisation are only detectable at the metabolic level (Bornmann 1974, Bohm 1977). However, the production of many secondary metabolites; including monoterpenes; is seen as a complex sequence of events comprising biosynthesis, transport, accumulation and excretion/storage. These processes may occur within individual cells; but it often appears that the participation of several tissues is necessary. For example, although monoterpenes are generally accumulated within specialised glands or cavities there is evidence to suggest that key intermediates in their biosynthesis may be largely produced in adjacent mesophyll tissues. This segmentation of metabolism within the plant poses several problems for the cell culture experimentalist. Firstly, in order to produce biosynthetically active cell cultures it may be necessary to initiate them from particular tissues within the plant such as where biosynthesis or accumulation occurs naturally. Secondly, if the interaction of several specialised tissues is necessary for secondary metabolite production within the intact plant, how can cell culture systems be developed to cope with this requirement? In general, the selection of a cell line from a high yielding plant will also potentially be high yielding (Townsley 1977, Widholm 1980, Yeoman et ^1 1980, Chaleff 1981) but cell cultures derived from various plant parts that differ in their secondary metabolism, usually show a uniform metabolism themselves. However, at least one important exception to this statement has been documented. The essential oil of Ruta graveolens consists of aliphatic hydrocarbons (Cg and C ^ ) and various terpenes; the former predominating in the shoots and the latter in the roots of the intact plant. Nagel fie Reinhard (1975) initiated callus cultures from stem sections and found that they produced hydrocarbons when cultured in the light, and the terpene fraction predominantly in the dark. In contrast, callus derived from root tissues only synthesised terpenes, irrespective of illumination. In the latter case the cultures functionality appeared to be restricted by the initial potential of the explant. The continuation of secondary metabolism in cultured explants of various species is well known from biochemical studies (Brown & Wetter 1977). In many instances tissues of the original explant remain metabolically active, with only slight changes occurring in its composition. However, newly developed callus tissues frequently fail to produce the materials of interest, unless there is some degree of accompanying cellular specialisation. The development of terpenoids has been documented for cultured stem sections of Parthenium argentatum (Arreguin & Bonner 1950), shoot apices of Foeniculum vulgare (Garcia-Rodriguez et al 1978) and explants of citrus pericarp (Billy & Paupardin 1971, Chablier & Paupardin 1973). All evidence suggests that the presence of a relatively large explant will affect the growth of adjacent callus in such a way as to cause biochemical and occasionally morphogenetic changes. In the above cases, de novo terpene accumulation was associated with small groups of parenchymatous cells within the callus, though whether biosynthesis was within the callus itself or the explant was not established on any occasion. The differentiation of certain organs during the morphogenesis of cell cultures often leads to a marked change in the spectrum of secondary products present. In practice, because of the interdependence of various tissues involved in secondary metabolism, it appears that varying degrees of morphogenetic differentiation, up to the regeneration of complete plantlets may be necessary to facilitate reproduction of the full, species-specific pattern of secondary metabolites in plant cell cultures. This phenomenon is best illustrated by reference to specific examples. 1) Young calli of H. brasiliensis failed to accumulate rubber latex until rootlets were initiated (Wilson & Street 1975). 2) The addition of a water soluble leaf extract to Parthenium argentatum stem/callus cultures promoted latex formation, suggesting that leaves may be the natural source of precursors (Arreguin & Bonner 1950). 