Aspects of Physiology and Trichome Chemistry in the Medicinal Plant
Aspects of Physiology and Trichome Chemistry in the Medicinal Plant
Tanacetum parthenium (L.) Schultz-Bip.
by
Kevin Bernard Usher
B.Sc, Okanagan University College, 1994
A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
DEPARTMENT OF BOTANY
We accept this thesis as conforming to the required standard
G.H.N. Towers, Supervisor (Botany, University of British Columbia)
.E.P. Taylor, Co^ipen/isor (Botany, University of British Columbia)
P.A. Bowen, Committee Member (Pacific Agriculture Research Center, Agriculture and Agri-Food Canada)
A.D./vKala^s, Commit$e4v1ember (Botany, University of British Columbia)
THE UNIVERSITY OF BRITISH COLUMBIA
September 2001
© Kevin Bernard Usher, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department
The University of British Columbia Vancouver, Canada
Date S" Oct , Zoo(
DE-6 (2/88) 11 ABSTRACT
This study investigated aspects of physiology and terpenoid chemistry in feverfew, a medicinal plant used for migraine therapy. The sesquiterpene lactone parthenolide accumulates in feverfew shoots and is thought to contribute to feverfew's antimigraine activity. The first part of this study examined the effects of nitrogen application and irrigation on shoot yield and shoot parthenolide concentration. Reduced shoot yield was observed under treatments of low nitrogen application and irrigation frequency. Leaf parthenolide concentration increased in plants grown with high nitrogen application rates and decreased with high irrigation rates.
In the second part of this study, shoot yield, parthenolide concentration and trichome distribution were examined in response to developmental changes. Days longer than approximately 12 hours induced flowering. Feverfew grown under days shorter than 12 hours for extended periods remained in a vegetative stage and their leaves accumulated parthenolide in concentrations up to 10x that of flowering plants.
Yield was lower in vegetative plants grown under short days but leaf to stem ratio was high.
Glandular trichomes are the site of parthenolide biosynthesis and storage. Leaf parthenolide concentration is related to glandular trichome densities on leaf surfaces.
Young leaves of vegetative plants have high trichome densities while young leaves of flowering plants have low trichome densities. Trichome densities decreased with leaf expansion and as density decreased, parthenolide concentration decreased.
The third part of this study investigated the two terpenoid biosynthetic pathways involved in parthenolide biosynthesis. Experiments using 14C and 13C labeled substrates revealed that both the mevalonate (MEV) and methylerythritol phosphate
(MEP) pathways contribute isoprene subunits to parthenolide biosynthesis. Two of the Ill three isoprene subunits in parthenolide were enriched after feeding shoots 1-13C- mevalonate and 2-13C-acetate, an enrichment pattern consistent with the MEV pathway.
Parthenolide's enrichment pattern after feeding 2-13C-pyruvate and 1-13C-Glucose was consistent with isoprene contributions from both pathways. After feeding 2-13C-pyruvate however, there was a higher proportion of 13C-enrichment from the MEP pathway than from the MEV pathway. TABLE OF CONTENTS
Abstract ii
Table of Contents iv
List of Tables vii
List of Figures viii
Acknowledgements x
Chapter 1: General Introduction 1.1. Plant Natural Products 1 1.2. Terpenoids 4 1.3. Sesquiterpene lactones 10 1.4. Feverfew: Historical use and modern medicine 13 1.5. Objectives 17 1.6. References 18
Chapter 2: Effects of irrigation and nitrogen application on feverfew shoot yield and parthenolide concentration 2.1. Introduction 26 2.2. Materials and Methods 2.2.1. General methods 30 2.2.2. Field irrigation trial 33 2.2.3. Field irrigation and nitrogen application trial 34 2.2.4. Greenhouse irrigation and nitrogen application trial 36 2.3. Results 2.3.1. Field irrigation trial: effects of irrigation frequency on parthenolide concentration and plant growth ... 38 2.3.2. Field irrigation and nitrogen application trial 39 2.3.3. Greenhouse irrigation and nitrogen application trial 42 2.4. Discussion 44 2.5. References 52 Chapter 3: Development and Regeneration 3.1. Introduction 55 3.2. Materials and Methods 3.2.1. General methods 58 3.2.2. Field growing medium and fertigation trial 60 3.2.3. Greenhouse growing medium and fertigation trial 62 3.3. Results 3.3.1. Field growing medium and fertigation trial 63 3.3.2. Greenhouse growing medium and fertigation trial 69 3.4. Discussion 72 3.5. References 82
Chapter 4: Developmental effects on glandular trichomes and leaf chemistry 4.1. Introduction 85 4.2. Materials and Methods 87 4.3. Results 4.3.1. Parthenolide variability during leaf development and flowering 89 4.3.2. Feverfew glandular trichome development, density, and concentration 94 4.4. Discussion 102 4.5. References 108
Chapter 5: Biosynthetic studies using 14C and 13C incorporation into Parthenolide
5.1. Introduction 110 5.2. Materials and Methods 113 5.2.1. 14C feeding experiments 114 5.2.2. 13C feeding experiments 116 5.2.3. Extraction methods, parthenolide isolation, and NMR analysis 117 5.3. Results 5.3.1. 14C labeling of parthenolide 119 5.3.2. 13C enriched parthenolide 120 5.4. Discussion 126 5.5. Conclusion 136 5.6. References 137 vi
Chapter 6: General Discussion 6.1. Overview 139 6.2. Future Research Directions 143 6.3. References .' 146
Appendix 148 vii LIST OF TABLES
Table 2.1. Parthenolide concentration of feverfew leaves 43 days and 87 days after transplanting. 38 Table 2.2. Average whole plant and organ dry weights of field grown feverfew. 39 Table 2.3. Leaf water status in field grown feverfew measured at 3 am (night), 12 pm (mid-day), and 6 pm (evening). 40 Table 2.4. Dry leaf parthenolide content measured over a 3 month period in the field. 41 Table 2.5. Leaf and flower parthenolide concentrations and total parthenolide content per plant of stems, leaves and flowers. 42 Table 2.6. Leaf parthenolide concentration in feverfew leaves grown in the greenhouse under irrigation and nitrogen treatments. 43
Table 3.1. Field experiment treatments and abbreviations. 61
Table 3.2. Treatments in the greenhouse trial and abbreviations. 63
Table 3.3. Dry weight and dry to fresh weight ratios of feverfew plants grown in the field and greenhouse. 64 Table 3.4. Leaf water potential and osmotic potential of greenhouse and field grown plants measured at 3 p.m. (light) and 4 a.m. (dark). 66 Table 3.5. Average leaf parthenolide concentration in greenhouse and field-grown plants. 67 Table 3.6. Parthenolide content at harvest in leaf, stem, and flower tissues, based on subsample analysis. 68 Table 4.1. Parthenolide concentration in leaves of different ages from vegetative and reproductive shoots. 92 Table 4.2. Trichome density and parthenolide concentration of leaves measured from the apex to the base ofthe stem in vegetative and reproductive plants. 100 Table 5.1. Incorporation of 14C labeled substrates into parthenolide. 119 Table 5.2. 13C-NMR assignments and enrichment of parthenolide after feeding 13C-enriched substrates. 124 viii LIST OF FIGURES
Figure 1.1. Mevalonate and methylerythritol phosphate routes to isoprene Biosynthesis. 5 Figure 1.2. The diversity of terpenoid biosynthesis showing examples of the compounds derived from this pathway 8 Figure 1.3. Major skeletal types of sesquiterpene lactones showing the common pathway through the germacranolides. 9 Figure 1.4. Scanning electron micrograph of non-glandular and glandular trichomes on the abaxial leaf surface of Tanacetum parthenium. 12 Figure 4.1. A glandular trichome derived from an epidermal cell with a subcuticular extracellular space where secretory cells secrete non-polar compounds for storage. 86 Figure 4.2. Greenhouse-grown feverfew leaf parthenolide concentration during development from vegetative to reproductive growth. 89 Figure 4.3. Leaf parthenolide concentration of greenhouse-grown feverfew overtime. 91 Figure 4.4. HPLC chromatograms of leaf and flower extracts from feverfew shoots in the vegetative and reproductive stages. 93 Figure 4.5. HPLC chromatograms of a composite disk flower trichome extract and a receptacle trichome extract. 94 Figure 4.6. Scanning electron micrographs of feverfew leaf surface showing glandular and non-glandular trichomes. 96 Figure 4.7. Scanning electron micrographs of feverfew flower glandular trichomes on the floret petals of the inflorescence. 97 Figure 4.8. Increased visibility of trichomes on slide preparations of leaf epidermal peals after drying. 99 Figure 4.9. Trichomes before and after treatment with dichloromethane. 101 Figure 5.1. The methylerythritol phosphate pathway and the mevalonate pathway to terpenoid biosynthesis. 111 Figure 5.2. 1H-NMR spectra of parthenolide. 121 Figure 5.3. Carbon numbering of parthenolide and the predicted conformation of the three isoprene units. 121 Figure 5.4. 13C NMR of parthenolide isolated from feverfew shoots fed with 2-12C-mevalonolactone or 2-13C-mevalonolactone. 122 Figure 5.5. 13C enrichment patterns in parthenolide after feeding enriched substrates. 125 Figure 5.6. Glucose catabolism. 127 Figure 5.7. 1-13C-D-glucose feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.8. 2-13C-acetate feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.9. 2-13C-mevalonolactone feeding experiment. Observed and predicted patterns of 13C enrichment. Figure 5.10. 2-13C-pyruvate feeding experiment. Observed and predicted patterns of 13C enrichment. ACKNOWLEDGEMENTS
I extend my gratitude to Professor G. H. Neil Towers for his guidance and support throughout this thesis. The unrestricted freedom to explore plant physiology and phytochemistry in the lab and field, and the liberty to explore where I felt the research led, was invaluable to me. I give my sincerest thanks to Dr. Pat A. Bowen for giving me an early start in my career as a scientist, for support and encouragement throughout my thesis and the invaluable guidance in crop physiology. Dr. Bowen was instrumental to the success of this thesis by providing the resources for field and greenhouse experiments. I also thank my co-supervisor Professor lain E. P. Taylor and committee member Professor Anthony D. M. Glass for their advice and support throughout my thesis and particularly in the preparation and writing of this dissertation.
Many thanks to the staff at the University of British Columbia, Department of
Chemistry NMR laboratory and at the Faculty of Pharmaceutical Sciences LC-MS laboratory.
I thank the many members of Dr. Towers research group for their involvement with my research and for the many wonderful memories. In particular I want to thank
Zyta Abramowski the cornerstone of Dr. Towers laboratory, Dr. Jon Page, Eduardo
Jovel, Ji Yang, Fiona Cochrane, Andres Lopez, and Keith Pardee. My gratitude to Heidi
Remple and Brenda Frey for the numerous hours maintaining my field and greenhouse crops in Agassi, B.C.
This research was supported by Natural Sciences and Engineering Reserch
Council of Canada in the form of operating and equipment grants to Professor G. H. N.
Towers, and by Agriculture and Agri-Food Canada at the Pacific Agri-Food Research
Centre who provided the laboratory and horticultural facilities under the supervision of
Dr. P. A. Bowen.
Finally, I thank my wife Kathy Usher, for her unending support and encouragement to pursue my interests and dreams. 1
Chapter 1
Introduction to Phvtochemistry and Feverfew
1.1. PLANT NATURAL PRODUCTS
It is estimated that 80% of the known natural products are of plant origin
(Robinson, 1980; Swain, 1974). We are becoming increasingly aware that secondary compounds have important survival functions within plants including roles as plant defensive mechanisms against herbivory and infection, allelopathic agents and protection from damage caused by UV radiation, oxidation, and free radicals. They also serve as photoreceptors and provide communication between plants and other organisms through chemical or visual stimuli (Harborne, 1993). Biologically active secondary metabolites interact with enzymes or other chemicals to elicit responses that result in physiological changes at the cellular level. Over the past century, as people discovered these properties, interest in plant natural products has increased. Humans have exploited the abundance of secondary compounds as cosmetics, medicines, recreational drugs, pigments, perfumes, and pesticides (Buchanin et al., 1980).
Consumption of natural products can protect against free radical damage, oxidation, and many diseases such as cancer. Recently, attention has focused on food plants due to findings that certain grains, fruits, vegetables, and nuts contain important non- nutritional secondary compounds that contribute to human health. Foods with beneficial pharmacological properties are called nutraceuticals. The full significance of health benefits provided by nutraceuticals is not yet known. Phytochemicals can also be 2 detrimental to human health. For example, the toxic glycoalkaloids a-solanine and «- chaconine are present in potato tuber skins. When the tubers are exposed to light they turn green and the glycoalkaloid concentration increases. Solanine is an established acetylcholine esterase inhibitor, a key component of the nervous system, and signs of neurological impairment have been recorded after ingestion of the toxin (Dalvi and
Bowie, 1983). Thus, the more that is known about natural products in plants, the better we can develop safe and effective pharmaceuticals, nutraceuticals, and foods.
Establishing the function of secondary metabolites in plants is often difficult.
These chemicals may have more than one function and closely related compounds may have completely different functions. Some investigators have suggested that plant secondary compounds are waste products of metabolism, storage compounds, or a means to keep primary pathways open in times of reduced growth (Swain, 1977). The large number and diversity of plant natural products cannot be dismissed this easily.
One of the underlying rationalizations for the alternative chemical defense theory of secondary metabolites is that plants are sessile and must be capable of defending themselves (Harborne, 1993). The rich diversity of secondary metabolites in the plant kingdom, and the ability of plants to continue generating novel compounds, may have contributed greatly to their evolutionary success (Rausher, 1992). Chemical defenses can exist throughout the plant in key locations such as specialized cells and compartments, or at specific stages in the life cycle. Biosynthesis of defensive compounds is regulated through either constitutive or inducible mechanisms or may be controlled temporally, changing the chemical complement over time (Gershenzon &
Croteau, 1990). Plants also protect themselves through physical barriers such as thick or waxy epidermis, hairs, thorns or spines that may be impregnated with chemicals that are unpalatable or toxic. Some plants use combinations of these defense mechanisms 3 (Denno & McClure, 1983). For example, glandular hairs are specialized extrusions on the epidermis containing toxic or repellent chemicals. The glandular trichomes rupture, releasing their contents on contact with insects or herbivores. After the attack is finished the chemicals may remain on the surface of the plant providing protection from infection or herbivore feeding. The trichomes thus provide both physical and chemical obstacles to phytopathic organisms.
The secondary chemistry of a plant species is often consistent among individuals. This is the foundation of chemotaxonomy, which uses species-specific chemicals in the identification of plants. However, natural product formation is inherently variable and this may confound attempts to determine chemotaxonomic relationships (Harborne, 1993; Cates, 1987). It seems that chemical variation ensures reproductive success through natural selection. By keeping the chemical mixture variable, plants increase probability of survival and evolutionary success. The factors influencing variation are genetically determined and but concentrations may be modulated by climate, predation, infection, competition, edaphic factors, nutrition, or other environmental stimuli (Denno & McClure, 1983).
The three most prevalent classes of plant secondary compounds are the alkaloids, phenolics, and terpenoids. The alkaloids are nitrogen containing, basic compounds, many of which are derived from the shikimate pathway. Phenolics are derived from a combination of the acetate and shikimate pathways and include flavonoids (Mann, 1986). Terpenoids are synthesized two ways in plants, the first is through the classical mevalonate pathway via acetate, the second is through the methylerythritol phosphate (MEP) pathway which uses pyruvate and glyceraldehyde-3- phosphate as substrates (Dewick, 1999; Rohmer, 1999; Eisenreich era/., 1998). The 4 terpenoids are of interest due to their broad range of biological activities and potential usefulness as medicines or agrochemicals.
1.2. TERPENOIDS
Terpenoids are ubiquitous and some are perceived as necessary for the existence of life (Rohmer, 1999). Glasby (1982) compiled a list of more than 10,000 structures known at that time. They form the largest group of natural products, now numbering between 15,000 and 22,000 characterized compounds (Rohmer et al., 1996;
Gershenzon and Croteau, 1991). In contrast, there are over 10,000 alkaloids characterized (Southon and Buckingham, 1989) and approximately 8,000 phenolics including the flavonoids (Harborne, 1988). Since ancient times terpenoids have been used in oils, perfumes, soaps, drugs, and pigments. The lower terpenoids, mono- and sesquiterpenes, are characteristically volatile and often have an odor (Gershenzon &
Croteau, 1990). For example, camphor is an aromatic monoterpene traded since the
11th century for its fragrance (Banthorpe, 1991).
Terpenoids are generally unsaturated, lipophilic, and predominantly cyclic compounds that may be highly oxygenated and contain various functional groups.
These compounds have been studied since the 19th century when Wallach put forward the isoprene rule of terpenoid biosynthesis (Fowler et al., 1999). The rule says that terpenoids are made up of repeating five carbon isoprene units joined together head to tail. Isopentenyl pyrophosphate (IPP) is the isoprene unit from which all terpenes are synthesized. As with all rules there are exceptions and in this case tail-to-tail and head- to-head condensation of isoprene units does occur, but are rare. The reactions of isoprene biosynthesis are shown in Figure 1.1. The details of the mevalonate pathway MEP pathway Mevalonate pathway o
-SCoA + OH + -SCoA
H3C H3C acetyl-CoASH
Pyruvate G-3-P O CoASH O
C02 SCoA HcC SCoA + H3C OH H20 HMGS oASH
3 OH O
HOOC SCoA • NADPH/H + 2 NADPH/2H+ HMGR NADP+ 2 NADP+ *d CoASH ^r
H3C. OH 3 OH
HOOC. >^ s OH
3R-mevalonic acid (MVA)
2 ATP 2 ADP <
3 OH
HOOC OPP
IPP CH2 5-PPMVA
OPP
DMAPP
Figure 1.1. Two routes to isoprene biosynthesis. The mevalonate pathway and the methylerythritol phosphate pathway converge at isopentenyl pyrophosphate. HMGS = hydroxymethylglutaryl synthase, HMGR = hydroxymethylglutaryl reductase, 5-PPMVA = 5-pyrophosphomevalonate, IPP = isopentenylpyrophosphate, DMAPP = dimethylallylpyrophosphate, G-3-P = glyceraldehydes-3-phosphate, DXP = 1-deoxyxylulose-5-P, MEP = 2-C-methyl-D-erythritol-4-P. 6 were worked out in the 1950's when it was discovered that in flax, rat liver and yeast preparations, acetyl-CoA was the precursor to mevalonic acid (MVA) and IPP (Fisher,
1999). Demonstration of radiolabeled mevalonic acid incorporation into various plant terpenoids reinforced the evolving principle that the mevalonate pathway was the route to all terpenoids. Since the elucidation of the MVA pathway in the 1950's there have been reports contradicting the universal role of MVA for terpenoid biosynthesis.