3) Undifferentiated onion cultures do not produce flavour and lachrymatory principles or precursors (Freeman 1974). The formation of roots led to an increase in the 3 trivial flavour components - s-allyl, s-methyl and s-propyl cysteine sulphoxides though lachrymatory potency and flavour impact remained weak (Turnbull & Collin 1978, Selby et al 1979). Development of shoots on "rooted" cultures led to a further increase in lachrymatory potency and the production of the key flavour compound s-propenyl cysteine sulphoxide (Turnbull & Collin 1978, Turnbull et al 1980) 4) Undifferentiated cultures of M. chamomilla (Reichling & Becker 1976, Reichling eit al 1978) Anethum graveolens and Pimpinella anisum contain high levels of a "simple" volatile which is atypical of the parent plant and which disappears with the formation of shoots (Becker 1970). Similar "volatiles" present in undifferentiated cultures and parenchymatous tissues of the intact plant have been identified as 5-carbon terpenoids in the present study. 5) Undifferentiated cultures of M chamomilla do not produce an essential oil whilst maintained in isolation. However, crown gall tumours induced on the intact plant produce a normal essential oil contained within secretory cavities (Reichling £t^ al 1978). 6) Organogenesis is necessary; and roots play an important role; in the biosynthesis of an essential oil in tissue cultures of Eucalyptus citridora (Gupta 1983). Morphological differentiation can occur to various levels of complexity within cell cultures and be independently controlled with varying degrees of success. Since studies of the intact plant have shown that the root system of M piperita does not participate in essential oil metabolism (this study, Dimitrova et al 1961, Murray & Lincoln 1970) other than as a possible depository for menthy1-glucoside after flowering (Croteau & Martinkus 1979), it seemed unlikely that rootlet regeneration would significantly enhance monoterpene accumulation in peppermint cell cultures. In practice, roots were predictably initiated on cultures relatively easily through the use of a medium containing a high auxin; cytokinin ratio (eg 2,4-D, 0.1 mg 1 BAP, 0.01 mg 1 ^"). However, as expected this in itself had no detectable influence on the monoterpene profile of cultured material. Shootlet formation and de novo embryogenesis were considered potentially more likely means of enhancing monoterpene accumulation in cell cultures. This approach has previously been widely employed with cell cultures of the Umbelliferae and Labiatae, where the characteristic plant biochemistry appears to be dependent upon morphogenetic specialisation. Cultures of Umbelliferae have the added attraction that they are also particularly responsive to relatively simple means of controlled morphogenesis (Steward & Ammirato 1970). Whilst cultures of Coriandrum sativum, Anethum graveolens, Pimpinella anisum, Conium maculatum, Apium graveolens, Carum carvi, Ruta graveolens and Matricaria chamomilla consist of free cells and unorganised clusters they do not possess the characteristic odour of the parent plant. However, once organisation commences; even though the structures are minute; aromatic principles are accumulated in newly differentiated glands and cavities, imparting cultures with the familiar odours of the complete plant (Krikorian 1964, Becker 1970, Reichling & Becker 1976). Mono- and sesquiterpenes, carbonyl compounds such as phthallides, and a number of alcohols are constituents of the aroma and flavour of celery. The main contributors to the characteristic flavour are the phthallides - 3,n-butylhexahydrophthallide, 3,n-butyl phthallide and sedanolide, all of which are absent from undifferentiated cultures (Al-Abta et^ al 1978). However, their production has been successfully initiated in both callus and suspension cultures by inducing embryogenesis through the withdrawal of 2,4-D in the presence of a low level of Kinetin (Williams 1976, Al-Abta et al 1979, Al-Abta & Collin 1978). 218 - The successful production of secondary metabolites frequently appears to depend upon obtaining the correct type and degree of differentiation, together with an environment which favours secondary metabolism. Often these factors are closely linked; such that although Hart ert al (1970) successfully regenerated plantlets of Pogostemon cablin, these failed to accumulate the characteristic sesquiterpenes of the parent plant until they had been exposed to specific variations (light and temperature) in the culture regime (Jones 1973). Qualitative changes in the terpenoid profile of light and dark-grown stem-derived Ruta cultures was also associated with morphogenetic developments. Light-grown cultures regenerated shootlets, in which typical lysigenous and schizolysigenous secretory glands were developed in leaves and at the periphery of the callus (Reinhard et al 1968, Peterson et al 1978). Under continued illumination the central gland cells underwent gradual lysis, forming a lumen into which essential oil was excreted from adjacent tissues. Cultures maintained in the dark failed to regenerate viable shoots, and a typical "root" oil was accumulated within groups of parenchymatous callus cells. Secretory cavities were not formed (Corduan & Reinhard 1972). In the present study, shoot regeneration from callus material proved difficult and unreliable (see also Lamba 1963), although a small number of plantlets were obtained from cultures transferred to M & S medium containing 0.1 mg 1 ^ 2,4-D and 1 mg 1 ^ FAP. Visually, the shoots appeared similar to those obtained from nodal explants in culture, though essential oil yield; at 0.9% of dry weight; was some 30% reduced. The regenerated shoots bore glandular structures, but in many instances the stalk cell was brown and necrotic, the secretory cells collapsed and lacking essential oil. In an attempt to improve the frequency and viability of shoot regeneration from M piperita cultures various additives were included within the culture medium. By replacing 2,4-D with NAA, or including GA^ in the medium (as advocated by Pillai & Hildebrandt (1969) and Ammirato (1974, 1977)) culture growth was visibly slowed and tissues were greener and more compact. However, even after 8 weeks growth, there were no signs of shoot regeneration, at a micro or macro leve1. By inhibiting the basipetal transport of endogenous auxins, 2,3,5-triiodobenzoic acid has been shown to overide apical dominance, and thereby encourage the formation of many shoots from cultured explants of Lycopersicum esculentum and Pelargonium spp (Cassells 1979). Although treated M piperita cultures appeared compact, green and healthy, there was no sign of de novo shoot formation. Similarly, TIBA can also initiate callose formation, causing cellular isolation and degeneration (Bouck & Galston 1967). Although such changes might facilitate the localised accumulation of monoterpenes in Mentha cell cultures there was no significant change in the profile of treated compared to control callus material. Activated charcoal has also previously been shown to promote morphogenesis in cultures of Daucus carota, Allium cepa (Fridborg & Erikson 1974) and Nicotiana tabacum (Anagnostakis, 1974) by absorbing leachable metabolites, including endogenous auxins. Whilst promoting root regeneration in M piperita cultures however, its use failed to stimulate shoot formation or embryogenesis. Are cultures genetically stable? Today the concept of the uniform, "true to type" clone is regarded with some suspicion when cell culture techniques have been applied in the multiplication or maintenance of cell lines (Chaleff 1981). This is based upon the rationale that most explants contain a variety of cell types, that genetic aberrations such as polyploidy frequently occur during the culture period (Wilson & Street 1976) and that the regulators used to maintain the growth of plant cell cultures often induce morphogenetic and biochemical changes (Hart et^ al^ 1970). These factors are a major potential drawback to the industrial application of cell cultures for the production of secondary metabolites - where consistent quality and yield are required; but represent an exciting opportunity for the development of new cultivars with novel secondary products, improved yield, disease and drought resistance for example. In the present study, it was intended to fingerprint the essential oil profiles of regenerated plant lets, as an indicator of culture stability were it possible to reliably regenerate such material. Unfortunately this was not achieved. However, reference to the literature provided several examples of cases in which tissue cultures and their derived plantlets of Pelargonium spp (Pillai & Hildebrandt 1969, Skirvin & Janick 1976), celery (Williams & Collin 1976, 1976a) and onion (Selby & Collin 1976) were morphogentically and biochemically varied, including changes in essential oil yield and composition. Currently, the only means of minimising genetic variation during cell culture is to minimise the number of in vitro cell divisions by slowing growth. The inclusion of low levels of ABA has proven partially successful in this respect with embryogenic cultures of Carum carvi (Ammirato 1974, 1977). When one considers that fast growing cultures are required for commercial processes, and that in the case of H brasiliensis up to 40% of cultured cells were polyploid within two such culture passages (Wilson & Street 1976) there remains considerable need for further work in this area. Problems and Perspectives Much of the work presented in this thesis suggests that monoterpene metabolism in plants and their derived cultures is primarily regulated by an overriding requirement to minimise the level of free monoterpenes within the tissues at all times. Evidence supporting this hypothesis includes:- 1. The accumulation of monoterpenes exclusively within specialised glands in the intact plant. 