Labeled MVA and acetate were not incorporated, or were incorporated in low levels, into monoterpenes, diterpenes, carotenoids and phytol, but were readily incorporated into sterols, triterpenes and sometimes into sesquiterpenes (Sagner et al., 1998;
Lichtenthaler et al., 1996). Rhomer discovered that 13C enriched acetate incorporation into bacterial terpenoids resulted in unexpected patterns of enrichment. Subsequent investigations led him to the discovery of an alternative pathway to terpenoid biosynthesis called the methylerythritol phosphate (MEP) pathway (Rohmer et al., 1993;
Flesch & Rohmer, 1989; Flesch & Rohmer, 1988). The precursors for this novel pathway are pyruvate and glyceraldehyde-3-phosphate (Figure 1.1). In eukaryotes the mevalonate (MEV) pathway functions in the cytosol while the MEP pathway functions in chloroplasts. (Arigoni et al., 1999; Paseshnichenko, 1998; Disch et al., 1998; Putra et al., 1998; Rohmer et al., 1996). It was suspected that compartmentalization was playing a role in terpenoid distribution at the cellular level, but until the elucidation of the
MEP pathway the plastidic contribution to terpenoid synthesis could not be determined
(Disch era/., 1998; Nabeta etai, 1998; Lichtenthaler et al., 1997a, 1997b).
The regulation of all terpenoids begins with regulating isoprenoid biosynthesis.
Hydroxymethylglutaryl reductase (HMGR) is the rate limiting step and regulation point in the MEV pathway controlling the production of IPP (Bach et al., 1990). All steps of the 7 MEP pathway have not been determined, including the rate-limiting step.
Methylerythritol phosphate is the first committed metabolite in this pathway so the rate- limiting step may be at the formation of, or downstream from, this compound. The mevalonate and MEP pathways converge at IPP after which all the reactions likely proceed via enzymes of the same or similar structure. However, there has not been an investigation into the possibility that the enzymes downstream from IPP may be different in the plastidic and the cytosolic pathways. After IPP is synthesized, it reacts with dimethylallyl pyrophosphate (DMAPP) forming a monoterpene. Sequential condensation of IPP units forms the higher terpenoids (Figure 1.2) (Towers & Stafford,
1990). The cyclase enzymes control the branch point of terpenoids into the subclasses such as mono- or sesquiterpenes. Once cyclization occurs, the molecule is committed and is usually not prenylated to form a higher class of terpenoid. For example, sesquiterpene lactones are composed of three isoprene units to form farnesyl pyrophosphate. Farnesyl cyclase converts farnesyl into a ten membered ring after which it is committed to sesquiterpene biosynthesis (Figure 1.3) 8
Mixed Biosynthesis Pure Biosynthesis (c„) Gutta rubber -Prenylatiorn— Polyisoprenoids -> (C„) Polyprenols T i Plasto-, ubi-quinones
Prenylation •<— DMAPP/IPP (C5) Isoprene
T Flavonoids, alkaloids ipp nucleicacid bases coumarins, proteins benzoquinones
i Monoterpenes -Prenylation <* GPP/NPP (^10) iridoids Pseudoalkaloids
Benzo-, naptha-quinones ipp alkaloids, dlavonoids cannabinoids
_^ . Sesquiterpene lactones -Prenylation FPP (C ^ 15' Abscisic acid FPP- Porphyrins IPP Sterols, brassins
(C30) saponins, sardenolides pseudoalkaloids
-> (C o) Gibberellins Esterification GGPP 2 20 pseudoalkaloids GGPP- Porphyrins IPP (C ) Carotenoids DMAPP = dimethylallyl pyrophosphate 40 IPP = isopenteny pyrophosphate GPP = geranyl pyrophosphate NPP = neryl pyrophosphate FPP = farnesy pyrophosphate t GGPP = geranylgeranyl pyrophosphate GFPP (C25) Sesterterpenes GFPP = geranylfarnesyl pyrophosphate
Figure 1.2. Terpenoid biosynthesis showing the diversity of this chemical class. A few examples of the compounds derived from this pathway are given.
Prenylation refers to the addition of the respective C5,Cio,Ci5, etc.. units to another non-terpenoid compound. Adapted from Towers and Stafford (1990). 9
Eremophilanolide
Figure 1.3. Major skeletal types of sesquiterpene lactones showing the common pathway through the germacranolides. 10 1.3. SESQUITERPENE LACTONES
The largest and most diverse class of terpenoids is the sesquiterpene lactones
(STLs). In 1979 the number of identified naturally occurring STLs was 950, by 1987 it had tripled to 3200 (Fischer, 1990) and presently may exceed 5000. There are over
200 skeletal types (Fowler et al., 1999) but the majority of known compounds fall into 9 major skeletal classes. The majority of sesquiterpene lactones are formed by an initial cyclization of farnesyl pyrophosphate to form a germacrene and then germacranolides, which are modified to form the other skeletal types (figure 1.3). The 12,6-lactonization is represented in figure 1.3. The 12,8-lactonization is also common and occurs in all these skeletal types (Fischer et al., 1979). The obvious control point for STL biosynthesis is at the branch point where farnesyl cyclization forms germacrene (figure
1.3). The biosynthesis of STLs has not been studied in enough detail to know how the pathways are regulated. The majority of STLs have been isolated from the Asteraceae where they are prevalent. There are many other plant families containing STLs including Acanthaceae, Amaranthaceae, Burseraceae, Bombacaceae, Coriariaceae, llliciaceae, Magnoliaceae, Menispermaceae, Lamiaceae, Lauraceae, Polygonaceae, and Winteraceae. STLs have also been found in the gymnosperms in the family
Cupressaceae as well as fungi, liverworts, and marine organisms (Fischer, 1990).
Interest in STLs has grown for two reasons: they are useful in chemotaxonomy, and they have a range of biological activities (Bruneton, 1995; Fischer et al., 1979).
Many STLs have been shown to be antiviral, antibacterial, antifungal, cytotoxic, and
nematocidal as well as having other pharmacological activities (Bos et al., 1998;
Beekman etal., 1998; Maruta et al., 1995; Woynarowski & Konopa, 1981; Hoffmann et al., 1977). They also have potential as herbicides and insecticides due to their allelopathic and antifeedant properties (Macias et al., 1999; Macias et al., 1996; Macias 11 et al., 1993; Macias et al., 1992). The STLs have a bitter taste so they contribute to flavour of foods. The bitter taste in the chicons of chicory roots is due to STLs (Peters et al., 1997). The STLs have met with limited application as medicines due to their inherent toxicity. The best known medical application of an STL has been the development of the antimalarial drug, artemisinin, from Artemisia annua. This chemical, in its purified form, is currently undergoing clinical trials. Artemisinin has very few known side effects unlike other antimalarials such as chloroquine and quinine.
Plasmodium falciparum and Plasmodium vivax , the parasites responsible for malaria, have acquired resistance to many pharmaceutical drugs in current use (Bruneton, 1995;
Klayman, 1985). From a chemical ecology perspective we know that organisms can adapt rapidly and develop resistance to chemicals. This is apparent in the antibiotic resistance developed by bacteria, which has scientists around the world searching for new antibiotics. In malaria treatment, P. falciparum has not become resistant to artemisinin. Its use however, has been as a traditional plant preparation and not a pure drug. It will be interesting to watch this drug as its use in pure form increases in western medicine to see if P. falciparum develops resistance to artemisinin.
One important functional group in STLs is the a-methylene-y-lactone which confers biological activity to these compounds. The methylene group can bind to sulphydryls such as those in cysteine residues of proteins to causes loss of function
(Heptinstall et al., 1988; Heptinstall et al., 1987). Sesquiterpene lactones can cause allergic eczematous contact dermatitis and can cross sensitize a person to other STLs, a direct effect of the lactone methylene (Spettoli et al., 1998; Lamminpaa et al., 1996;
Burry, 1980; Rodriguez et al., 1977; Schulz et al., 1975). Other functional groups also give STLs increased biological activity such as a 2,3-double bond, epoxides, peroxides, 12 and cyclopentenone groups (Yuuya et al., 1999; Beekman et al., 1998; Goren et al.,
1996; Woerdenbag, 1986; Elissalde etai, 1983).
STLs are not free in plant cells but are sequestered and cannot cause toxic damage. STLs are thought to be defensive compounds and appear to be located in strategic areas of the plant, such as laticifers, canals, and trichomes or exuded to external surfaces of the plant. The position and form of these structures can be important in the effectiveness of defensive mechanisms. They are generally near or on structures likely to be attacked by plant eating organisms. The forms of trichomes for example include glandular hairs, trigger hairs, root hairs, and scales that provide physical barriers and sometimes toxic chemical barriers.
Trichomes on leaf surfaces can be divided into three categories, simple
(unbranched), complex (branched), and glandular (Behnke, 1984). Glandular trichomes are easily accessible and easily removed or manipulated for biosynthetic studies (Tellez et al., 1999; Gershenzon et al., 1992; Gershenzon et al., 1987; Croteau & Johnson,
1984). A scanning electron micrograph of glandular and simple trichomes on the leaf surface of T. parthenium (Figure 1.4) shows the abundance of these structures.
A
B
Figure 1.4. A) Simple and B) glandular trichomes on the abaxial leaf surface of Tanacetum parthenium. 13 1.4. FEVERFEW: Historical use and Modern medicine
People have been using plants as medicines since antiquity. In his "Archidoxa" of the "Arcanum" written early in the 16th century, Paracelsus referred to the need to discover the active component of a remedy or the "secret" of a treatment (Di Stephano,
1951). The search for active constituents in plants began in the 1780s with Scheele's work on organic acids (referred to by Sneader, 1985). In the early part of the 19th- century investigation into several well-known medicinal plants led to the discovery of a number of biologically active alkaloids. Morphine, atropine, papaverine, and codeine were a few of the alkaloids discovered and subsequently became cornerstones of modern medicine and remain among the most important pharmaceutical compounds isolated from plants (Foye et al., 1995; Sneader, 1985). The sesquiterpene lactones have not been of the same medicinal value as the alkaloids, but they also have not been studied as intensively. Many traditional herbal drugs are from the Asteraceae and contain STLs (Spring era/., 1999; O'Hara era/., 1998; Heinrich et al., 1998; Bruneton,
1995). Feverfew (T. parthenium) is a plant in the Asteraceae containing sesquiterpene lactones which have been shown to be effective for migraine prophylaxis in clinical trials
(Murphy era/., 1988).
Feverfew is a species native to Western Europe and has been used medicinally for centuries. It now occurs in many places around the world due to its popularity as a garden plant and herbal remedy and its propensity to become weedy. It's classification has been changed several times starting with a move from the genus Rudbeckia to
Matricaria assigned by Linnaeus, then to Leucanthemum parthenium (L.) Gren and
Godron, to Pyrethrum parthenium (L.) Sm., then Chrysanthemum parthenium (L.)
Bernh., and finally, as it is known today, to Tanacetum parthenium (L.) Schultz Bip.
(Tutin et al., 1976). It is known as mutterkraut in Germany, feddygen fenyw in Wales, 14 Santa Maria in Latin America as well as federfoy, nosebleed, midsummer daisy, featherfew, flirtwort, and bachelor's buttons. One of the earliest recorded uses of feverfew is found in Culpepper's Complete Herbal written in 1649, where it was recorded as a cure for agues and headache. Feverfew has been called the aspirin of the 18th century (Berry, 1984). In England there was a resurgence of interest in feverfew when in the early 1970's newspapers reported feverfew as a cure for migraine and arthritis. Thousands of people started taking this plant even though nothing was known about its pharmacology. English scientists began researching feverfew and it quickly became one of the best-studied medicinal plants on the market. It was found that feverfew leaves taken daily reduce both intensity and frequency of migraine attacks
(Pattrick et al., 1989; Murphy et al., 1988; Johnson etal., 1985). The active compounds are believed to be STLs and the one in highest concentration is parthenolide.
Parthenolide has been found at concentrations of up to 2% of dry weight in aerial organs (Heptinstall et al., 1992b; Awang et al., 1991). Thus the focus of pharmacological research on feverfew has been on parthenolide (Knight, 1995; Abad et al., 1995). However, differing bioassay results have been reported for pure parthenolide, different plant extracts, and fresh vs. dry plant preparations (Barsby et al.,
1993; Groenewegen & Heptinstall, 1990; Ross et al., 1999; Bejar, 1996; Barsby et al.,
1992) . The problem in identifying the pharmacological agent(s) of feverfew may lie in the approach. Many scientists look for a single compound responsible for the pharmacological activity. However, it is quite possible that the activities of this and other herbal drugs lie within the mixture of compounds present in the plant. More than
25 STLs belonging to three classes have been isolated from feverfew leaves (Maries,
2000), as well as biologically active flavonoids (Williams et al., 1999; Smith & Burford,
1993) and monoterpenes (Knight, 1995). Parthenolide, however, was investigated 15 because it is easy to isolate, is available in large quantities in feverfew, and has a range of biological activities.
In vivo, feverfew inhibits platelet aggregation (Losche et al., 1987) and the ADP- or adrenaline-induced release of serotonin (Bejar, 1996; Maries et al., 1992). It inhibits the degranulation of granulocytes (Hayes & Foreman, 1987; Elissalde et al., 1983), inhibits the release of enzymes involved in the inflammatory process (Jain & Kulkarni,
1999; Hehner er al., 1999; Makheja & Bailey, 1981), exhibits a protective effect on vascular endothelial cells (Voyno-Yasenetskaya et al., 1988) and blocks voltage- dependent potassium channels (Barsby et al., 1993). Many of these effects are related to current knowledge about the physiology of migraine but direct evidence of specific chemicals in feverfew responsible for migraine prophylaxis has not been established and may not be until migraine physiology is understood.
The quality of many plant drugs is assessed by the content of specific chemicals.
There are two approaches to rating quality. The first is to use chemotaxonomy which is the use of chemical traits in the identification of a plant species. The second is to determine the presence and quantity of the compounds responsible for the attributed physiological response or medicinal properties. However, these two methods are often confused and chemotaxonomic markers become promoted as medicinal compounds.
Because the use of chemical profiles for species identification does not assure medicinal quality of a plant, the best method for quality control is a combination of these two approaches (Bruneton, 1995). In feverfew, parthenolide is used as a taxonomic marker and is thought to be active against migraine. In this case the chemotaxonomic marker and an active compound are the same. Most over-the-counter (OTC) herbal drugs have not undergone rigorous clinical testing to determine which chemicals are the bioactive principles and whether the remedies are effective or dangerous. In addition, 16 secondary compounds in plants vary within species which makes it difficult to evaluate the medicinal quality of an unpurified product. There are reports of substantial chemical variation in commercial preparations of OTC plant drugs, including feverfew (Heptinstall et al., 1992a; Groenewegen & Heptinstall, 1986). This has raised concern over both the efficacy and safety of unproven and untested herbal remedies. In order to reduce variability of the chemicals of interest and to retain a consistent reliable product it's important to understand the cause of these chemical variations.
The use of parthenolide as a marker compound to authenticate feverfew products is common and it is suggested by Health Canada that feverfew products contain a minimum of 0.2% parthenolide (Bruneton, 1995). However, without the use of another chemical marker or additional taxonomic information, the authentication of feverfew using parthenolide may be misleading because there are a number of plants which produce parthenolide (Hendricks & Bos, 1990; Fischer era/., 1979; Hoffmann et al., 1977; Wiedhopf et al., 1973) which could potentially be mistaken for, or used as an adulterant in commercial feverfew preparations. One of these plants, Tanacetum vulgare (common tansy) contains parthenolide and has been used as an adulterant in feverfew (Heptinstall et al., 1992a)(Smith, 1994; Mitich, 1992; Hendricks & Bos, 1990).
T. vulgare is a common weed species and contains the monoterpene thujone which is a potent neurotoxin causing epileptic and tetanic type seizure (Bruneton, 1995). Not only do we need to ensure that authentic plant species are being used in the correct dosage, but tests for possible contaminating plant material (weeds or adulterants), such as common tansy in feverfew crops, are also necessary.
The premise of this thesis is that by determining the causes of parthenolide variation, the location of synthesis and parthenolide accumulation within feverfew, and the biosynthetic origins of parthenolide, we can better understand sesquiterpene 17 metabolism in plants. This knowledge may be used to grow higher quality crops by better understanding how STL levels vary in plants. This information is important if regulations are to be established for achieving safe herbal drugs. Finally, elucidation of the MEP pathway in sesquiterpene biosynthesis must be established before successful manipulation of the biosynthetic pathways through molecular techniques, in tissue culture and through horticultural methods can be utilized to their fullest potential.
1.5. OBJECTIVES
I am interested in the fifteen carbon terpenoids called sesquiterpenes and how development, climate, edaphic factors, water and nitrogen affect variability in these compounds. My thesis is that these external factors affect plant growth, in addition to variability, localization, and biosynthesis of the sesquiterpene, parthenolide, in the medicinal plant Tanacetum parthenium (L.) Schultz Bip. and that biosynthesis occurs through two independent pathways.
The research in this thesis examined characteristics of trichome chemistry and plant growth in the medicinal plant Tanacetum parthenium (feverfew). There were three areas of investigation; (1) The influence of water, nitrogen, photoperiod, growing media, and regeneration after harvesting on chemical variation and plant growth in both greenhouse and field culture, (2) Parthenolide localization and chromatographic profiles of glandular trichome contents in leaves and flowers throughout plant development, and
(3) The isoprene biosynthetic route to parthenolide, a closely related compound to germacrene and the first committed step in STL biosynthesis. 18 1.6 REFERENCES
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Effects of irrigation frequency and nitrogen application on feverfew shoot yield and parthenolide concentration
2.1. INTRODUCTION
Concentrations of terpenoids may vary within a plant species. Changes in secondary metabolism in response to environmental factors appear to be species- specific traits (Gershenzon, 1984). Abiotic stress, herbivory, and infection induced responses are well known and often affect mono- and sesquiterpene concentrations.