2. When monoterpenes are found in unspecialised plant tissues they are at a low level, or are present as water soluble derivatives (e.g. monoterpene-glycosides). 3. Undifferentiated plant cell cultures produce only trace amounts of a limited range of monoterpenes, although they appear to generate relatively large amounts of 5-carbon compounds which are related to known terpenoid precursors. These same compounds - 2-methyl butan-l-ol and 2-methyl butan-l-al; have also been found in the internal tissues of intact plants. It is hypothesised that their volatilisation from cell cultures; where there is no facility for localised monoterpene accumulation; may help to maintain a low endogenous terpene concentration. - 222 - 4. Morphogenesis; even the formation of relatively simple embryoids; is frequently correlated with the accumulation of lower terpenes and characteristic secondary metabolites in plant cell cultures. 5. Addition of substrates such as DMAPP and MVA fails to stimulate monoterpene biosynthesis in undifferentiated cell cultures. This would be consistent if monoterpenes regulate their own biosynthesis via feedback repression (see Gray & Kekwick 1972). 6. Cultured plant cells rapidly metabolise exogenous monoterpenes, related compounds temporarily accumulating in the culture medium. Monoterpene concentrations above 5 raillimolar appear to be cytotoxic, causing visible necrosis of cultures. This particular phenomenon imposes a number of theoretical and practical contraints on the utilisation of plant cell cultures for the production or modification of monoterpenes. These can be summarised by the following general requirements which will need to be satisfied prior to commercial exploitation (Puhan & Martin 1971, Lee & Scott 1979, Ruttloff 1982). 1. The rates of cell growth and product biosynthesis must be increased significantly. 2. The cultured cells must be genetically stable, ensuring a constant yield of consistent quality. 3. The product(s) should ideally be excreted into the medium, or at least be accumulated to high levels by the cells with catabolism minimised. 4. Cell culture viability must be significantly extended. 5. Production costs; including the medium, necessary precursors and extraction/concentration processes should be low enough to make the process profitable. Experiments with various suspension cultures suggests that their growth rate can be accelerated considerably by improving culture conditions (Kato £t al 1972, Yasuda et al^ 1972) and the selection of particular cell lines (Townsley 1977, Williams & Collin 1976a). Although some authors have also demonstrated increased recovery of secondary metabolites from specific cell lines (Widholm 1980, Mok eX al 1976, Yeoman e_t jil 1980) their physiological and biochemical manipulation have been less well studied, particularly when combined with the controlled modification of culture environment. The potential of this combined approach is illustrated by the early work of Nishi (1974) who treated a population of Daucus carota cells with the mutagen; N-methyl-N'-nitro-N-nitrosoguanidine. A "high carotenoid" strain was subsequently selected, and the accumulation of lycopene found to parallel the log phase growth of the culture, Data of this type forms a solid foundation upon which the further manipulation of cultures is possible. For example, one attractive venue appears to be the rapid production of biomass (growth phase) which can then be subsequently manipulated under a "production phase" to yield large quantities of the specified products. Liquid nitrogen appears to be the most suitable method for preserving the genetic stability of stock cell cultures (Nag & Street 1975) . However, there is currently no reliable means of controlling the nuclear or cytoplasmic variations which may occur during the scale-up and routine growth of cell cultures (Dougall 1980a). ft - 224 - The likelihood of such aberrations appears to be promoted by rapid growth and the inclusion of plant growth regulators • (Pillai 1969, Skirvin 1976). Conversely the addition of low concentrations of ABA, has been shown to