There are no simple explanations or rules to account for the variability of essential oil and terpenoids in response to environment. For example, increased nitrogen resulted in higher essential oil content in sage (Salvia officinalis) (Rohricht et al., 1996) but a lower content in sweet basil (Ocimum basilicum) (Adler et al., 1989). Essential oil content increased under moderate water stress in basil (Simon et al. 1992) but decreased in Cymbopogon sp. (Vivek et al., 1998) and peppermint (Charles et al.,
1990). Terpenoid profiles and concentrations of essential oils in Artemisia annua and
Oreganum vulgare varied depending on the geographic location and climate in which they were grown (Kokkini et al., 1994; Wallaart et al., 2000). Sesquiterpene lactone content, particularly parthenolide, is highly variable in feverfew (Fontanel et al., 1990;
Hendriks et al., 1997; Heptinstall et al., 1992; Awang et al., 1991) but the cause of this variation has not been investigated. In an effort to elucidate some factors influencing parthenolide biosynthesis, experiments presented in this chapter were designed to examine water and nitrogen effects on parthenolide content in feverfew. 27 In the field, light intensity, photoperiod, temperature, and rainfall are determined by season and climate, but it is possible to manipulate some of these environmental
parameters. Plasticulture is an intensive agriculture system which increases air and soil temperatures in the field by covering both the soil and crop with vented plastics, which may result in increased crop yield and quality (Brown and Channell-Butcher,
1999; Ricotta and Masiunas, 1991). Trickle or drip irrigation is often used with
plasticulture and is an efficient delivery system for both nutrients and water. Fertigation
is the application of soluble fertilizer with irrigation water to deliver nutrients directly to the root zone (Barua et al., 2000). Combining these systems provides a variety of
advantages to farmers and scientists alike. Black or dark green plastic mulch used to
cover the ground leads to soil warming, eliminates weeds, and reduces labour costs
(Ricotta and Masiunas, 1991; Bonanno, 1996). Heating the soil early in the season
and covering the crop with miniature tunnels of clear plastic, advances the growing
season and results in earlier maturation, higher yields and quality ( Dubois, 1978;
Galambosi and Szebeni-Galambosi, 1992). Fertigation enables accurate manipulation
of nutrient and water delivery and for these resources plasticulture with fertigation in the
field is similar to greenhouse hydroponic systems but maintains the advantages of field
edaphic factors.
Greenhouses are used for growing high value crops and have been
indispensable for research. Crops can achieve high levels of production, quality, and
cleanliness when cultured in greenhouses. These benefits come from artificial lighting,
climate control, and soil-less nutrient delivery systems. Greenhouse hydroponics
provides a variety of benefits including easy manipulation of the nutrient supply, pH,
and salt concentration, and uniform application of nutrient solution to the plants. One
primary advantage of greenhouses in colder climates is the ability to grow plants 28 throughout the winter. When the climate is right, however, the field has a distinct advantage over greenhouses. In the Fraser Valley, British Columbia, common cloudy conditions reduce irradiance levels. When there is a further reduction of 30% of incident light reflected and absorbed by greenhouses, the light which reaches the leaves is significantly lower in greenhouse than in field crops. Greenhouse growers often use sterile and soil-less medium devoid of soil microflora. Soils most often contain beneficial microorganisms, provide unlimited space for root growth, and generally have a high capacity for nutrient and water retention (Vince-Prue and
Cockshull, 1981; Downs and Hellmers, 1975).
Plant growth depends on temperature, CO2, light, nutrition and water availability.
Nitrogen forms a large part of plant element composition and is the fourth most abundant element in plants after carbon, oxygen and hydrogen (Salisbury and Ross,
1991). Generally, plant growth increases with nitrogen (N) supply but excessive N supply can inhibit growth. Many species alter their terpenoid composition in response to N availability but the complement of lower terpenoids in plants is species-specific.
Nitrogen-deprived Heterotheca subaxillaris has been found to have greater monoterpene and sesquiterpene content than nitrogen-rich plants (Mihaliak and
Lincoln, 1989). The terpene content of Abies grandis did not respond to N fertilizer
(Muzika, 1993). Sesquiterpenes in basil (Ocimum basilicum) increased with increasing nitrogen but decreased with excessive nitrogen supply (Youssef et al., 1998). The terpene-rich essential oil content of geranium (Pelargonium graveolens) was greatest at moderate N fertilizer application levels (100 kg/ha) but declined with either excess or low N supply (Rao et ai, 1990). STLs vary among species and among individuals within a species in response to nitrogen and water supply. For example, STL accumulation in chicory (Chichorium intybus L.) roots is dependent on the cultivar, 29 growing location and N supply (Peters et al., 1997). In general there appears to be an optimum nitrogen level for maximum essential oil and STL content. The variation observed in secondary metabolism, and particularly terpenoids, among genotypes and when grown in different environments and under different cultivation methods may be a
concern because many of these compounds are toxic and they are often consumed in foods and medicine. High levels of some terpenoids may be detrimental to health.
Water is necessary in plants as a medium for transporting nutrients and organic
compounds, it is the reaction medium in plant cells and tissues, it is involved in holding the plant upright and turgid, it enables growth through expansive osmotic forces of the
vacuole, and it is a source of molecular oxygen and hydrogen in chemical reactions.
Reduced water supply to plants can result in a variety of responses. The stress caused
by reduced water supply leads to decreased synthesis of cytokinin, and increased
synthesis of abscisic acid. This may result in decreased growth, stomatal conductance,
and photosynthetic rate (Kramer and Boyer, 1995; Letchamo, 1996). The effects of
water stress on secondary chemistry are not well understood and may vary between
species. There seem to be no consistent effects of water stress on terpenoid content in
plants. Water stress in mint has been shown to increase cyclic monoterpenes in the
essential oil, whereas the essential oil in well-watered plants contain predominantly
acyclic monoterpenes (Gershenzon, 1984). The sesquiterpene and diterpene content
of a desert sunflower (Helianthus ciliaris) increased, by double, under water stress
(Gershenzon, 1984), whereas essential oil in lemon grass (Cymbopogon flexuosis) was
not affected by mild water stress (Singh, 1999), and in marigold (Tagetes pagula) there
was a reduction in the sesquiterpene containing volatile oil (Razin and Omer, 1994).
Plant growth is reduced by limitations or excesses of resources such as
nutrients, light, and water. Thus from an economic perspective, the potential benefits 30 of a stress-induced increase in synthesis of valuable phytochemicals are worth
investigating. If biomass is reduced substantially while commercially important
secondary compounds increase, then the economics of processing and marketing high versus low potency materials must also be considered.
Reported in this chapter are the results of three experiments that explored the
effects of irrigation frequency and nitrogen fertilizer on parthenolide content and growth
of feverfew. The first was a field experiment to determine the effects of irrigation frequency. The second experiment, also in the field, examined the interactions of
nitrogen fertilizer supply (3 levels) and irrigation frequency (2 levels). The third
experiment was conducted in the greenhouse, and also determined the interactions of two levels of irrigation and two levels of nitrogen fertilizer in a hydroponic system.
2.2. MATERIALS AND METHODS
2.2.1. General methods
Propagation of field transplants
Feverfew was grown from seed supplied by 5-B Produce (Langley, B.C.), a
commercial grower in the Fraser Valley. The seeds were sown into flats containing a
peat and perlite mixture, in rows spaced 5 cm apart and covered with 3 mm of perlite.
Germination was apparent one week after sowing and the seedlings were grown for 30
days before transplanting into trays with 100 ml cells. The repotted seedlings were
grown in the greenhouse, fertilized weekly for 4 weeks with commercial 20-20-20
(N:P:K) fertilizer at 2 g/l (50 ml/plant), then were grown for another 4 weeks without
fertilization, at which they reached an average height of 10 cm. Immediately after
transplanting, the crop was watered. 31 Field culture methods
Feverfew field trials were conducted in 1996 and 1997 in Agassiz, B.C. The soil is a Monroe silt loam with 5.5 to 5.8% organic matter, classified as a Eutric Eluviated
Brunisol. In the year before each planting liquid dairy manure was applied in the spring and a rye cover crop was seeded in mid summer and incorporated by ploughing the following April. In south-coastal British Columbia, approximately 1000mm of rainfall perculates through the soil in the fall and winter leaching all nitrate N from the soil rooting zone.
The field was prepared each year for transplanting by cultivation followed by the formation of raised beds that were 30 cm high, 1.1 m wide and 1.8 m apart from center to center. A drip irrigation line was laid down the centre of the beds to deliver water and dissolved fertilizer. The drip emitters were spaced 12 cm apart and an in-line pressure regulator maintained pressure at 10 - 12 psi. The raised bed and the irrigation line were covered with 2 mm, UV resistant dark green polyethylene mulch
(IRT-76; AEP industries, South Hackensack, N.J.) applied with a tractor-drawn applicator. The plants were spaced 45 cm apart within the row and 45 cm between staggered rows. For transplanting, a small slit was cut into the plastic and the seedlings were planted through the slit and into soil. A mini-tunnel system was erected over the new crop to create a greenhouse effect. The mini-tunnels consisted of 2mm clear polyethylene stretched over 0.8 m tall wire hoops spaced 2 m apart. The tunnels were ventilated with 8 cm holes along the tunnel tops. The tunnels were removed in mid-July. Air and soil temperature was monitored hourly using thermistor sensors attached to a data logger (Campbell Scientific). Air temperature was measured at the canopy level and 10 cm below the soil surface. When the temperature was above 35
°C in the tunnels, the sides were lifted 30 cm at every second hoop to increase 32 ventilation and reduce the temperature. Soil matric potential was measured at a depth of 21 to 24 cm midway between two plants in a row using a tensiometer capped with a
rubber septum and measured using a tensimeter (Soil Measurement Systems, Tucson,
Ariz.) pressure sensor. Soil matric potential was measured every three or four days or
more often when warm, dry conditions warranted more frequent monitoring.
Subsampling and harvesting for yield measurement
The plants in each plot or experimental unit (EU) were harvested at the same time. An experimental unit is equal to one replicate of one experimental treatment.
The plants were cut 12 cm above the ground. One or more plants from each EU were taken as subsamples for organ partitioning. The remaining plants were weighed fresh.
The subsamples were partitioned into leaves, stems, and flowers for measurement of
dry to fresh weight ratios and number of flowers. Subsamples were dried at 50 °C for
three days to measure tissue dry weights.
Parthenolide extraction and quantification
As specified in the respective sections, fresh or dry feverfew leaves, stems, and
flowers were extracted separately with dichloromethane for 30 seconds. Both fresh
and dry leaves were not ground and only the leaf surfaces were extracted. This
extraction method was used to selectively extract non-polar compounds on the leaf
surfaceand in glandular trichomes. After the dichloromethane extraction the solvent
was vacuum filtered through Whatman No. 1 filter paper. The leaves were extracted
two additional times (30 seconds each) with 50 ml aliquots of dichloromethane and
vacuum filtered. The three extracts were combined into a round bottom flask and
evaporated to dryness under vacuum at 30 °C in a rotary evaporator (rotovap). The
extracts were resuspended in 2 x 10 ml methanol, agitated in a sonicating water bath, 33 transferred quantitatively to a 25 ml glass vial and sealed. Approximately 1 ml of the extract was used for HPLC analysis.
The HPLC system used for analyses was a Waters 600E controller, 790 photodiode array detector, 770 autosampler, operated with Millennium software. The
column was Waters C18 reverse phase, 150 mm x 3.9 mm. The mobile phase was isocratic wateracetonitrile (55:45) for ten minutes at a flow rate of 1 ml/minute. A ten point standard curve was prepared in duplicate from a parthenolide standard (97% pure) purchased from Sigma-Aldrich. The regression line passed through zero and R2 was 0.998. Quantitation was based on peak area and retention time.
Statistical analysis
Treatment effects on all plant response variables were analyzed using analysis of variance.
2.2.2. Field irrigation trial
Experimental design
Transplanting was on 26 May, 1996. Two irrigation treatments were randomized in each of four blocks for a total of eight EUs. Six raised beds consisted of two outer beds as guard rows and four inner beds, each one designated a block. The beds were oriented north/south. Each EU was half the length of a bed with two rows, each with six plants. The two plants at the row ends were guard plants. Guard plants were used to reduce the effects of growing on the edges of the plots and were not used for measurements. Therefore, each EU had a total of eight plants for measurements.
Fertilizer and irrigation schedule
Fertilizer was weighed, dissolved in warm water to make 20 L of concentrated solution. The pH of the concentrate was adjusted to 6 before it was injected into the 34 irrigation line over 45 minutes. Fertilizer application rate was based on bed surface area and calculated in kilograms per hectare (kg-ha"1). The crop was fertigated every two weeks according to the fertigation schedule in Appendix 1. There were two
irrigation treatments that started one week after transplanting. Water was applied
according to tensiometer measurements at a threshold of -20 kPa for the high irrigation frequency and at -80 kPa for the low irrigation frequency treatment or with the
scheduled fertigation. The low and high irrigation rates were applied for three months.
Leaf sampling for extraction
The fourth fully expanded leaf from the shoot apex was sampled from each of two shoots per plant every three weeks. At each sampling, the sixteen leaves from
each EU (two from each plant) were pooled into one sample for chemical analysis. The
pooled samples were dried, weighed, extracted with dichloromethane and analysed by
HPLC, as described in Section 2.2.1.
The crop was harvested 23 August, 1997. Shoot organs were partitioned into
stem leaf and flower and dry weights measured.
2.2.3. Field irrigation and nitrogen application trial
Experimental design
Transplanting was on 16 May, 1996. The treatments consisted of a factorial
combination of three rates of nitrogen fertilizer and two irrigation rates, applied in a
randomized block design with four blocks. The nitrogen fertilizer rates were per year
totals of 0, 50, and 100 kg-ha"1 applied in split applications every two weeks. The
nitrogen source was ammonium nitrate. High and low frequency irrigation treatments
were applied as in 2.2.2 according to tensiometer measurements taken every two or
three days. There were eight raised beds orientated north/south. The two outside 35 beds contained guard rows. A block consisted of 1.5 beds. Each bed split into four
EUs. The treatments were applied at random to EUs within a block. Each EU contained 12 plants in two rows of six. The two plants at the end of the rows in each
EU were guard plants. The guard rows and guard plants were not included in data collection, leaving 8 plants for data collection in each EU. There were four replicates and treatments were randomized within a replicate block.
Fertilizer and irrigation schedule
Fertilizers to be applied (Appendix 1) were dissolved in warm water to make 20
L of concentrate. After adjusting the pH to 6, the concentrated fertilizer solution was injected into the irrigation line. All nutrients, with the exception of ammonium nitrate, were applied at the same rate. Fertilizer application rate was based on the bed surface area. The bimonthly fertigations were 45 minutes long. Irrigation was applied when the soil matric potential was below - 20 kPa for high frequency irrigation rate, and below
-80 kPa for the low frequency rate. Irrigation was applied for 70 minutes.
Leaf water potential and leaf osmotic potential
Leaf water potential and leaf osmotic potential measurements were recorded three times over 20 hours, at 3 am, 12 pm, and 6 pm. The measurements were taken on a cloudless day/night, July 25, 70 days after transplanting. Water potential measurements were taken with a portable pressure bomb. Leaves with petioles, approximately 12 cm long, were cut and immediately placed into the pressure bomb with petioles extruded. Six leaves from each EU were measured. The pressure was recorded (bars) when sap emerged from the cut petiole. The same leaves were frozen in preparation for osmotic potential measurements. The leaf samples were thawed and their liquid contents pressed into a vial for measurement. The osmotic potential of the 36 liquid was determined using a freeze-point depression osmometer (Advanced
Instruments).
Leaf sampling for chemical analysis
Leaves were sampled every three weeks. The leaf samples were the fourth leaves from the apices of two shoots per plant. The 16 leaves sampled per EU were pooled together for analyses. Each leaf was cut in half and the petiole removed. One- half of each leaf was weighed fresh and then dried and reweighed to obtain dry to fresh weight ratio. The second half of the leaf was weighed and extracted fresh. The dry weight for the second leaf half was then estimated from its fresh weight and the dry to fresh weight ratio of the first leaf half. The extraction method and HPLC analysis is described in Section 2.2.1.
2.2.4. Greenhouse irrigation and nitrogen application trial
Propagation
Feverfew was grown as described in Section 2.2.1 with the following exceptions.
The seedlings were transferred from the cell trays to 8 x 8 x 8 cm rockwool cubes. On
21 January, 1997 the plants in rockwool cubes were transferred onto sawdust-filled, white polyethylene bags (pillow bags). The pillow bags were approximately 1 meter long and 18 cm in diameter. The bags were laid horizontally and holes cut on the top surface for plant roots to penetrate, and the bottoms were slit for drainage.
Greenhouse culture system
The sawdust pillow bags were supplied nutrient solution via trickle irrigation. A pump submersed in a 500 L nutrient tank pumped dilute solution through half-inch polyethylene tubing to a small diameter polyethylene "spaghetti" tubing. A plastic spike attached to the end of the spaghetti tubing held it at the base of the plant. The flow 37 delivered 35 ml/min/per drip line. The flow through fertigation system allowed excess nutrient solution out through slits at the bottom of the bag.
Experimental design
This experiment had four treatments, which were a factorial combination of two levels of nitrogen and two irrigation rates, as follows (concentrations refer to ammonium nitrate in the nutrient solution):
1. 170 ml of 0.91 mM N solution per day
2. 170 ml of 0.45 mM N solution per day
3. 170 ml of 0.91 mM N solution plus 70 ml water per day
4. 170 ml of 0.45 mM N solution plus 70 ml water per day
Each treatment was assigned at random to six EUs in six blocks. Each pillow bag was one EU and had two plants spaced 30 cm apart. In all there were 48 plants in 24 EUs in six blocks. Guard plants were placed around the perimeter of the experiment and were not included in data collection. Pillow bags were spaced 60 cm between rows and plants spaced approximately 45 cm within rows.
Fertilizer and irrigation schedule
The formula of the nutrient solution is outlined in the Appendix. Each N solution was held in a 500 L tank. The EC was adjusted by adding NaCl to the lower N concentration tank to match that of the higher N tank which was 2 mSv. The pH was adjusted to 6.0 in both tanks using sulphuric acid. All nutrients other than ammonium nitrate were the same concentration in both tanks. Nutrient delivery was controlled with an ARGUS environmental control system. The nutrient solution was applied four times per day to deliver 170 ml/plant/day. Nutrients were delivered at the same time for the same duration in all treatments. There was a secondary irrigation system for watering the high irrigation rate which delivered an additional 70 ml of water/plant/day. 38 Sampling methods
Leaf samples were collected every three weeks and included two leaves from each plant, both the fourth leaf from the apex of two shoots. The four leaves sampled from each EU were pooled into one sample for chemical analysis. The leaves were cut in half and analysed fresh and dry on a dry weight basis according to the procedure described in Sections 2.2.1 and 2.2.3.
2.3. RESULTS
2.3.1. Field irrigation trial: effects of irrigation frequency on parthenolide
concentration and plant growth
Irrigation rate had a small but significant affect on leaf parthenolide concentration and shoot yield. Parthenolide content was similar between treatments at
43 days, but at 87 days it was higher in leaves of plants grown at the lower irrigation rate. Under the higher irrigation frequency, leaves maintained a constant parthenolide concentration between the two times. Shoot dry weight yield was 8% higher in response to the higher irrigation rate after 87 days.
Table 2.1. Parthenolide concentration of feverfew leaves 43 days and 87 days after transplanting. Units for parthenolide concentration are mg parthenolide / gram dry leaf and plant dry weights are in grams.