significantly reduce morphological aberrations in embryogenic cultures of Carum carvi (Ammirato 1977), whilst barely affecting the growth rate. Clearly, this is an area where there is still a great need for • fundamental studies. Investigation of the third problem, concerning the release of cellular metabolites into the culture medium may be important in avoiding possible negative feedback or repression due to the 41 excessive accumulation of the final product within cultured cell. This approach may be particularly relevant in the case of lower terpenes, since they are frequently excreted into intercellular or subcuticular cavities in the intact plant, and also appear to be excreted following their metabolism by cell • cultures. (Jones 1974, How 1970, Itokawa 1977, Lang et al 1977). There is evidence to suggest that the production of certain metabolites can be significantly increased if it is possible to alter the permeability of the cellular membrane. For example, a surface-active agent has been successfully used by Tanaka et^ a_l (1974) for the purpose of extracting up to 20% of dry weight of glutamine from cultures of Symphytum officinale. An interesting alternative approach to enhancing the accumulation of monoterpenes was reported by Robinson (1978) who ^ attempted to select cell lines from Rosa damascena cultures which were deficient in terpenoid degrading enzymes and also exhibited a relatively high tolerance to natural monoterpenes. Unfortunately the detailed results of his work were never published. t - 225 - Since frequent sub-cultures are time consuming, expensive and increase the risk of contamination, there is a great need to extend the viability of cells under fermentation conditions. Basic studies which have considered the problems of cell sedimentation, culture growth on vessel walls and cell flotation together with their effects on growth rates have been reviewed by Wagner & Vogelmann (1977). The high viscosity of cultures due to the large volume and relative rigidity of plant cells has been partly overcome by the use of low shear aeration/agitation methods. However, there has been little real success to date in extending culture viability and maintaining high cell densities by biochemical means. Gathercole & Street (1967), King (1976), Westcott & Henshaw (1976), Withers (1976) and Lang & Horster (1979) have identified the accumulation of phenolics as being associated with slowing of cell growth, and Siegel & Enns (1979) adopted an original approach when they attempted to prolong cell culture viability by the use of PVP. However, it has been shown in the present study that whilst this particular material extends the viability of cultures by slowing growth, it does not lead to an increased yield of biomass during the culture period. The fifth requirement is related to the economic problems of industrial production, which is itself dependent upon the demand for the product and the total production costs (Goldstein e£ a\_ 1980). Since a large proportion of the production cost relates to the medium, power for aeration and temperature regulation, these factors must be minimised. Ideally, cell cultures will be maintained on a cheap carbon source such as molasses, starch or alcohol, and by reducing the time between culture initiation and product formation other costs will also be reduced. For example, the introduction of a semi-continuous process may not only reduce sterilisation costs and the need to continuously replenish culture stocks, but also allow a programmed change in the medium composition, thereby providing a possible opportunity to exert close control over the biosynthetic activity of the cells. - 2 2 6 - Undoubtedly, one of of the most attractive commercial applications of plant cell culture in the short and medium term » appears to lie in the development of novel varieties of crops destined for fragrance, flavour and pharmaceutical production (Karasawa e£ jal 1977). The genetic variability which appears to be intrinsically • associated with rapidly dividing cell cultures may provide a means of rapidly expanding their gene pool. Since most flavour and fragrance crops are cultivated from traditional unselected populations there would appear to be good opportunities for selecting new cultivars with improved yield, a profile of novel I compounds or disease and drought resistance. Many such crops are currently vegetatively propagated in order to maintain "true-to-type" stocks of predictable and recognisable biochemical composition. Provided there is minimal • carryover of