Leaf parthenolide concentration (mg/g) Plant dry weight (g)
Irrigation frequency 43 days 87 days 87 days
Low 5.10 5.39 236.6
High 4.49 4.46 258.0
Significance NS * *
* Significance (p < 0.05) NS No Significance (p > 0.05) 39 2.3.2. Field irrigation and nitrogen application trial
Biomass was affected by irrigation rate but not by nitrogen treatments (Table
2.2). There was a 9% reduction in shoot dry weight (leaves, stems and flowers
combined) in response to the low compared to high irrigation rate (p=0.017). In a
mature plant, stems contributed more to total dry weight than did flowers and leaves
combined (Table 2.2). Leaf dry weight was 14% lower (p = 0.049) under the lower frequency irrigation treatment. Stem dry weight was 10% lower (p=0.012) under the
lower frequency irrigation rate. Dry to fresh weight ratios of leaves, stems, and flowers were not affected by irrigation treatments.
Table 2.2. Average whole plant and organ dry weights. Samples were harvested 87 days after transplanting.
Dry weiqht yield (q/plant) Nitroqen rate (N)(n=8) stem leaf flower total 0 Kg/ha 130.4 34.89 71.65 236.9 50 Kg/ha 133.4 37.52 72.10 243.0 100 Kg/ha 136.6 39.89 71.33 247.8 significance NS NS NS NS Irriqation rate (I) (n=12) low 127.0 34.57 70.09 231.6 high 139.9 40.30 73.29 253.6 significance ** * NS *
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05
Leaf water potential, osmotic potential, and turgor were highest at 3 a.m. and
decreased through the day at 12 pm and 6 pm (Table 2.3). Plants under the lower
frequency irrigation treatment had lower water potentials, osmotic potentials, and turgor
pressure at all three measurement times than did plants under high frequency 40 irrigation, specifically in the late afternoon and at night. The calculated turgor was approximately zero under low frequency irrigation at 6 pm. The N treatments affected leaf water status. The 50 kg/ha N treatment had higher water potential and turgor than both the 0 and 100 kg/ha treatments at 12 pm. At 6 pm the water potential was lower in the 50 kg/ha N treatment resulting in a turgor pressure of 0 MPa.
Table 2.3. Leaf water status in field grown feverfew measured at 3 am (night), 12 pm (mid-day), and 6 pm (evening). Leaf water potential was measured in the apoplast and osmotic potential was measured from the expressed cell contents. Turgor pressure was calculated by subtracting osmotic from water potential. All values reported in MPa.
Water potential Osmotic potential Turgor pressure
Nitrogen rate 3 am 12 pm 6 pm 3 am 12 pm 6 pm 3 am 12 pm 6 pm
0 Kg/ha -0.453 -1.19 -1.36 -1.15 -1.33 -1.44 0.696 0.139 0.077
50 Kg/ha -0.447 -1.04 -1.44 -1.18 -1.26 -1.41 0.733 0.224 0
100 Kg/ha -0.447 -1.16 -1.41 -1.20 -1.31 -1.49 0.750 0.148 0.085
significance NS NS NS NS NS NS NS
Irrigation rate
Low -0.496 -1.15 -1.55 -1.19 -1.31 -1.49 0.697 0.161 0
High -0.402 -1.11 -1.25 -1.16 -1.29 -1.41 0.755 0.180 0.160
significance NS ** NS NS NS
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05
Leaf parthenolide concentration increased during early development and then decreased as the crop matured to the reproductive stage (Table 2.4). After the feverfew crop was harvested on 24 July (87 days after transplanting) it was regenerated and leaf parthenolide concentration increased. Leaf parthenolide varied over time but was consistent between treatments. There were significant treatment 41 effects due to irrigation frequency and nitrogen rate in samples taken 69 days after transplanting just prior to harvest. The low irrigation frequency and 50 Kg/ha nitrogen resulted in the highest leaf parthenolide concentration. The regenerated growth showed significant nitrogen treatment effects after 28 days of regeneration. Applying no N resulted in a lower parthenolide concentration than did the other nitrogen treatments. After 53 days of regeneration lower frequency irrigation resulted in lower parthenolide content.
Table 2.4. Dry leaf parthenolide content measured over a 3 month period in the field. Sampling dates are indicated and days after transplanting are bracketed. Units are mg parthenolide/gram dry leaf.
Leaf parthenolide concentration (mg parthenolide/q dry leaf) First crop Regenerated crop 14-Jun 26-Jun 09-Jul 24-Jul 21-Aug 15-Seo Nitrogen rate (32) (44) (58) (73) (101) (126) 0 Kg/ha 3.96 5.50 2.84 2.71 2.82 4.49 50 Kg/ha 4.25 6.10 3.09 3.40 3.72 4.51 100 Kg/ha 4.34 5.46 2.81 2.45 3.74 4.39 Significance NS NS NS * * NS Irrigation frequency Low 3.93 5.52 3.01 3.16 3.32 3.99 High 4.43 5.85 2.82 2.55 3.53 4.93 Significance NS NS NS * NS * * significance p<0.05 ** significance p<0.01 NS no significance p>0.05
The parthenolide concentration in dried flowers was more than twice that in dried
leaf lamina (Table 2.5). Stems contained very little parthenolide. Approximately 80%
of the parthenolide in a flowering plant was contained in the flowers. Leaves contained
about 18% followed by stems at 2%. The field treatments affected both the flower and 42 leaf parthenolide content. Leaf parthenolide content was 20% higher under the lower irrigation rate than the higher rate. Among the three nitrogen rates, 50 Kg/ha N applied resulted in the highest leaf parthenolide content. As applied N was increased under the lower irrigation rate, flower parthenolide content decreased, but under the higher irrigation rate, increasing N application resulted in increased flower parthenolide content (Table 2.5).
Table 2.5. Leaf and flower parthenolide concentrations and total parthenolide content per plant of stems, leaves and flowers. Samples were harvested from mature flowering plants grown in the field.
mg Parthenolide per gram mg Parthenolide per plant
Nitrogen rate Leaf Flower Stem Leaf Flower Total
0 Kg/ha 2.71 8.36 19.57 92.59 594.0 706.1 50 Kg/ha 3.40 8.39 20.02 139.9 603.0 762.9 100 Kg/ha 2.45 8.40 20.49 99.40 597.6 717.5 Significance * NS NS NS
Irrigation frequency
Low 3.16 8.63 19.05 117.8 604.4 741.3 High 2.55 8.13 21.00 103.4 591.9 716.3 Significance * NS NS NS
* significance p<0.05 NS no significance p>0.05
2.3.3. Greenhouse irrigation and nitrogen application trial
Irrigation and N fertilization effects on leaf parthenolide developed two months after treatment initiation. The mean leaf parthenolide concentration was 8.4 mg/g dry weight at transplanting. As the crop matured, young flowering tops were removed to repress flowering and promote crop homogeneity for 54 days. During this time, the mean leaf parthenolide concentration increased to 37 mg/g dry weight and was unaffected by treatments (Table 2.6). During the next 16 days treatment effects began 43 developing and were maintained as the crop matured to flowering. At 70 days, leaves on plants fed with 1mM N had 40 mg parthenolide /g dry weight while those on plants fed 0.5 mM N had less than 30 mg parthenolide/g dry weight after 70 days.
Subsequently, flowering was allowed to proceed, and parthenolide concentration decreased to 1.8 mg/g under low N and 13 mg/g under high N fertilization. The N treatment effect on leaf parthenolide was significant after two months and was
maintained through the transition from vegetative to reproductive growth. Irrigation did
not have significant effects on parthenolide content and there was no interaction
between irrigation and N rate that effected parthenolide content. The higher irrigation treatment was continued for an additional 40 days after the lower irrigation treatment was stopped.
Table 2.6. Leaf parthenolide concentration in feverfew leaves grown in the greenhouse under irrigation and nitrogen treatments. Days from treatment initiation are in brackets. Flowering tops were picked off plants 31 days, 40 days, and 54 days after treatments were applied. Parthenolide concentration is mg parthenolide/g dry leaf
Days after transplanting for sample collection 29 50 71 98 Nitrogen rate 0.45 mM 8.44 18.7 35.9 27.9 1.77 0.91 mM 8.44 21.7 37.3 39.6 12.6 significance NS NS NS ** ** Irrigation freguencv Low 8.44 21.8 37.2 34.5 High 8.44 18.5 36.0 33.4 5.80 significance NS NS NS NS NS * significance p<0.05 NS no significance p>0.05 44
2.4. DISCUSSION
The experiments in this chapter showed that low irrigation frequency in the field
had a small but significant effect on both plant yield and leaf parthenolide content. In
the two field trials, yield was 8% (Table 2.1) to 9% (Table 2.2) lower three months after
a low irrigation rate was initiated. Dry matter partitioning among organs of mature
feverfew in Table 2.2 revealed that most of the dry weight (55%) was in stems followed
by flowers (29%) and leaves (16%). The average leaf dry weight per plant under the
low irrigation rate was 14% less (p = 0.049), and stem dry weight was 10% less
(p=0.012) than plants irrigated at the higher frequency. Treatment effects in the 1997
field irrigation trial were probably reduced due to the high rainfall that summer.
Water stress can reduce plant growth and yield but has variable affects on
terpenoid chemistry (Hanson and Nelson, 1980; Gershenzon, 1984). Under water
stress, nutrient acquisition and mobility is reduced, primary metabolism slows down,
and specialized stress responses may occur such as increased abscisic acid synthesis,
and increases in the synthesis of osmoregulators like proline, and betaine (Losche,
1996). Reduced stomatal conductance and metabolism are the main causes of
reduced biomass underwater stress (Losche, 1996). Leaf water potential is a measure
of water status of a plant and is regulated by stomatal conductance which in turn
determines rate of CO2 uptake. Low C02 uptake can reduce carbon assimilation and
biomass. In the field study, feverfew responded to low irrigation frequency with
reduced biomass in leaves and stems. Feverfew under low irrigation frequency had
higher water potentials and osmotic potentials, specifically in the late afternoon (6 pm)
when transpiration was likely at a peak and during the night (3 am) when the stomata
and the plant xylem equilibrate with the soil. As indicated by the lower plant dry weight,
stomatal conductance was probably lower in response to the lower irrigation frequency 45
and reduced C02 uptake and carbon assimilation. Low turgor pressure was calculated for low frequency irrigation treatments which also lead to reduced growth and wilting.
Reduced CO2 uptake and low turgor are both possible precursors for the yield
reduction that was observed in stem and leaf yields under low irrigation treatments.
Unlike stems and leaves, flower dry weights were not significantly affected by irrigation
rate. The flowering process is a primary sink for carbon and often does not show the
same response to reduced water availability as leaves and stems which exhibit
reduced growth and development.
Nitrogen is a component of all plant enzymes and many metabolites and thus is
an important plant nutrient which is often a limiting factor in field crops. Feverfew is a
weedy species commonly found growing on roadsides and poor soils. The nutrient
requirements of feverfew are probably low since it seems able to thrive in unfertilized
wastelands where it survives well and reproduces under low N conditions. In the field,
nitrogen application did not significantly affect feverfew dry weight yields and there
were no visible signs of nitrogen deficiency such as leaf yellowing or reduced growth
when no N was applied. A plausible explanation is that residual nitrogen in the field
was sufficient for feverfew growth and therefore additional nitrogen did not greatly
contribute to yield.
Nitrogen affected both water potential (Table 2.3) and turgor measured at 12pm.
When soil water is limited, plants can adjust the leaf osmotic potential to maintain
turgor at low water potentials. Plants utilize osmoregulators like proline, and betaine
which contain nitrogen (Losche, 1996). Nitrogen availability may influence synthesis of
osmoregulators in feverfew. There was an effect of N fertilizer rate on water potential
at mid-day (12 pm) which resulted in a higher water potential for plants fertilized with 50
kg/ha N compared to 0 and 100 kg/ha N treatments. An explanation for this result may 46 be that 50 Kg/ha N is sufficient for growth while maintaining the salinity of the soil lower than at 100 kg/ha N. Higher salinity in the soil caused by excess nitrogen could decrease soil water potential reducing the plants ability for water uptake. Therefore, soil with higher nitrogen content and higher salinity may result in lower leaf water potentials in response to the soil osmotic potential. This in turn would result in lower turgor which is the result obtained for 100 Kg/ha compared to 50 kg/ha N treatments.
Tugor pressure at 12 pm under the 0 Kg/ha treatment was the lowest of all treatments and was primarily due to the low leaf water potential. Feverfew under 0 kg/ha N treatment may not have adequate N and therefore must keep stomates open to fix carbon for root growth, and to maintain the transpirational stream for nitrogen acquisition. Interestingly the treatments with significant differences in water relations are the same as those with differences in parthenolide concentrations.
The role of secondary chemistry in plants under water stress remains, for the most part, unknown. Stress induced increases of secondary compounds may provide antioxidant potential to combat increased oxidation due to water stress (Losche, 1996).
Stress induced susceptibility to pathogens may elicit increased chemical defences.
Primary metabolism may be affected while secondary metabolism remains unchanged resulting in higher relative concentrations of the secondary compounds. Finally, secondary metabolism may decrease in response to water stress as a conservation mechanism to minimize waste of resources (Gershenzon, 1984; Gershenzon and
Croteau, 1991). There have been many studies of the effects of water stress on essential oil content in a number of species showing various and sometimes contradictory results, but few that specifically examine sesquiterpene lactones. In the
1996 and 1997 field trials, feverfew's parthenolide content 73 days after planting (DAP)
(Table 2.4) and 87 DAP (Table 2.1) respectively were higher under low irrigation rates 47 while shoot biomass decreased. The average leaf parthenolide concentration was 17%
(table 2.1) to 20% (figure 2.5) higher under low irrigation treatments compared to high
irrigation treatments after three months. These treatment effects took more than two
months to develop. In addition, only the plants in full flower showed this treatment
effect. At harvest, leaf parthenolide concentration in regenerated vegetative plants was
higher under the high irrigation frequency compared with the low frequency. Nitrogen
moves in the soil with water. Under low irrigation treatments N may build up in the soil
because it is less prone to leaching compared with higher irrigation treatments. This
can result in higher N availability under low irrigation compared to high irrigation
treatments. If the plant takes up more N under low irrigation frequency a higher rate of
enzyme production may result allowing greater STL production. Another explanation
for higher parthenolide concentration in low irrigation treatments was briefly mentioned
above where parthenolide and STL synthesis may not have been directly affected by
water or nitrogen availability but was an indirect result of reduced biomass caused by
these factors. For example if biosynthesis remained constant but leaf size was
reduced due to limited resources then leaf STL concentration may be higher. The
response of feverfew to mild water stress was similar to that found with Mentha piperita
in which essential oils (including sesquiterpenes) increased by 37% and was
accompanied by decreased biomass (Charles et al., 1990). In field grown Ambrosia
maritime and Achillea millefolium, decreased growth in response to reduced irrigation
was correlated with decreased leaf and flower sesquiterpene lactone concentration
(Sidky, El-Mergawi, 1997; El-Kholy, 1984; Simon er al., 1992). In these types of
investigations however, problems arise when differentiating between changes in
biomass and secondary chemistry since it is often unclear if biosynthetic rate of the 48 secondary compound or plant biomass are the main effect when they both may change in response to treatments.
Irrigation and nitrogen affected both flower and leaf parthenolide. At harvest 72
DAP leaf parthenolide was highest under the 50 kg/ha N treatment regardless of irrigation rate (Table 2.5). Similar results were obtained by Rao (1990) in a study of essential oil yields of geranium (Pelargonium graveolens) which were 24% higher at moderate nitrogen levels (100 kg/ha) compared with excess or limited nitrogen treatments. In a study of basil (Ocimum basilicum), sesquiterpenes increased with increasing nitrogen application but decreased when nitrogen was excessive (Youssef et al., 1998). The main effects of irrigation and nitrogen on flower parthenolide concentration were insignificant, but an interaction between the two factors had an effect on parthenolide concentration resulting in opposing effects of N when different irrigation rates were applied.
Nitrogen treatments on feverfew grown in a greenhouse had significant effects on leaf parthenolide. Leaf parthenolide was the highest in response to lower irrigation level and higher nitrogen treatments 71 and 98 DAP, a result similar to the field trials.
However, in contrast to the field trials leaf parthenolide substantially increased as the crop matured. This increase was enhanced by the removal of young flowering tops to repress flowering. Treatment effects on leaf parthenolide were significant after two months, and were sustained through the transition from vegetative to reproductive growth. In the third month while the plants were in a vegetative stage, the highest leaf parthenolide concentration (maximum 5%) was recorded. Plants grown under high N rate had 30% greater leaf parthenolide (4%) than plants treated with a low N rate
(2.8%). This is in contrast to the field trials, in which parthenolide decreased as the crop developed and never attained levels greater than 1%. After flowering was allowed 49 to proceed in the greenhouse trial, the crop continued its development to the
reproductive stage during which leaf parthenolide substantially decreased.
Preflowering leaf parthenolide was very high in this greenhouse trial compared to that
reported elsewhere. Leaf parthenolide from vegetative plants was reported to be much
lower than in leaves from flowering plants (0.33% versus 1.27%) (Awang, 1991).
Feverfew leaf parthenolide concentration in flowering plants was reported as high as
2.77% in apical leaves, but generally ranges between 0.3% - 1.5% in mature leaves
(Hendriks et al., 1997; Dolman et al., 1992; Brown er al., 1996). The majority of
research on feverfew appears to have been performed in greenhouses, and results of detailed field studies have not been reported. It would be unfair to compare directly the field and greenhouse trials presented in this chapter because they were done at different times under different conditions. However, in the greenhouse the significant
decrease in parthenolide content after being held in a vegetative state for 3 months and then allowed to flower suggested there was a large effect correlated with the
developmental stage that might also explain patterns observed in the field.
Experiments that explore developmental affects on parthenolide and yield are
presented in chapter 4.
Feverfew is marketed with an assurance of minimum parthenolide concentration.
Parthenolide content was proposed as a marker compound to assure feverfew
authenticity. It has been incorrectly presented as a quality assurance and sometimes
as the primary active principle. Not all the active components have been identified and
parthenolide is found in many other plants (see Chapter 1). Farmers are paid by dry
weight of the herb regardless of parthenolide concentration and therefore high levels of
field production with low overhead costs are important regardless of chemical quality.
Yield and parthenolide variability have been discussed separately but could be 50 considered together to determine crop performance based on parthenolide concentration of the whole plant. The weight contributions of different organs are
important when considering parthenolide content. For example, the field crop was
harvested two months after transplanting when the plants had approximately 80%
mature flowers. In whole plants, flowers contributed most parthenolide (80%) per plant followed by leaves (18%) and stems (2%). However, stems contributed more than 55% total dry weight followed by flowers (29%) and leaves (16%). Low irrigation rate
reduced leaf and stem dry weights, but not flower dry weight which was the major
contributor of plant parthenolide. Therefore, total parthenolide was higher in plants
receiving lower irrigation rates and 50 kg/ha N. By growing plants under specific
conditions it should be possible to achieve the most favourable organ proportions and
chemical composition if yield is not the first priority.