exogenous growth regulators which might interfere with subsequent growth and product quality (Hart et £1^ 1970, Moshonas & Shaw 1980); plant cell culture should make a significant contribution in the near future. Indeed it is encouraging that the basics of in vitro multiplication have already been successfully developed for several essential oil crops including geranium (Cassells 1979, Pillai & Hildebrandt 1969, Skirvin & Janick 1976), Sandalwood (Bapat & Rao 1979) Lavandula (Quazi 1980) and Eucalyptus (Gupta 1983). ^ However, when one considers the enormous acreage of high technology agriculture that are devoted to crops such as Peppermint and Spearmint, it is perhaps hardly surprising that the first commercial applications of plant tissue culture are most likely to be in offering new varieties of such species for cultivation by relatively traditional means. - 227- Plate 14. Open Field Cultivation of Peppermint in the USA, still a highly efficient process >• 6. SUMMARY MONOTERPENE METABOLISM OF MENTHA AND ITS CELL CULTURES Summary Vegetative plants of Mentha piperita contain approximately 1.5 percent of essential oil by dry weight. The leaves contain more than ninety-five percent of the oil; of which at least 90 percent appears to be located within the epidermal oil glands. Oil glands develop at an early stage in shoot ontogeny and rapidly accumulate essential oil. The underlying mesophyll tissue is compact and appears classically developed for the symplasmic transport of oil or its precursors into the epidermal glands. The composition and yield of oil was observed to change during leaf development. Initially present in large amounts were r-pulegone and 1-menthone. As development proceeded their level decreased, with a concomitant increase in the concentration of menthol isomers. Finally, acetylation and glycosylation of menthols occurred. The possible role of terpene-glycosides as water soluble, precursors of essential oil was investigated. Low levels were found to occur naturally in vegetative plants and tissue cultures. The liberation of terpene alcohols from glycoside bound forms may be controlled by the distribution and compartmentation of glycosidase enzymes. However attempts to discover the localisation of B-glucosidase, using the diazotization of para-acetoxy mercurianiline were unsuccessful. Callus and suspension cultures were grown on Murashige & Skoog medium, with the addition of 3 percent (w/v) surose, 1 mgl ^ 2,4-D and 0.1 mgl ^ BAP. Culture growth was heterogeneous. Cultures did not produce an essential oil characteristic of the parent plant, but trace amounts of the steam volatile compounds; 2-methyl butan-l-ol, 2-methyl butan-l-al, alpha-terpinene, 1-menthol and its acetate were detected in cell cultures during their growth. Light did not affect the monoterpenes present in tissue cultures, but with intact plants oil yield was increased by light, due in part to an increase in the leaf-to-shoot ratio. Exposure to stress factors; paraquat, 2-chloroethyl phosphonic acid and abscisic acid; failed to initiate the accumulation of increased levels of monoterpenes in Mentha cell cultures. Application of beta-ionone and retinol; reported to deregulate an early rate-limiting stage in terpenoid metabolism and improve the incorporation of precursors; failed to increase the concentration of free or bound monoterpenes in suspension cultures. The hypothetical precursors of monoterpenes; dimethyl allyl alcohol, mevalonic acid and beta-methyl crotonate; were metabolised by cells of log phase suspension cultures, but did not lead to enhanced accumulation of free or bound monoterpenes. Cells of log phase suspension cultures have been shown to possess the ability to interconvert enzymically, a number of monoterpenes normally found in Peppermint oil. In cases where 1-menthol was formed there was also a subsequent accumulation of menthol glycoside. -231- 12. Essential oil accumulation was associated with morphological differentiation in intact plants. Attempts to induce controlled shootlet regeneration in cell cultures by the use of active carbon, triiodobenzoic acid, gibberellin A.,, chlorocholine 6 ^ chloride, naphthalene acetic acid, N -Benzyladene, furfuryl adenine, indol-3yl-acetic acid and indolebutyric acid, alone and in combination failed to yield consistent results. In summary, monoterpene biosynthesis appears to be primarily associated with actively growing tissues in the intact plant, important precursors originating within the mesophyll. The accumulation of essential oil is exclusively