Variability of secondary compounds within a species is well established and
appears to be under both genetic and environmental control. For example, STL
accumulation in chicory (Chichorium intybus L.) root was dependent on the cultivar,
growing location and nitrogen supply (Peters et al., 1997). Parthenolide content in
feverfew has been examined in plants grown in tissue culture (Brown et al., 1993,
Brown et al., 1996), grown in different regions and climates (Maries et al., 1992), and
in commercial preparations (Awang et al., 1991; Heptinstall et al., 1992). Parthenolide
variability has been attributed to many factors such as processing methods, chemical
degradation (Smith and Burford, 1992), fillers and adulterants (Awang, 1991), varietal
differences (Awang, 1989) and extraction and analytical methods (Brown et al., 1996).
Aside from this investigation, there have not been studies on the effect of the growing
environment on feverfew. Indirect evidence that growing conditions affect parthenolide
variability comes from the literature which reports large variation in feverfew 51 parthenolide content in leaves (0 % - 2.8 %), flowers (0.5% - 2.3%), parthenolide concentrations that are very low in Canadian-grown plants (Awang et al., 1991;
Heptinstall and Awang, 1998), and even undetectable in Mexican- and Yugoslavian- grown feverfew (Maries et al. 1992). If high parthenolide concentration and high plant yields are desirable, these experiments show that we can grow high quality feverfew in one part of Canada in spite of previous claims that our climate is unsuitable. 52 2.5. REFERENCES
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Feverfew production under field and greenhouse conditions
3.1. INTRODUCTION
While feverfew reduces the frequency and intensity of migraine headaches, both migraine physiology and STL (parthenolide) actions are complex and not understood.
Parthenolide and other STLs are considered the primary candidates for the antimigraine activity due to their vasoregulatory properties. There is indirect evidence that parthenolide has antimigraine activity but until direct evidence is presented for parthenolide, and we have evaluated the antimigraine action of other feverfew constituents, the medicinal quality of feverfew should not be assessed only on parthenolide concentration. Once the active compounds are established, greenhouse production may give the flexibility to manipulate chemical content through cultivation methods.
The controlled environment of greenhouses makes it possible to grow a wide range of fruits, vegetables, herbs, flowers and houseplants throughout the year and in areas with harsh climates. Greenhouse experiments allow for strict control of nutritional factors and environmental parameters. Although greenhouse research is important for many of the common medicinal plants, most commercial production is outdoors or plants are harvested wild. Differences between the greenhouse and field may be temperature, light quality, nutrient and water availability, wind, rain, growing media, root restriction, and length of growing season, each of which may in turn cause significant physiological and chemical changes in a plant. Most of these factors affect stomatal 56 conductance and leaf water potential, which directly affect yield, physiology and alter chemical composition.
In Chapter 2, I reported that field-grown feverfew with low water potential had a lower dry weight yield and higher leaf parthenolide concentration when plants were at full flower, a result also found in Cymbopogon martinii under irrigation treatments where low irrigation resulted in reduced growth and higher essential oil yields (Shabih et al.,
1999). Two causes of low water potential are reduced water availability and high temperature leading to a high vapor pressure deficit. For an herbaceous plant like feverfew in which leaves have commercial value, water relations are important because they directly affect leaf yield. Therefore, the difference in water relations between the greenhouse and field may be important in determining whether greenhouse production can be used to improve yield and alter chemical concentrations and/or composition of feverfew.
Feverfew is commercially grown in the field but has potential as a greenhouse crop. The cost of greenhouse operation is high and often restricts greenhouse growers ability to produce high-value crops. To gain a market advantage greenhouse producers select crops with extremely high fresh market value or grow seasonal crops during the out-of-season periods when they can't be grown outdoors. Currently feverfew may not be grown economically in the greenhouse because its market value is low, it grows well in the field, and it can be dried and stored for long periods. Feverfew is generally sold as dried crushed shoots, therefore visual quality of the raw plant material does not contribute to value like other quality parameters such as chemical composition.
Greenhouse production is only viable if high quality, high yield, or fresh product are required. There is evidence that fresh leaves have different pharmacological activity compared with dried leaves, so the value of feverfew may be very different if sold as a 57 fresh product. In a study testing the effects of feverfew leaf extracts on aortic ring relaxation and contraction, fresh extracts caused relaxation while dried extracts caused contraction (Barsby et al., 1993). In contrast to fresh leaves, the dried leaves did not contain STLs. More research is required but this may be evidence toward the preferential use of fresh feverfew leaves.
In this Chapter I report that field-grown feverfew had greater dry weight yields than the greenhouse. However, total shoot yield may not be as important as the yield of specific organs such as leaves or flowers. Leaves have been the traditional source and mixed preparations of leaves, flowers and stems may be of lower quality (efficacy) than leaves alone. One way to change quality is by altering the proportion of leaf, stem and flower tissues. An economical way for producers to achieve this is to grow feverfew in a way that provides the best ratio of shoot organs. Another, more labor intensive way to increase quality is separating leaves from flowers and stems. The removal of stems would reduce yield but may increase potency.
Reducing variability in chemical composition of medicinal plants is important in the herbal drug industry since variability makes it difficult to administer the correct dosage. Water relations, yield, and chemical composition are three related attributes in plants that vary between field and greenhouse production. The research presented in this chapter examines the potential for manipulating leaf, stem, and flower proportions in the greenhouse and field by manipulating growing conditions. In this study I used feverfew as a model to investigate the relationship between yield and chemical content
(STLs) in plants and how they are influenced by greenhouse and field environments. In this chapter comparisons are made between the greenhouse and field by conducting experiments in both environments at the same time with comparable treatments.
Response of yield, parthenolide concentration, and water status to media treatments 58 and to production environment (field or greenhouse) were determined. Specifically three primary questions were addressed: Does growing medium affect yield and parthenolide concentration of feverfew leaves, stems, and flowers? How do potted plants in the field or greenhouse compare with plants grown in field soil? What is the effect of crop regeneration on yield, parthenolide concentration, and water status in feverfew grown in the greenhouse?
3.2. MATERIALS AND METHODS
3.2.1. General methods
Propagation of feverfew seedlings
Feverfew was grown from seed collected from a previous feverfew trial at the
Pacific Agri-Food Research Center in Agassiz, British Columbia, Canada. The seeds were sown (April 6th) into flats containing a peat and perlite mixture, in rows spaced 5 cm apart and covered with 3 mm of perlite. Germination was one week after sowing and the seedlings were grown for 30 days in the flats before being transplanted into cell trays with 4 x 4 x 4 cm cells filled with a mixture of peat moss, sawdust, and compost in a 1:1:0.5 ratio. Seedlings for the field experiment were moved outside into cold frames
June 10th and transplanted to the field June 14th (72 days after seeding). Seedlings for the greenhouse trial were transplanted June 14th (72 days after seeding). On June 30th
(16 days after transplanting) the growing tips were removed from plants in both the greenhouse and field trials.
Harvesting and Shoot Regeneration
Seventy-seven days after transplanting, the greenhouse and field grown crops were harvested. The shoots were pruned to crowns 10 cm above the base ofthe plant.
This left sufficient leaf material on the crown which promoted quick regeneration. The 59 field experiment was terminated after the harvest. The greenhouse experiment was continued by regenerating the plants that were pruned to crowns. New growth was observed one week after pruning. The second crop was harvested 95 days after the first crop was harvested (172 days after transplanting). The total shoot fresh weights were measured for each experimental unit (EU).
Subsampling
Shoot subsamples for organ partitioning were selected randomly from plants in each EU. The subsamples were separated into leaves, flowers, and stems. Their fresh and dry weights were measured and the number of flowers counted.
Sampling
Leaf samples for chemical analysis were harvested from the greenhouse and field grown crops 0, 36, and 77 days after transplanting. Two additional samples were taken from the regenerated greenhouse crop 128 and 173 days after transplanting (50 and 95 days after the first harvest). Leaves sampled were the fourth leaf from a shoot apex taken from four shoots per plant. Leaves from plants in each experimental unit
(EU) were dried in an open container at 40 °C for 6 hours and then weighed.
Water potential and osmotic potential
Prior to the crops being harvested at the end of summer and end of autumn, samples for leaf water potential and osmotic potential measurements were collected.
Leaf water potential was measured at 2 pm and 4 am using a portable pressure bomb.
Leaves with petioles that were approximately 10 cm long were cut and immediately placed into the pressure bomb with petioles extruding. The pressure was recorded when sap extruded from the cut petiole. The same leaves were frozen for osmotic potential measurements. The osmotic potential of the expressed sap from the thawed 60 leaves was measured with a freeze point depression osmometer (Advanced
Instruments).
Surface extraction
Leaves were extracted by dipping three times with dichloromethane for 30 seconds each time. The extract was vacuum filtered through Whatman No. 1 filter paper. The filtrate was evaporated to dryness under vacuum at 30 °C using a rotary evaporator. The extract was quantitatively transferred to a vial with 20 ml HPLC grade methanol for chemical analysis.
Chemical analysis: parthenolide extraction and quantification
Methods for parthenolide extraction and quantification are described in Chapter
2.2.2.
Statistical analysis
Statistical analysis was performed using SAS and SYSTAT software. Analysis of variance with P<0.05 was used to determine significance between treatments.
3.2.2. Field fertigation frequency and growing medium trial
Experimental design
The experiment was a randomized block design with five treatments and four blocks. The five field treatments are outlined in Table 3.1. Treatments were selected to compare field production methods with greenhouse production methods. The plasticulture treatment represented field production with plants directly in the field soil.
In this treatment, there were ten plants per experimental unit (6 experimental plants and
4 guard plants on the row ends), spaced 45 cm between the rows and 45 cm within the rows. The fertigated plasticulture treatment was the same as previous plasticulture trials (Chapter 2) except that the fertigation system was through individual drip lines, 1 61 per plant, instead of drip tape under the plastic. The remaining four treatments represented greenhouse hydroponics and consisted of plants grown in 35 cm pots.
Each pot was spaced 30 cm apart. There were three pots in each EU and each pot contained 2 plants spaced 20 cm apart. There were a total of six raised beds. The two outside beds were guard beds and the inner four beds were the blocks within which the five treatments were arranged randomly. The four blocks each with five treatments resulted in a total of 20 experimental units. All experimental plants were sampled for physiological and chemical measurements.
Table 3.1. Field experiment treatments and abbreviations.
Treatment descriptions Abbreviations
Plasticulture, low fertigation Plasticulture, Low
Soil filled pots set into the ground, low fertigation Inset Pots, Low
Soil filled pots above ground, high fertigation Soil Pots, High
Soil filled pots above ground, low fertigation Soil Pots, Low
Sawdust filled pots above ground, high fertigation Sawdust Pots, High
Field culture system
Seedlings were transplanted to the field after propagation in the greenhouse (see
Section 3.2.1). A fertigated plasticulture system was used in combination with pot- culture. One of the treatments utilized plasticulture with plants grown in field soil. The other treatments used pot-culture. Each pot was set either into the ground or on top of the ground on dark green polyethylene covered raised beds. Pots above the ground in the field had a white polyethylene sleeve covering the black pot exterior to minimize high pot temperatures. A clear polyethylene tent was erected over the crop to reduce exposure to wind and rainwater. 62 Fertigation schedule
The fertigation system in the field was designed to mimic the greenhouse nutrient delivery system. A nutrient solution was drawn into the irrigation line via a Mazzi injection system, which draws nutrient solution by a hydraulic vacuum into the irrigation line. There were two rates of nutrient solution (high fertigation and low fertigation).
Nutrient solution was delivered either four (low rate) or eight (high rate) times daily, for two minutes each. The flow rate averaged 35 ml/min, which delivered 280 ml (low) or
560 ml (high) nutrient solution per day per plant. All treatments were fertigated with the formulation outlined in the Appendix.
3.2.3. Greenhouse irrigation frequency and growing medium trial
Greenhouse culture system
Feverfew seedlings were transplanted 20 cm apart into sawdust or soil treatments in the greenhouse. Sawdust filled pillow bags (cylindrical shaped, polyethylene, 1 m long x 20 cm diameter) were used for one treatment. Pots, 35 cm diameter and 35 cm deep, were used for the other three treatments (two soil and one sawdust). The plants were grown on a bench one-meter high, under natural day-length.
Experimental design
The greenhouse trial was designed to match closely the field trial described in section 3.2.2. Treatments were arranged randomly in blocks from west to east. Three treatments received a high rate of nutrient feed and one soil treatment received a low rate of nutrient feed (table 3.2). There were ten experimental units per treatment. With the exception of the pillow bag treatment, the other three treatments matched those in the field. The experiment lasted for 6 months. This was 3 months longer than the field trial. All plants were used for physiological and chemical measurements. 63
Table 3.2. Treatments in the greenhouse trial and abbreviations.
Greenhouse treatment descriptions Table abbreviations
Sawdust filled pots, high fertigation Sawdust Pots, High
Sawdust filled pillow bags, high fertigation Sawdust Pillows, High
Soil filled pots, high fertigation Soil Pots, High
Soil filled pots, low fertigation Soil Pots, Low
Greenhouse nutrient solution and feeding schedule
The fertilizers to be applied were dissolved in a tank containing 500 L of water.
The electrical conductivity ofthe nutrient solution was adjusted between 1.8 to 1.9 mSv using sodium chloride and the pH was adjusted to 6 with sulphuric acid. Two pumps were submersed in the tank, each attached to 1.8 cm diameter polyethylene tubing that delivered nutrient solution to the experiment. Nutrient solution was delivered from this line to each plant through smaller drip line (0.15 mm i.d.) at a rate of 35 ml/minute. The pumps and timing of nutrient solution delivery were regulated by an Argus control system. Nutrient solution was delivered four times per day for the low rate treatment or eight times per day for the high rate treatments. There was one drip line per plant.
Each feed delivery was for 2 minutes to provide 280 ml (low rate treatment) or 560 ml
(high rate treatment) solution per day. The nutrient feed formula is in the Appendix. 64 3.3. RESULTS
3.3.1 Field media and fertigation trial
The field trial compared plasticulture with culture in pots containing soil and sawdust media. The plants grown in plasticulture and in pots set into the ground had greater yields than plants grown in pots above-ground (Table 3.3). Plants in the plasticulture treatment had the highest stem proportion and shoot dry weights 2-3 times
Table 3.3. Dry weight and dry to fresh weight ratios of feverfew plants grown in the field and greenhouse. Plants were partitioned into leaves, stems and flowers. The field and first greenhouse crops were harvested when the plants were in full flower, 142 days after seeding and 77 days after transplanting. The second greenhouse crop, regenerated from crowns, was harvested 95 days after harvesting the first crop. The regenerated greenhouse crop was in the vegetative developmental stage when harvested November 30.
Dry weight yield (g) Drv:Fresh weight # flowers/ gdry Field Leaf Stem Flower Total Leaf Stem Flower shoot Plasticulture, Low 36.7 70.3 48.1 155 0.156 0.239 0.238 7.39 Inset Pots, Low 16.3 26.6 23.3 66.3 0.165 0.258 0.234 7.70 Soil Pots, High 9.56 17.1 11.4 38.0 0.228 0.295 0.265 7.74 Soil Pots, Low 10.7 12.9 12.3 35.8 0.241 0.337 0.275 7.57 Sawdust Pots, High 9.72 15.5 11.6 36.8 0.208 0.290 0.249 7.07 Significance ** ** ** ** ** ** NS NS
3reenhouse (1st harvest) Sawdust Pots, High 15.5 18.4 33.6 67.5 0.149 0.239 0.311 10.5 Sawdust Pillows, High 18.5 20.6 39.9 78.9 0.158 0.229 0.309 10.9 Soil Pots, High 18.5 21.2 30.1 69.9 0.156 0.251 0.365 9.33 Soil Pots, Low 20.9 18.9 24.9 64.7 0.172 0.290 0.344 8.55 Significance NS NS * * * ** NS NS
Breenhouse (2nd harvest) Sawdust Pots, High 10.8 11.6 22.4 0.093 0.0873 Sawdust Pillows, High 22.2 19.0 41.2 0.110 0.0990 Soil Pots, High 16.4 17.5 33.9 0.102 0.0823 Soil Pots, Low 20.4 21.9 42.2 0.111 0.106 Significance * * * * NS
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05 65 greater than plants in the pot-culture treatments. Soil-filled pots set into the ground had greater aerial organ yield than the above-ground treatments. The pattern of leaf, stem, and flower proportions was consistent among treatments; stems (35%-45%) contributed most of the plant dry weight followed by flowers (30-35%) and leaves (24-26%).
Plant water status was assessed using water potentials, osmotic potentials, and dry to fresh weight ratios. The water and osmotic potentials were significantly lower in the two above-ground soil-filled pot treatments, and the low rate fertigation treatment had the lower water potentials of the two (Table 3.4). The calculated cell turgor was higher at night, particularly in the three above-ground treatments, but during the day turgor was significantly less and was negative in low rate fertigation soil and sawdust pots (Table 3.4). The plasticulture and the inset-pot treatments had the highest water and osmotic potentials during the day. Turgor at night in these treatments was lower than that of other treatments. The dry to fresh weight ratios (Table 3.3) were significantly lower in the plasticulture and the inset soil-filled pot treatments indicating the higher water content of these plants. The flower dry to fresh weight ratio was the same as the stem dry to fresh weight ratio in the plasticulture treatments, unlike in the other treatments where stem dry to fresh weight ratios were greater than that for flowers and leaves. All three of the above-ground soil or sawdust pot-culture treatments had low water potentials, low osmotic potentials, high dry to fresh weight ratios and the lowest dry weight yields. Overall, stems had the highest dry to fresh weight ratio followed by flowers and leaves. 66
Table 3.4. Leaf water potential and osmotic potential of greenhouse and field grown plants measured at 3 p.m. (light) and 4 a.m. (dark). Turgor was calculated by subtracting osmotic potential from water potential. Potentials and turgor are reported in MPa.
Location and Treatments Water potential Osmotic potential Turgor Field Dark Light Dark Light Dark Light Plasticulture, Low -0.288 -1.35 -1.12 -1.27 0.83 0 Inset Pots, Low -0.275 -1.21 -1.04 -1.30 0.77 0.09 Soil Pots, High -0.300 -1.56 -1.23 -1.52 0.93 0 Soil Pots, Low -0.770 -2.05 -1.66 -1.85 0.89 0 Sawdust Pots, High -0.283 -1.59 -1.20 -1.43 0.92 0 Significance * ** ** ** * *
Greenhouse (1st harvest) Sawdust Pots, High -0.722 -1.71 -1.57 -1.85 0.850 0.14 Sawdust Pillows,High -0.772 -1.76 -1.55 -1.90 0.775 0.14 Soil Pots, High -0.783 -2.04 -1.56 -1.96 0.777 0 Soil Pots, Low -0.974 -2.17 -1.53 -2.08 0.558 0 Significance ** * NS NS * NS
Greenhouse (2nd harvest) Sawdust Pots, High -0.619 -1.55 -1.37 -1.65 0.760 0.10 Sawdust Pillows,High -0.567 -1.59 -1.28 -1.70 0.714 0.11 Soil Pots, High -0.752 -1.83 -1.36 -1.76 0.607 0 Soil Pots, Low -0.862 -1.73 -1.21 -1.68 0.548 0 Significance * NS NS NS NS
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05 67
Table 3.5. Average leaf parthenolide concentration in greenhouse and field-grown plants. The first leaf sample was a crop average for both the greenhouse and field taken just prior to transplanting. The first sample was 7.4 mg parthenolide/gram dry leaf. Both field and greenhouse crops were harvested 73 days after transplanting (DAP. The greenhouse crop was regenerated. Samples 123 DAP and 168 DAP were taken from regenerated plants. Units are mg parthenolide/g dry leaf.
Location and Treatments Time of Sampling First qrowth Second growth Field 32 DAP 73 DAP 123 DAP 168 DAP Plasticulture, Low 6.00 1.74 Inset Pots, Low 7.65 2.73 Soil Pots, High 6.99 2.03 Soil Pots, Low 6.30 1.89 Sawdust Pots, High 6.11 3.32 Significance NS *
Greenhouse Sawdust Pots, High 6.09 2.13 24.1 23.7 Sawdust Pillows, High 5.37 2.84 22.3 27.6 Soil Pots, High 6.66 4.61 24.2 27.2 Soil Pots, Low 7.71 2.08 20.8 27.7 Significance NS * NS NS
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05 68 Table 3.6. Parthenolide content (mg/plant) at harvest (73 DAP) in leaf, stem, and flower tissues, based on subsample analysis. Units of measurement are mg parthenolide/g dry weight.
Location and Treatments Parthenolide content per plant
Field Leaf Stem Flower Total Plasticulture, Low 69.5 10.5 385 460 Inset Pots, Low 43.7 3.99 187 234 Soil Pots, High 19.7 2.56 91.0 113 Soil Pots, Low 20.3 1.93 98.0 120 Sawdust Pots, High 30.9 2.32 93.0 126 Significance ** ** ** **
Greenhouse (1st harvest) Sawdust Pots, High 17.7 2.76 269 289 Sawdust Pillows, High 23.9 3.08 319 346 Soil Pots, High 49.0 3.19 241 293 Soil Pots, Low 29.7 2.83 200 232 Significance * NS * *
Greenhouse (2nd harvest) Sawdust Pots, High 261 1.75 263 Sawdust Pillows, High 631 2.85 634 Soil Pots, High 454 2.63 457 Soil Pots, Low 592 3.28 595 Significance ** NS **
* significance p<0.05 ** significance p<0.01 NS no significance p>0.05
Parthenolide concentration of feverfew leaves was assessed from samples taken three times during a 73-day period (Table 3.5). The initial sampling was done just prior to the application of treatments and parthenolide concentration was 7.4 mg/g dry leaf.
As the summer progressed, plants developed from the vegetative to the flowering stage. Parthenolide concentration remained nearly unchanged (avg. 6.8 mg/g dry leaf) over the first 36 days. However, when the crop was at the peak of flowering (73 DAP) leaf parthenolide concentration decreased significantly (avg. 2.5 mg/g dry leaf). This 69 pattern of decreased parthenolide concentration as the crop matured was observed in experiments discussed in Chapter 2. Parthenolide concentration was greatest in plants grown in the sawdust filled pots (3.3 mg/g dry leaf) and in plants grown in the soil filled pots set into the ground (2.8 mg/g dry leaf), both of which had low water and osmotic potentials. Analysis of shoot organs and the parthenolide content per plant showed that flowers produced 74% to 84% ofthe total plant parthenolide, leaves contributed 15% to
25%, and stem produced 1.4% to 2.3% (Table 3.6). High dry weight yields of flowers and leaves in response to the plasticulture treatment resulted in very high whole-plant parthenolide concentrations, even though leaves under this treatment produced the lowest parthenolide content by dry weight. Total average plant parthenolide content ranged from 450 mg per plant under the plasticulture treatment to 125 mg per plant from above-ground, soil-filled pots. Regardless of irrigation frequency or medium, total plant parthenolide in leaves, stems, and flowers was very similar from the three above- ground pot treatments.
3.3.2 Greenhouse growing medium and fertigation trial
There were four treatments in the greenhouse trial, three of which were also applied in the concurrent field trial. A fourth greenhouse treatment was the use of sawdust filled pillow bags, a treatment which had been successfully used in another experiment (Chapter 2) resulting in high leaf parthenolide concentration. Flowers accounted for the most dry matter (37% - 50%), followed by leaves (21% - 31%) and stems (21% - 27%) (Table 3.3). The sawdust treatments produced more flowers and therefore a 25% higher flower dry weight than soil treatments. Plants in the greenhouse had 10% - 30 % more flowers than field plants. 70 Feverfew is a long day plant, requiring short periods of darkness for flowering. In my experience, flower induction has occurred under days longer than approximately 12 hours. Crop regeneration in the greenhouse was under short days which held plants in a vegetative stage for the three months until the experiment was terminated. Leaf and stem yields were similar between the regenerated crop and the first growth (Table 3.3).
The yields from the first crop were not significantly affected by treatments but from the regenerated crop, the sawdust-pot treatment had lower stem and leaf yields compared with that in the other three treatments.
Dry to fresh weight ratios were measured to assess plant water content. The dry to fresh weight ratios in flowers were higher (avg. 0.34) than in the stems (avg. 0.26) and the leaves (avg. 0.16) of plants grown in the greenhouse (Table 3.3). The field grown plants had lower dry to fresh weight ratios for flowers (avg. 0.25) than for stems
(avg. 0.29). In the greenhouse crop, dry to fresh weight ratios in leaves and stems were greater in response to the low level fertigation in soil filled pots compared to the other treatments. The dry to fresh ratios in the regenerated crop was 35% lower in leaves, and 70% lower in stems compared with that of the first crops from the greenhouse and field. Unlike the flowering plants, the regenerated vegetative plants had dry to fresh weight ratios that were greater in vegetative leaves (avg. 0.105) than in stems (avg.
0.09)).
Leaf water and osmotic potentials show that plants from the low-level fertigation soil treatment resulted in the lowest mid-day and night water potentials at -2.2 MPa and
-0.95 MPa respectively (table 3.4). These results resemble the field trial where the low- fertigation soil-filled pot treatment had the lowest water potentials at mid-day and at night. The soil treatments had lower water potentials than did the sawdust treatments.
Turgor was very low for plants in the soil-filled pots during the day and the low-irrigation 71 treatment resulted in significantly lower turgor at night than did the other treatments. In the regenerated crop, water potential was higher than the first growth but lower than in the field trial. Water potentials in the regenerated crop were the lowest in response to the low water treatment. Overall, plants in the greenhouse trial had lower water potentials than those of the field trial. In the first greenhouse crop, mid-day and night leaf water potentials averaged -1.95 MPa and -0.83 MPa, respectively, whereas the field trial mid-day and night water potentials averaged -1.63 MPa and -0.53 MPa, respectively.
Leaf parthenolide concentration changed with plant development. Leaf parthenolide concentration in the first greenhouse crop showed the same relationship with development as in the field trial where leaf parthenolide concentration decreased as the plants matured (Table 3.5). After crop regeneration and growth under short days however, leaf parthenolide concentration was 10 times higher in the vegetative plants compared to the flowering plants of the previous crop. This same relationship was observed in the work described in Chapter 2 (Table 2.6) where parthenolide levels became high when plants were held in a vegetative stage in the greenhouse. There were no significant treatment effects on parthenolide concentration in the regenerated feverfew crop.
Comparing dry weights of plants from the field and greenhouse experiments, the regenerated crop had dry leaf weights within the same range as those from both the field trial and the first growth of the greenhouse trial under comparable treatments
(Table 3.3). Greenhouse flower yields were two to three times higher than field flower yields from plants from the potted treatments. The high ratio of flower to stem dry weight in the greenhouse crop did not occur in the field. Feverfew grown in plasticulture produced higher yields than it did under all other treatments in the field and 72 greenhouse. Most of the parthenolide from flowering plants was in flower tissue. For plants in the vegetative stage most of the parthenolide was in leaf tissue (Table 3.6).
Total plant yield of regenerated greenhouse plants was lower than the first greenhouse crop yield, and approximately 30% of the yield from the plasticulture treatment. Due to high leaf parthenolide in the regenerated greenhouse plants, the whole-plant parthenolide concentration was greater than for the large flowering plants of the field plasticulture treatment. Of the four greenhouse treatments used to produce the regenerated crop, the sawdust-filled pillow bag and the soil-filled pot, with low-level fertigation produced the most parthenolide per plant.
3.4. DISCUSSION
Historically (>400 years) feverfew has been harvested from outdoor sources such as gardens or from the wild. It is field-grown commercially but much of the research on its phytochemistry has been conducted on plants grown in greenhouses
(Heptinstall et al., 1998). Feverfew from both the greenhouse and field has been used in clinical trials, pharmacological studies, and for comparisons of commercial products
(Heptinstall et al., 1998; Awang et al., 1991) but the effects of the two environments on plant chemistry have not been studied until now. Clinical trials showing feverfew efficacy in migraine prophylaxis used either greenhouse or field-grown plants (Johnson et al., 1985; Murphy et al., 1988) and in some cases the origin was not mentioned
(Pattrick et al., 1989). Likewise, the developmental stage at which feverfew leaves were harvested is also not mentioned. In this study, leaf parthenolide concentration during vegetative growth under short days in the greenhouse was 2-4 times higher than had been previously reported by Awang et al. (1991), a result consistent between two greenhouse experiments (see also Chapter 2). In both the field and greenhouse 73 experiments presented here, leaf parthenolide concentration decreased as the crop matured from the vegetative to reproductive stage. Developmental stage appears to be an important determinate in STL biosynthesis.
The experiments described in this chapter were designed to examine differences between greenhouse and field production methods and explore the effect of environment on plant physiology and STL metabolism. Experiments presented in
Chapter 2 were conducted in the greenhouse and field but different growing media and at different times of the year made direct comparisons inappropriate. The field and greenhouse trials presented in this chapter were designed with matching treatments, and conducted at the same time of year to eliminate the difference of daylength, growing media, and pot size between the two locations.
Limited capacity for root growth in the confined or compacted pots, large diurnal fluctuation of root temperature of above ground pots, and differences in drainage between treatments are three factors which may have caused yield difference between field plasticulture and potted treatments. In the annual species Abutilon theophrasti and
Setaria faberii, limitations in physical space for root growth resulted in reduced vegetative growth and lower reproductive output. Abutilon theophrasti decreased allocation to reproductive tissues relative to vegetative tissues whereas Setaria faberii responded with earlier flowering and higher reproductive output in smaller soil volumes
(Mcconnaughay and Bazzaz, 1991). Thus the developmental response to root restriction depends on the species but yield usually decreases. For example, root restriction in Gossypium hirsutum caused reduced shoot biomass and leaf area
(Thomas and strain, 1991), and with Salvia splendens reduced growth was positively correlated with container volume (Vaniersel, 1997). Compacted soil can have a similar effect on root restriction and can affect drainage. Feverfew grown in plasticulture had 74 50% - 75% higher dry weight yields than potted plants in both field and greenhouse
(Table 3.3) in a season where yields were not optimal. Since above ground environments were the same among treatments in the field, the difference may have been caused by an effect on the roots.
Above ground potted treatments had higher shoot dry weight yields in the greenhouse than in the field at the first harvest (Table 3.3). Yields were nearly double in the greenhouse, with flower weights contributing most to the higher yield. Stems contributed most to dry weight of field-grown plants. One of the differences between the field and greenhouse was temperature, which fluctuated in the field between 35°C and 10°C while in the greenhouse ranged between 38°C and 18°C. In Chrysanthemum sp., warmer temperatures result in earlier flowering (Larsen, 1982). In Chamomilla recutita, fresh weight yield increases in response to temperature (Fahlen et al., 1997).
Another difference between field and greenhouse climates is air movement. Wind
alters stomatal conductance, which in turn alters C02 uptake affecting carbon assimilation and growth (Brenner et al., 1995; Cordero, 1999). Wind also increases lignification of stems which is consistent with the higher stem dry weights in field-grown compared to greenhouse-grown plants. Some of the energy required for stem lignification might otherwise be redirected to growth and flowering in the greenhouse.
Soil-filled pots, particularly under low-level fertigation, resulted in lower leaf water and osmotic potentials, lower turgor, and higher dry to fresh weight ratios. Plant water content was low in these potted soil treatments compared to sawdust treatments, which may have been caused by a low matric potential in soil compared to that in sawdust. If that was the case, a greater water potential gradient existed for plants grown in soil resulting in lower water content in plant tissues. The low-level fertigation soil treatment 75 resulted in a lower osmotic potential which may have been a response to the higher gradient.
Lower water and osmotic potentials coincided with lower dry weight yields in the potted soil treatments. Dry to fresh weight ratios revealed that the water content was higher in greenhouse plants compared to field plants (Table 3.3). Generally, plants with high water potential and low dry to fresh weight ratio had higher dry weight yields.
Other studies have found that low water potential usually results in high dry to fresh weight ratio and low yield (Flenet et al., 1996; Kimura et al., 1994). In the field, flower dry to fresh weight ratios were lower than stem dry to fresh weight ratios (Table 3.3 and
Chapter 2), but the opposite was found in greenhouse plants.
As with most plants used in traditional medicine, chemical quality is difficult or impossible to assess because there is little research on active constituents and their physiological roles. Therefore, when we know how specific chemical concentrations vary there is little information on which to asses the pharmacological quality. In an effort to establish some regulation for quality control of herbal remedies, Health and
Welfare Canada proposed that feverfew products contain a minimum of 0.2 % parthenolide (Heptinstall and Awang, 1998). This guideline is widely used in Canada to ensure species authenticity and a standard of quality. Parthenolide was probably chosen because it has been the focus of pharmacological research and found to be a vasoregulator (Barsby et al., 1991; Groenewegen and Heptinstall, 1990). The antimigraine activity of feverfew is thought to be mediated by the effect of parthenolide on serotonin (Maries et al., 1992; Biggs et al., 1982; Groenewegen et al., 1992).
Parthenolide is an early metabolite in sesquiterpene lactone (STL) biosynthesis (Fischer et al., 1979) and most other STLs in feverfew are likely derived from parthenolide or its immediate precursor. Since most STLs have pharmacological activity, parthenolide 76 might be a useful indicator of total STL biosynthesis. For example, low parthenolide in flowering plants may be a result of metabolism and hence a higher concentration of downstream STLs.
One important question to producers of medicinal plants is: What conditions result in the highest crop quality and yield? Methods of quality assessment may include visual appearance, purity of selected organs such as leaves, and chemical composition.
For feverfew, these quality standards have not been set. Currently, feverfew shoots are ground and sold compressed or encapsulated, so visual quality or the proportion of leaves, stems, and flowers is not identifiable by the consumer and thus may not be considered important. High leaf and low flower/stem composition in commercial products should be an indicator of higher quality since leaves appear to have been selected historically and they have thus become the focus for pharmacological and clinical research. The present study found that it would be easiest to produce plants with high leaf to stem ratio, no flowers, and high leaf parthenolide concentration in the greenhouse under short days. Ideally, the chemical compliment and concentration are the most important measure of quality in a medicinal plant. If parthenolide and other
STLs are the only group of active compounds in feverfew that relieve migraine, flowers rich in STLs may be an important ingredient in feverfew preparations for migraine prophylaxis. Selection for high quality feverfew crops in terms of STL concentration in leaf and flower while maintaining high yields is possible. This study shows that organ proportions and parthenolide concentration in feverfew can be manipulated by modifying the environment. This work provides information on how quality can potentially be maximized without genetic modification or significantly increasing overhead costs. 77 Many commercial preparations use the whole aerial portion of feverfew. It was interesting to look at parthenolide concentration of the whole plant based on its component organs to see the environmental effect on both yield and parthenolide concentrations. Plants in the reproductive stage had the majority of parthenolide in the flowers (74% to 84%), followed by leaves and very little in stems even though stems represented 25% to 50% of the plant dry weight. This is consistent with results of another study in which flower heads were found to contain the highest parthenolide concentration (Brown et al., 1996). Among the three comparable treatments in the greenhouse and field, shoot parthenolide content was considerably higher in greenhouse plants than in field plants. The high greenhouse flower yield contributed most to the higher total parthenolide concentration in greenhouse plants. Flower parthenolide concentration based on dry weight was consistently high among treatments. The leaf parthenolide contribution to the whole plant was also higher in the greenhouse.
For commercial preparations, the significance of harvesting at the correct developmental stage is important for quality and possibly efficacy. Commercial preparations from flowering plants may contain high proportions of flowers, which are not traditionally used for migraine therapy. A feverfew preparation rich in flowers could contain very high parthenolide levels among other STLs. Preparations containing a low proportion of leaves to flowers and stems have not been tested for side effects or efficacy, which may be important because flowers are reported to contain different terpenoids than the leaves (Banthorpe et al., 1990; Dolman et al., 1992). More research is required on the pharmacology of feverfew to identify all the antimigraine compounds before we will know which organs are the safest and most efficacious to use. 78 Regeneration of feverfew in the greenhouse resulted in a 40% lower dry weight yield than the first crop, primarily due to the lack of flowers. In my experience, feverfew is a long day plant requiring periods of darkness shorter than 12 hours to flower. The regenerated plants remained in a vegetative stage due to the short photoperiod.
However, dry weight yields of stems and leaves in the second crop were similar to the first crop. Plants grown in the sawdust pillows had the highest yields and were the only treatment to produce more leaf tissue than stem tissue. In addition, the first crop grown in the greenhouse in sawdust-filled pillow bags had lower yield than the regenerated crop. The pillow bag is long and tubular and contains the same volume of sawdust as the pot but has greater surface area. This may result in higher root temperatures and contribute to the higher shoot yield found in plants grown in the pillow bag treatment.
Regrowth from the established crown should have resulted in quicker maturation and growth due to the resources available in the roots.
The dry to fresh weight ratios of the regenerated crop reveal two interesting features of the vegetative plants. Leaf dry to fresh weight ratios were greater than stem dry to fresh weight ratios, and both were low compared to those of the first crop indicating the second growth had high water content. This may have resulted because stems of a vegetative plant are not as woody as stems from flowering plants. Stem and leaf dry to fresh weight ratios were highest in response to the low-level fertigation soil- filled pot, the same affect as was found in the first greenhouse crop. Sawdust-filled pillow bags are used in commercial greenhouses throughout the Fraser Valley, B.C. to grow a variety of vegetables. The results for yield and dry to fresh weight ratios indicate pillow bags are a viable way of producing feverfew and may have potential in greenhouse production of other herbaceous medicinal plants. Plant regeneration in the greenhouse under short days produced plants with a low proportion of stem tissue and, 79 in the pillow bag treatment, higher leaf yields. In British Columbia, sawdust is readily available as a forestry byproduct. It provides good aeration, adsorbs minerals, has a high water holding capacity while allowing excellent drainage, and is inexpensive and biodegradable. These attributes make sawdust an excellent media for growing some species of plants including feverfew.