associated with morphological differentiation both within % the intact plant and cell cultures. Callus and growth suspension cultures exhibited a similar terpene profile as parenchymatous cells of the intact plant, predominantly the 5-carbon compounds 2-methyl butan-l-ol and 2-methyl butan-l-al. The • regulation of monoterpene biosynthesis appears to be tightly regulated in vivo, and various attempts to increase the yield or diversity of products in cell cultures were inconclusive. The best opportunities for the commercial application of plant cell culture techniques with essential oil crops appears to lie in the development of new varieties for subsequent cultivation by relatively traditional means. BIBLIOGRAPHY Ahlgrimm E.D. (1956) The biogenesis of secondary products of metabolism in Mentha x piperita and varieties of Fagopyrum. Planta 47 pp 255-298 Akers C.P., Weybrew J.A., Long R.C. (1978) Ultrastructure of glandular trichomes of leaves of Nicotiana tabacum L. cv Xanthi. Amer. J. Bot. b5 (3) pp 282-292 Al-Abta S., Galpin I.J., Collin H.A. (1979) Flavour compounds in tissue cultures of celery. Plant Sci. Letts. 16> pp 129-134 Alfermann A.W., Reinhard E. (1978) Production of natural compounds by plant cell culture methods. Proc. Int. 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SEB Special Symposium on Plant Biotechnology % - 269- APPENDICES - 270- APPENDIX 1 Naphthol ASBI B-D-Glucopyranoside ~ Substrate Stock Solution 1 . 20 mg of substrate dissolved initially into 2mls MeoH and dilute to 10 mis with Mcilvaine buffer pH 5. * 2 . dilute 1 : 1 0 prior to use (0.2 mg ml ^ substrate cone11) with 20% MeoH prepared in buffer pH 5. Diazotization of P-acetoxymercanic aniline 1. lg acetoxymercuric aniline dissolved in 50% acetic acid at room temperature to 25 ml final volume. Chill on ice for in excess of 45 minutes. 2. 2g NaNO^ dissolved in 50ml distilled water Chill on ice for in excess of 45 minutes. 3. Take 25 mis from (1) and make up to 50mls with (2) keep on ice and mix well Leave for 15 mins, until brown precipitate forms. 4. Filter (Whatmans No 1) and discard residue. Collect the filtrate on a chilled vessel. (STABLE 12 HOURS) V 5. Dilute (4) 1:10 with 0.1M phosphocitrate buffer and adjust pH to 5.0. (USE WITHIN 15 MINUTES) - 271- APPENDIX 2 Plant Cell Culture Media Fonnalations Murashige & Skoog Gamborg B5 COMPONENT Mgl" 1 mM Mgl- 1 mM NH4 N03 1650 20.6 -- KNO3 1900 18.8 2500 25 CaCl2.2H20 440 3.0 150 1.0 MgS04.7H20 370 1.5 250 1.0 KH2P04 170 1.25 -- (NH4)2S04 - - 134 1.0 NaH2P0 4.H20 — 150 1. 1 MICRONUTRIENTS Mgl""1 uM Mgl- 1 uM KI 0.83 5.0 0.75 4.5 H3BO3 6 .2 100 3.0 5.0 MnS04 .4H20 22.3 100 -- MnS0 4.H20 - - 10 60 ZnSO4 .7 H.3O 8.6 30 2.0 7.0 Na2Mo0 4.2H20 0.25 1.0 0.25 1.0 CuS04.5H20 0.025 0 .1 0.025 0 .1 Co C12 .6H20 0.025 0 .1 0.025 0. 1 Na2EDTA 37.3 100 37.3 100 FeS04.7H2 27.8 100 27.8 100 MICRONUTRIENTS Mgl* 1 uM Mg]."1 uM Nicotinic Acid 0.5 4.0 —_ Thiamine HC1 0.1 0.3 10 3 Pyridoxine HC1 0.5 2.4 1.0 4.8 Glycine 2 0.3 - - Casein Hydrolysate 10 - - - myo-Inositol 100 555 100 555 Murashige T. & F. Skoog (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15 pp 473-■497. Gamborg O.L., Miller R.A., Ojima K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 5() pp 148-151 « MICRODETERMINATION OF ESSENTIAL OILS IN PLANT TISSUES Hudson M.J. & Goldsworthy A» Botany Dept. Imperial College, London UK In order to study the distribution of essential oils within the cells and tissues of natural botanical material and plant cell cultures, a rapid and detailed microanalysis, performed by "direct-volatilisation gas-liquid chromatography" is employed. The contents of individual oil glands can be withdrawn into the lumen of a fine glass capillary, which is then transferred into the injector of the gas chromatograph. By controlled heating, the volatile components are driven off and swept onto the GC column by the flow of carrier gas. The technique has been successfully applied to studying:- i) the variation and changes in essential oil content/composition in various glandular and non-glandular tissues of intact plant in the Genus Mentha. ii) the correlation between morphological specialisation in Mentha spp tissue cultures and the accumulation of "free" essential oi The oil profiles obtained with this technique should closely reflect the in vivo ratios of components. Abstract No 805 from the Annual meeting of the American Society of Plant Physiologists and the Canadian Society of Plant Physiologists. June 14-18, 1981 Published in Supplement to Plant Physiology 1981, volume 67 No 4.