In the greenhouse experiment presented in Chapter 2, feverfew grown in a sawdust medium produced a high parthenolide concentration compared to plants grown in the field and compared to other reported results. The high parthenolide concentration could not be attributed to any particular feature in the greenhouse since many differences existed between greenhouse and field conditions. The experiments in
Chapter 3 were designed partly to determine if sawdust medium was promoting high leaf parthenolide concentrations. It is now clear that growing feverfew in the sawdust medium did not account for the high parthenolide concentrations observed in prior experiments. Instead, developmental status of the plants is an important determinant in the accumulation of parthenolide. The regenerated greenhouse crop was held in a vegetative stage and it produced high leaf parthenolide concentrations regardless of sawdust or soil media. Experiments in this thesis consistently show plants sampled in the vegetative stage had the higher leaf parthenolide than flowering plants. In the greenhouse and field experiments conducted in the summer, parthenolide concentration was within the range of reported values (<1%) early in the season when plants were still vegetative. In the regenerated crop, the extended growth period (3 months) of the vegetative stage may cause a build-up of parthenolide to high concentrations, a result not observed in the vegetative stage of field trials. It is possible that the onset of flowering triggers a reduction in parthenolide biosynthesis or an increase in parthenolide catabolism, resulting in lower concentrations as plant development proceeds. 80 The primary contributor to whole plant parthenolide concentration in the regenerated crop was the leaves with only small amounts from the stems. The higher levels of total plant parthenolide in regenerated plants compared with flowering plants of field-grown and greenhouse-grown crops were surprising because the yield was low in the regenerated crop. The plasticulture treatment was the only one that produced similar amounts of parthenolide, primarily due to high biomass but low parthenolide concentration. One major difference between regenerated and first crops was that leaves were the primary source of parthenolide in the regenerated plants, whereas in the first greenhouse crop and field-grown crops the primary source of parthenolide was flowers. The regenerated greenhouse crop had more than 10x the leaf parthenolide concentration than the flowering crops. The organ source of parthenolide may be important in considering product efficacy because there are different chemicals found in the trichomes of each organ (see next chapter). Leaves from vegetative plants contain primarily parthenolide but leaves and flowers from flowering plants contain complex mixtures of compounds and parthenolide may not be the most abundant.
The differences in parthenolide concentrations and dry weight yields between the regenerated crop and flowering crops may have been affected by regeneration or by photoperiod. The effects of photoperiod are investigated in Chapter 4. Daylength or photoperiod has a variety of physiological effects in plants. These effects in the plant are mediated by photoreceptors which sense changes in light duration, intensity, and wavelength. Much work focused on how day length affects flowering and how light perception differs in species. As already mentioned, flowering in feverfew is induced by long days. In some way the critical photoperiod for flowering, or the flowering process itself plays a role in modifying the parthenolide concentration of leaves. Normally plants germinate in the spring when days get longer. This results in the plant remaining in a 81 vegetative stage for only a short period, developing quickly toward flowering. The changing STL composition in feverfew may be part of an adaptation strategy against herbivores and pathogens to ensure reproductive success.
Pharmacological research (Groenewegen and Heptinstall, 1990; Groenewegen et al., 1986; Heptinstall et al., 1987; Losche et al., 1988) and clinical trials (Johnson et al., 1985; Murphy et al., 1988) have been conducted with plants grown either in the greenhouse or field and often location or developmental stage is not mentioned. From the results presented in this chapter, there are significant differences in the effects of greenhouse and field conditions on parthenolide concentration, dry weight yield, and organ proportions. Reports of chemical investigations and clinical trials should thus state the developmental stage of the plant and whether it was greenhouse or field grown. 82 3.5. REFERENCES
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Heptinstall, S., and Awang, D.V.C. (1998) Feverfew: a review of its history, its biological and medicinal properties, and the status of commercial preparations of the herb. In: Phytomedicines of Europe, chemistry and biological activity pp. 158- 175, American Chemical Society, Washington. Eds. Lawson, L. and Bauer R.
Heptinstall, S., Groenewegen, W. A., Spangenberg, P., and Loesche, W. (1987) Extracts of feverfew may inhibit platelet behaviour via neutralization of sulphydryl groups. Journal of Pharmacy and Pharmacology 39:459-465.
Johnson, E.S., Kadam, N.P., Hylands, D.M., and Hylands, P.J. (1985) Efficacy of feverfew as prophylactic treatment of migraine. British Medical Journal 291:569- 573
Kimura, M., Ichimura, M., and Tomitaka, Y. (1994) Effects of watering on growth, yield, essential oil concentration and evapotranspiration of sweet basil (Ocimum basilicum L). Journal of Tropical Agriculture. 38:65-72.
Larsen, R. (1982) The effect of night temperature on flower initiation and early differentiation of Chrysanthemum morifolium. Swedish Journal of Agricultural Research. 12:95-102.
Loesche, W., Groenewegen, W.A., Krause, S., Spangenberg, P., and Heptinstall, S. (1988) Effects of an extract of feverfew (Tanacetum parthenium) on arachidonic acid metabolism in human blood platelets. Biomedica et Biochimica Acta 47:S241-243
Loesche, W., Michel, E., Heptinstall, S., Krause, S., Groenewegen, W.A., Pescarmona, G.P., and Thielmann, K. (1988) Inhibition of the behaviour of human polynuclear leukocytes by an extract of Chrysanthemum parthenium. Planta Medica 54:381- 384
Maries, R.J., Kaminski, J., Arnason, J.T., Pazos-Sanou, L., Heptinstall, S., Fischer, N.H., Crompton, C.W., Kindack, D.G., and Awang, D.V. (1992) A bioassayfor inhibition of serotonin release from bovine platelets. Journal of Natural Products 55:1044-1056
McConnaughay, K.D.M., and Bazzaz, F.A. (1991) Is physical space a soil resource. Ecology, 72:94-103 84 Murphy, J.J., Heptinstall, S., and Mitchell, J.R. (1988) Randomised double-blind placebo-controlled trial of feverfew in migraine prevention. Lancet 2:189-192
Pattrick, M., Heptinstall, S., Doherty, M. (1989) Feverfew in rheumatoid arthritis: a double blind, placebo controlled study. Annals of Rheumatoid Disease 48:547- 549
Thomas, R.B., Strain, B.R. (1991) Root restriction as a factor in photosynthetic acclimation of cotton seedlings grown in elevated carbon-dioxide. Plant Physiology 96:627-634
Vaniersel, M. (1997) Root restriction effects on growth and development of salvia (Salvia splendens). Hortscience 32:1186-1190 85
Chapter 4
Developmental effects on glandular trichomes and leaf chemistry
4.1 INTRODUCTION
Various environmental cues affect plant metabolism. Light is an important environmental stimulus that plants respond to physiologically via photoreceptors, which initiate signal transduction pathways (Salisbury, 1982). Periodic stimulation of some a photoreceptors such as phytochrome affect periodic processes like flower initiation.
Both photoperiod and the developmental changes associated with flowering may affect trichome chemistry and density (Circella et al., 1995; Hendriks et al., 1996; Voirin et al.,
1990). Trichomes develop on the aerial organs of most plants from a single protodermal cell (Szymanski et al., 2000). They are specialized structures that protrude from the epidermis and can be uni- or multi-cellular, and glandular or non- glandular. One or more types of trichomes may occur on any one species and can be useful attributes for taxonomic identification. Glandular trichomes typically produce, accumulate, and secrete chemicals that generally are terpenoids, phenolics, and resins
(Behnke, 1984). The glandular trichome in Figure 4.1 is the type present on the epidermis of a feverfew leaf. It has cells that produce compounds that are secreted into a subcuticular space for storage. The contents of the glands provide the plant with a chemical defense against insects, herbivores, and pathogens (Behnke, 1984). Both glandular and non-glandular trichomes provide another defensive function by forming physical barriers to insects. They also create a boundary layer of air that reduces water loss and dampens temperature fluctuations. Trichome traits conserved within a species include time of trichome initiation, developmental patterns, and spacing (Szymanski et 86 al., 2000). Trichomes are important for plants as the first line of defense against phytopathic organisms and therefore their involvement in chemical ecology may be critical. Sesquiterpene lactones (STLs) are produced in feverfew trichomes but little is known about biosynthesis and regulation within these structures. Biosynthesis of sesquiterpenes and monoterpenes has been investigated in the glandular trichomes of
Artemisia annua (Tellez et al., 1999), Helianthus annuus (Spring et al., 1992), and
Mentha species (McConkey et al., 2000). In these plants biosynthesis occurred in the underlying secretory cells of the subcuticular space.
f*' Glandular Trichome
Leaf epidermis
Figure 4.1. A glandular trichome derived from an epidermal cell with a subcuticular, extracellular space where secretory cells secrete non-polar compounds for storage.
Feverfew contains a mixture of more than 25 STLs in the glandular trichomes.
Parthenolide concentration is generally higher than the other STLs but feverfew chemotypes have been identified in which parthenolide was not found (Heptinstall,
Awang, 1998). Variation in STL composition of feverfew leaves is common. In
Chapters 2 and 3, I reported that parthenolide concentration in feverfew is affected by development as the plant matures from vegetative to reproductive growth. The mechanism for this variation is unknown but changes in trichome density, leaf 87 metabolism, developmental stage, or photoperiod may affect changes in parthenolide concentration. Experiments presented in this chapter explore how trichome chemistry changes with development and organ type, age and location.
4.2 MATERIALS AND METHODS
General procedures
Fresh plant material for spectroscopy was grown at UBC, Vancouver, B.C., in a controlled greenhouse environment. Growing and sampling methods for plants used to collect data reported in figure 4.2 were the same as those described in the Materials and Methods section of Chapter 2.2. The growing and sampling methods used in the other experiments, and to obtain the tissues for microscopy, are described in Chapter
3.2. The standard extraction procedures and HPLC analysis used are described in
Chapter 2.2. All solvents used for extraction and HPLC analysis were HPLC-grade
(Fisher).
Trichome isolation and extraction
Flower trichomes were removed individually from disk floret petals, composite inflorescence receptacles and leaves using a fine 27-gauge syringe needle and a dissecting microscope at 300 x magnification. Excised trichomes were extracted in methanol and filtered using a 0.45 u.m PTFE syringe filter. The filtrate was dried under nitrogen, and resuspended in 1 ml methanol for HPLC analysis.
Microscopy
A new method for observing glandular trichomes on leaf surfaces was developed for this experiment. Four to eight epidermal peels from a single leaf were mounted on a slide with water and covered with a glass cover slip. The slide preparations were placed in an oven at 40 °C for approximately 30 minutes until the epidermal peels were 88 dry before trichome densities were determined. Trichome images (Figures 4.9 and
4.10) were made usine a Zeiss Axioplan 2 imaging light microscope equipped with a
DVC digital video imaging camera. Scanning electron micrographs were obtained with a Cambridge 250T scanning electron microscope (SEM). Preparations of fresh leaves and flowers for the SEM were mounted on a SEM stud and coated with gold under vacuum in a Nanotech Semprep 2 sputter coater.
Trichome density and parthenolide content of leaves
Slide preparations of the dried epidermal peels were observed under a light microscope at 400 x magnification. The field of view was calibrated using a micrometer.
One half of each leaf was used for epidermal slide preparations and trichome density measurements while the other half was extracted and analyzed for parthenolide concentration using HPLC. Trichome density was measured on a minimum of eight and a maximum of fifteen locations on the epidermal peels of each half leaf. Leaves from vegetative and flowering plants were sampled at 10 positions from the top to the bottom of the plant. The leaf samples were harvested from 8 plants, 4 flowering and 4 vegetative.
Leaf rinsing
Six fresh feverfew leaves of about the same age from a flowering plant were cut in half. The first half was dipped three times in 300 ml water for one second each and the other was not dipped. Both halves were dried in an oven at 40 °C, seperately extracted with dichloromethane and parthenolide concentration analyzed by HPLC.
The water into which the leaf halves were dipped was extracted with dichloromethane. 89 4.3. RESULTS
4.3.1 Parthenolide variability during leaf development and flowering
Feverfew grown under a short photoperiod (< 12 hours) in the greenhouse produced vegetative shoots for approximately 60 days after transplanting (DAT). Leaf parthenolide concentrations increased significantly over this two month period (from 7.5 to 36 mg parthenolide/g dry leaf) (Figure 4.2). The plants matured to the flowering stage under longer photoperiods (>12 hours of light / day) and leaf parthenolide concentrations decreased (34 to 5.8 mg parthenolide/g dry leaf).
1 29 50 71 98 days after transplanting
Figure 4.2. Greenhouse-grown feverfew leaf parthenolide concentration during development from vegetative to reproductive growth. The shaded area is the period when internodes elongated and the plants started flowering. Error bars are standard error ofthe mean. 90 In a second experiment where feverfew was grown under photoperiods >15 hours per day, the onset of reproductive growth occurred approximately 30 DAT.
Again, the leaf parthenolide concentration decreased with the transition from vegetative to reproductive stages (from 7.4 to 3 mg parthenolide/g dry leaf respectively) (Figure
4.3). Half of the plants were harvested when flowering was at a peak at 77 DAT. New shoots sprouted from the remaining crown under a photoperiod less than 12 hours.
These new shoots remained in an extended vegetative stage. The other half of the plants were not harvested and were allowed to continue flowering. The new shoots that developed from the crown on these flowering plants remained in a vegetative stage while shoots with inflorescence continued to mature but did not produce new flowers.
Leaf samples taken 127 and 172 DAT from the regenerated vegetative plants had significantly higher parthenolide concentrations than leaves from flowering plants (26 compared to 3.6 mg parthenolide/g dry leaf respectively) (Figure 4.3). Likewise, leaves from the new vegetative shoots growing from the flowering plants had higher parthenolide concentrations than the leaves from flowering shoots of the same plant.
Parthenolide concentration varied with leaf age (Table 4.1). The growing tip sampled from vegetative plants consisted of the shoot apical meristem and small, unopened leaves. Parthenolide concentrations in the growing tips were 13 mg/g dry weight which is similar to the concentration found in flowers (11 mg/g dry weight) but were not the highest concentration in the plant. In vegetative shoots, parthenolide concentration was highest in leaves at the middle of the shoot whereas on flowering shoots the greatest concentration was in the older (basal) leaves and was less in younger leaves (top of plant). 91
Figure 4.3. Leaf parthenolide concentration of greenhouse-grown feverfew over time. On day 77 half the crop was harvested and regenerated and the other half was not harvested. Plants not harvested remained in a flowering stage. 92
Table 4.1. Parthenolide concentration in leaves of different ages from vegetative and reproductive shoots.
Developmental stage
Vegetative** Reproductive
Leaf position* (mg parthenolide / g dry leaf)
growing tip / flower head 12.6 ±2.0 10.7 ±0.67
1&2 18.6 ±0.8 0.80 ±0.16
5&6 27.6 ±3.0 2.49 ± 0.90
9&10 33.4 ± 2.2 4.27 ±0.73
13&14 24.2 ±2.9 3.64 ± 0.99
19&20 9.4 ±2.0 6.88 ± 1.2
* leaf position from top to bottom of shoot ** mg parthenolide / g dry leaf ± standard error of the mean
HPLC chromatograms of leaf surface extracts revealed that vegetative shoots had a high concentration of parthenolide and a peak area greater than the other chromatogram peaks at all leaf ages (Figure 4.4). However, shoots in the reproductive stage had lower parthenolide concentration and lower peak areas than other chromatogram peaks in both young and old leaves. Older leaves of vegetative shoots had a lower proportion of parthenolide, as did younger leaves of the reproductive shoots, relative to the other peaks in the chromatograms. 93
0.14 ~ o.io 0.12^ 0.08- 0.10-1 e a 0.06-1 0.08^ S 0.06^ 0.04-1 s 0.04-3 0.02 i 0.02^ u1 0.00—1 0.00^
0. 00 10.00
0.14. 0.10 B 0.12.
0.08_| 0.10^
0.06- 0.08^ 0.06. 0.04- 0.04- 0.02- 3 0.02. < 0.00- 1L: 0.00. tz 0, 60 10.00 0.00 io!oo 0.06 0.04 0.02 o.oo 1 0.00 i r 10.00 -p. / • x io!oo 0.00 Time (mm.) Time (min.) 94 Figure 4.4. (Previous page) HPLC chromatograms of leaf and flower extracts from feverfew shoots in the vegetative and reproductive stages. Leaves were counted from the shoot apex beginning with the first fully expanded leaf and counting toward the base of the plant. Leaves from shoots in the vegetative stage (A-D), flowers (E) and leaves from shoots in the reproductive stage (F-H). A) Growing tip and unopened leaves. B) First and second fully expanded leaves. C) Ninth and tenth leaves. D) Ninteenth and twentieth leaves. E) Mature flowers. F) First and second fully expanded leaves. G) Ninth and tenth leaves. H) Seventeenth and eighteenth leaves. The chromatograms of whole flower surface extracts showed a complex mixture of compounds including parthenolide (Figure 4.4-E). Parthenolide was the major peak in HPLC chromatograms from flower receptacle trichomes but not in the trichomes from disk flower petals (Figure 4.5). Figure 4.5. HPLC chromatograms of extracts from isolated trichomes of composite disk flowers (A) and the receptacle (B). 4.3.2 Feverfew glandular trichome development, density, and content Two types of trichomes were present on feverfew leaves. Glandular, approximately 20 x 12 urn, and ribbon-like approximately 150 to 300 urn long (Figure 4.6). The glandular trichomes were multi-cellular capitate-sessile glands and appeared 95 to have two extracellular compartments with the cuticle forming the outer surface. The developing bi-lobed head can be seen at three different stages in Figure 4.6.a and different developmental phases together with non-glandular ribbon-like trichomes are shown in Figure 4.6.b. Leaf veins had mostly non-glandular hairs with a low density of glandular trichomes. Figures 4.6 c and 4.6.d show leaves at lower magnification with both types of trichomes present and high densities of non-glandular trichomes on the midrib and veins. Methanolic extracts of the non-glandular hairs did not contain parthenolide or other compounds detected as HPLC peaks normally found in extracts of the feverfew leaf surface and glandular trichomes. Glandular trichomes were also present on disk floret petals around the opening of the floret (Figure 4.7). The floret trichomes (50u.m x 30 urn) were always glandular, were much larger than leaf trichomes (20 pm X 12 urn) and had a shape similar to leaf trichomes but with irregular bulges. Both glandular trichomes and non-glandular trichomes were abundant on the green inflorescence receptacle. They were a similar size and shape as the leaf trichomes and were the primary location of parthenolide in the flowers. 98 SEM was the best method to observe the feverfew trichomes although they could be seen with a light microscope or a dissecting microscope. However, using the latter two methods, glandular trichomes on fresh leaf epidermis are not always easily visible due to their translucent appearance (Figure 4.8a). In addition, due to the uneven surface of the fresh epidermis, not all trichomes were visible in one plane of focus. When epidermis slide preparations were allowed to dry in the oven or at room temperature, the oil-filled trichomes remained intact and could be easily distinguished in one plain of focus (Figure 4.8b). This method for counting trichomes was fast and inexpensive when compared with SEM and did not appear to alter trichome size or density. Leaf glandular trichome density was measured using a light microscope and the dried epidermal peel method. There were consistent patterns in the relationships among trichome density, parthenolide concentration, and leaf age in vegetative and reproductive shoots (Table 4.2). In both vegetative and reproductive plants, leaf parthenolide concentration increased with increasing trichome density. Leaf trichome density of vegetative shoots was high on young leaves and decreased with leaf age. Conversely the trichome density in reproductive shoots was lowest in young leaves and increased with leaf age. Plants in the reproductive stage had lower leaf trichome densities than plants in the vegetative stage. 100 Table 4.2. Trichome density and parthenolide concentration of leaves measured from the apex to the base of the stem in vegetative and reproductive plants. Vegetative Reproductive Leaf position* Trichome density** Parthenolide*** Trichome density** Parthenolide*** 1 21.5 ± 1.6 40.6 ± 1.8 0.113 ±0.007 0.598 ± 0.086 2 13.3 ±0.57 35.6 ± 1.6 0.327 ± 0.006 0.849 + 0.072 3 11.1 ±0.61 35.9 ± 0.6 0.653 ±0.01 0.910±0.12 4 12.5 ±0.77 36.2 ±2.1 2.39 ±0.07 2.69 ± 0.43 5 13.1 ±0.52 39.3 ± 1.3 1.94 ±0.08 3.12 ±0.38 6 10.1 ±0.52 26.1 ±1.7 1.66 ±0.05 3.31 ±0.47 7 6.48 + 0.21 21.7 ± 1.2 2.51 ±0.08 6.59 ±0.26 8 5.56 ±0.19 17.2 ±0.58 2.87 ±0.06 5.12 ±0.34 9 3.89 ±0.23 21.5 ±2.8 3.10±0.1 4.57 ±0.21 10 6.19 ±0.39 27.5 ±0.91 1.72 ±0.09 3.18 ±0.68 * Leaf position 1 was the youngest leaf (top of the shoot) and position 10 was the oldest. ** number of trichomes / mm2 ± standard error of the mean *** mg parthenolide / g dry leaf ± standard error of the mean 101 Dipping feverfew leaves in water resulted in a 15 % decrease in parthenolide concentration. Prior to surface extraction with water, leaf parthenolide concentration was 3.68 ± 0.17 mg parthenolide / g dry leaf and after extraction was 3.12 ± 0.21 mg parthenolide / g dry leaf. Examination of the trichomes after the water extraction showed that they were not damaged. In contrast, leaf surface extraction with dichloromethane (DCM) ruptured glandular trichomes (Figure 4.10). • -fit . ~ • •s." t-^afc " " <* »Jfc Figure 4.9. Trichomes before (a) and after (b) treatment with dichloromethane. Trichomes are circled with a dotted line. 102 4.4 DISCUSSION Photoperiod controls flowering through photoreceptors which induce the developmental shift from vegetative to reproductive growth. Altered secondary metabolism may result from the cascade of events that occur during the developmental shift. This phenomenon is exhibited in Artemisia annua, Origanum majorana and some Mentha sp. which require a long photoperiod to flower. In Artemisia annua the concentration of the STL artemisinin was greatest in the vegetative stage and decreased during flowering (Liersch et al., 1986). Lower terpene concentrations in O. majorana occured under long photoperiods, a result of the developmental transition from vegetative to reproductive growth (Circella et al., 1995). Likewise, essential oil content in Mentha sp. (M. arvensis, M. citrata, and M. cardiaca) was highest under short photoperiods and during a prolonged vegetative stage. However, in the Mentha sp. both the chemical composition and concentrations in the essential oil changed with photoperiod (Farooqi et al., 1999). Essential oils contain many compounds and their individual concentrations can vary without significantly affecting total oil yields. In another study of feverfew, the total concentration of essential oil in the leaves decreased as the plant matured. In this same plant however, the camphor concentration increased while the crysanthenyl acetate concentration decreased with maturation (Hendriks et al., 1996). The variable mixtures of chemicals in essential oils go unnoticed when only the total essential oil content in a plant is measured. Investigation of the individual compounds in essential oils allows a better understanding of how individual chemicals in the oils are accumulated, stored, and metabolized in response to environmental and developmental cues. In many feverfew varieties parthenolide is the most abundant STL and is accumulated, stored, and probably synthesized in glandular trichomes. This 103 accumulation is altered in response to long photoperiods and developmental changes. Since parthenolide is probably an early intermediate in the biosynthesis of the more complex STLs present in feverfew, the study of parthenolide metabolism and regulation may provide insights into the role of STLs and help to explain their dynamics. The high accumulations of parthenolide early in development with subsequent decreases, and the increased abundance of other STLs later in development suggests this is a point of regulation in STL metabolism. Experiments presented in this chapter show that feverfew grown under short photoperiods remained in a vegetative stage as leaf parthenolide concentration increased by a factor of ten, comprising 4% of the leaves by dry weight. Under long photoperiods the plants flowered and leaf parthenolide content decreased. The transition from vegetative to reproductive growth was also followed by altered chemical composition in the leaves. The change may have been due to parthenolide metabolism into other STLs. It is not clear whether photoperiod directly affected STL metabolism in feverfew or if the altered chemistry was a biproduct of the developmental changes. It is possible that photoperiod has a direct effect on STL metabolism. A photoperiodic effect on monoterpene enzyme activity in peppermint (Mentha x piperita) was reported by Voirin et al. (1990). Long photoperiods were required for the hydroxylase enzyme to convert menthone to menthol (Voirin et al. 1990). The concentration of specific chemicals in an essential oil may fluctuate independently if they are regulated by seperate factors. This, occurs in essential oils of both feverfew and Mentha sp. due to photoperiodic, abiotic and developmental factors (Ghosh and Chatterjee, 1993; Jeliazkova et al., 1999; Maffei et al., 1986; Hendriks et al., 1996). Many environmental factors may change throughout plant development. Secondary metabolism will almost certainly be influenced by these changes. 104 Experiments in this thesis showed that nitrogen, irrigation, and photoperiod influenced feverfew leaf chemistry. Regardless of these treatments there were consistent parthenolide concentration gradients within the plant, such as parthenolide concentration that increased with leaf age. This suggests that there is a regulated pattern of parthenolide biosynthesis which can be modified by environmental factors and development. In vegetative plants the highest concentrations were in leaves at the middle and toward the bottom of plants while in the reproductive stage the highest parthenolide concentration was in leaves at the base of plants which also contained significant concentrations of other compounds. The lower leaves of flowering shoots originated from vegetative shoots which may explain the higher parthenolide concentrations. Similar developmental patterns were found in Origanum majorana where terpene concentrations were higher in older leaves (Circella et al., 1995). In Mentha species (M. spicata, M. longifolia, M. rubra), menthol and menthone were higher in the third to fifth leaf pairs compared to younger leaf pairs (Fahlen et al., 1997). Enzyme activities, protein levels, and the rate of monoterpene biosynthesis in peppermint (Mentha x piperita) glandular trichomes were determined early in leaves 12 to 20 days old which indicated monoterpene biosynthesis was developmentally regulated at the level of gene expression during early leaf development (McConkey et al., 2000). Terpene biosynthetic enzyme concentrations and activities in feverfew may be determined early and then modified with development from vegetative to reproductive growth. The mechanism or level of control of environmental modulators, whether the target is genetic or enzymatic, is largely unknown for sesquiterpene biosynthesis. Glandular trichomes are very small and the use of a microscope is necessary for measurements. Scanning electron microscopy (SEM) was the best method for 105 visualization but is time consuming and expensive (Duke and Paul, 1993). Another method involves acid staining to enhance the visibility of trichomes under the light microscope (Kelsey and Shafizadeh, 1980). I developed a quick method which does not require staining or SEM. Using epidermal peals mounted on a slide with water and then dried, glandular trichomes remained intact and were highly visible in one plain of view under a light microscope. A comparison of observations made using the SEM and dried epidermis preparations showed trichome density and size were not affected by the latter procedure. Using this new technique I was able to measure the size and densities of trichomes. The relationship between leaf parthenolide concentration, leaf age, and development may be explained by trichome densities (Brun et al., 1991). On a single mature leaf, trichomes are a uniform size and shape and contain similar amounts of parthenolide. In mature leaves there were high parthenolide concentrations when glandular trichome densities were high. Both parthenolide concentrations and trichome densities on leaves of vegetative plants were significantly higher than on leaves of flowering plants. This pattern was also found in leaves from Mentha verdae which had higher trichome densities before flowering and the lowest during flowering. The Mentha trichome densities were positively correlated with essential oil concentration (Maffei et al., 1986). In the absence of trichomes in glandless Artemisia annua mutants, STLs were not synthesized (Tellez ei* al., 1999). Certainly trichome density affects terpenoid concentration in a variety of plants but the question remains whether trichome density is a mechanism of control for leaf terpenoid concentrations. Trichome patterns are determined in the initial stages of leaf emergence, therefore the number of trichomes per leaf is determined at leaf emergence (Szymanski et al., 2000). As the leaf grows and expands both the size and the distance between trichomes increases. Trichome 106 density decreased during leaf expansion but trichome size increased, resulting in a maintenance or increase of essential oils per unit area. Any environmental effect on trichome density probably occurs before or during leaf emergence. Observations from trichome measurements presented in this chapter indicate that mature leaves did not have new trichomes developing. During feverfew's vegetative stage, older leaves had higher parthenolide concentrations than predicted from their trichome densities. This may be explained if trichomes had burst on older leaves exuding their contents onto the leaf surface resulting in lower trichome densities. In this case the trichome contents may have remained on the leaf surface. In some plants, glandular trichomes develop a weak area or pore at the apex to facilitate their rupture (Afolayan and Meyer, 1995; Ascensao and Pais, 1987). This suggests an inherent mechanism to distribute trichome contents on the leaf surface. Although there were no obvious weak areas or pores on feverfew's glandular trichomes, there may be a size limit that when exceeded results in rupture. If feverfew trichomes naturally rupture on older leaves and exude their contents to the leaf surface, water may be sufficient to rinse the exposed parthenolide away. In an experiment where feverfew leaves were rinsed with water, trichomes were not ruptured by the water but there was a 15% decrease in leaf parthenolide concentration after rinsing. The ecological significance for the release of trichome contents onto the leaf surface may be in deterring feeding insects or killing pathogens. Glandular trichomes on disk florets of the inflorescence were significantly larger than leaf trichomes. If trichomes were to rupture at a critical size, the flower trichomes must have different size characteristics. Individual trichomes sampled from petals of disk florets contained low parthenolide concentrations. This seemed to contradict results from whole flower extracts that had high parthenolide concentrations. Most of 107 the parthenolide in flowers was in the dense arrangements of glandular trichomes on the receptacle and only small amounts were present in the disk or ray florets. The disk floret petal trichomes were a different size, had an irregular shape with a lumpy appearance and their chemistry was different in comparison to leaf glandular trichomes. These three characteristics suggest that the disk floret petal trichome and leaf trichomes are different types and develop differently. The density and chemistry of glandular trichomes is variable and the environment affects this variability. It is believed that plants produce glandular trichomes and their chemical arsenals as defensive mechanisms. However, environmental factors affecting trichomes may indicate that trichomes have functions other than defense. For example, Lycopersicon hirsutum had higher trichome densities when grown under short photoperiods (Weston et al., 1989). In this case, there may be a physiological function for trichomes under short days such as cold tolerance in preparation for autumn and winter weather (Behnke, 1984). Only leaves, which develop during or after the environmental effect would exhibit the change in trichome density. With the isolation of new glandless mutants it may be possible to study the role of trichomes in relation to the plant's environment. 108 4.5 REFERENCES Afolayan, A.J., and Meyer, J.J.M. (1995) Morphology and ultrastructure of secreting and nonsecreting foliar trichomes of Helichrysum aureonitens (Asteraceae). 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(1997) Effects of light-temperature regimes on plant growth and essential oil yield of selected aromatic plants. Journal of the Science of Food and Agriculture 73:111-119 Farooqi, A.H.A., Samgwan, N.S., and Sangwan, R.S. (1999) Effect of different photoperiodic regimes on growth, flowering and essential oil in Mentha species. Plant Growth Regulation 29:181-187 Ghosh, M.L., and Chatterjee, S.K. (1993) Physiological and biochemical indexing of synthesis of essential oil in Mentha spp. grown in India. Acta Horticulturae. 331:351-356 Hendriks, H., Bos, R., and Woerdenbag, H. J. (1996) The essential oil of Tanacetum parthenium (I.) Schultz-bip. Flavour and Fragrance Journal 11:367-371. Heptinstall, S., and Awang, D.V.C. (1998) Feverfew: a review of its history, its biological and medicinal properties, and the status of commercial preparations of the herb. In: Phytomedicines of Europe, chemistry and biological activity, pp. 158-175 American Chemical Society, Washington. Eds. Lawson, L. and Bauer R. Jeliazkova, E.A., Zheljazkov, V.D., Craker, L.E., Yankov, B., and Georgieva, T. (1999) NPK fertilizer and yields of peppermint, Mentha x piperita. Acta Horticulturae 502:231-236 Kelsey, R.G., and Shafizadeh, F. (1980) Glandular trichomes and sesquiterpene lactones of Artemisia nova (Asteraceae). Biochemical Systematics and Ecology 8:371-377 109 Liersch, R., Soicke, H., Stehr, C, and Tullner, H.U. (1986) Formation of artemisinin in Artemisia annua during one vegetation period. Planta Medica 5:387-390 Maffei, M., Gallino, M., andSacco, T. (1986) Glandular trichomes and essential oils of developing leaves in Mentha viridis lavanduliodora. Planta Medica 3:187-193 McConkey, M.E., Gershenzon, J., and Croteau, R.B. (2000) Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiology 122:215-223 Salisbury, F.B. (1982) Photoperiodism. Horticultural Reviews 4:66-105 Spring, O., Rodon, U., and Macias, F.A. (1992) Sesquiterpenes from noncapitate glandular trichomes of Helianthus annuus. Phytochemistry 31:1541-1544 Szymanski, D.B., Lloyd, A.M., and Marks, M.D. (2000) Progress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis. Trends in Plant Sciences 5:214-219 Tellez, M.R., Canel, C, Rimando, A.M., and Duke, S.O. (1999) Differential accumulation of isoprenoids in glanded and glandless Artemisia annua L. Phytochemistry 52:1035-1040 Voirin, B., Brun, N., and Bayet, C. (1990) Effects of daylength on the monoterpene composition of leaves of Mentha piperita. Phytochemistry 29:749-755 Weston, P.A., Johnson, D.A., Burton, H.T., and Snyder, J.C. (1989) Trichome secretion composition, trichome densities, and spider mite resistance of ten accessions of Lycopersicon hirsutum. Journal of the American Society for Horticultural Science 114:492-498 110 Chapter 5 Biosvnthetic studies using 14C and 13C incorporation into parthenolide 5.1. Introduction Terpenoids are present in all living organisms. There are over 25,000 terpenoids reported in the plant kingdom (Eisenreich et al., 2001). These compounds are diverse in function. They include carotenoids which serve as light-harvesting and light protecting pigments, sterols which modulate membrane properties, the phytol side chain of the chlorophylls and a profusion of insect attractants and repellents. Important pharmaceuticals and nutraceuticals are synthesized from terpenoids including taxol the cytostatic diterpene from Taxus brevifolia, lycopene and lutein which are oncoprotective agents, and the antimalarial sesquiterpene artemisinin from Artemisia annua. In the 1950s investigations into isoprenoid and sterol biosynthesis resulted in the elucidation of the mevalonic acid (MVA) pathway. Subsequently the biogenetic isoprene rule was formulated which states: 1) Terpenoids are composed of repeating five carbon isoprene units united in a head-to-tail fashion, 2) Isoprene units are derived from acetate in the activated forms of acetyl-CoA and acetoacetyl-CoA, and 3) Mevalonic acid is an essential intermediate. It was thirty years before the discovery of an alternative pathway to isoprenoid biosynthesis in the early 1990s. The methylerythritol phosphate (MEP) pathway utilizes glyceraldehyde-3-phosphate and pyruvate to form the basic isoprene unit. The MVA and MEP pathways converge at the formation ofthe isoprene unit, isopentenyl pyrophosphate (IPP) (figure 5.1). The MEP pathway is also referred to as the alternative pathway, the Rohmer pathway, or acronyms derived from the intermediate compound deoxyxylulose-5-phosphate such as DOX-P, DOX, and DXP. Ill MEP pathway Mevalonate pathway o SCoA SCoA CH3 OH DXP HOOC SCoA • NADPH/H + 2 NADPH/2H+ HMGR 1^*- NADP+ 2 NADP CoASH H3C OH 3 OH HO HOOC. >^ / OH OP 3R-mevalonic acid (MVA) OH MEP 2 ATP -ATP 2 ADP < -ADP C H H3C OPP . 3 OH HOOC OPP IPP CH. 5-PPMVA ,OPP H,C OPP DMAPP IPP CH, Figure 5.1. The methylerythritol phosphate pathway and the mevalonate pathway to terpenoid biosynthesis. HMGS = hydroxymethylglutaryl synthase, HMGR = hydroxymethylglutaryl reductase, 5-PPMVA = 5-pyrophosphomevalonate, IPP = isopentenylpyrophosphate, DMAPP = dimethylallylpyrophosphate, G-3-P = glyceraldehydes-3-phosphate, DXP = 1-deoxyxylulose-5-P, MEP = 2-C-methyl-D- erythritol-4-P. 112 In plants, the MVA pathway occurs in the cytoplasm and is responsible for synthesis of triterpenes including sterols, and the prenyl side chain of ubiquinones (Eisenreich et al., 2001). The MEP pathway is localized in plastids (Araki et al., 2000). The plastidic terpenoids, which include isoprene, monoterpenes, diterpenes, carotenoids, the prenyl side chains of plastoquinones and chlorophylls are all synthesized through the MEP pathway (Rohmer, 1999). Isopentenyl pyrophosphate (IPP), farnesyl pyrophosphate (FPP), and geranyl pyrophosphate (GPP) are present, and may be synthesized, in both compartments (Rohmer, 1999). Both the compartmentation and utilization of different substrates by the two pathways explains the low incorporation of 14C labeled acetate or mevalonate via the mevalonate pathway into plastidic terpenoids such as phytol and plastoquinone. The low levels of incorporation however, indicate participation of MVA derived terpenoids in the plastids and that transport or diffusion must occur between the two compartments. The MVA pathway is widespread but not ubiquitous. It is found in the Archaea and certain bacteria, yeasts, fungi, algae, higher plants, some protozoa and animals. Despite the elusive history of it's discovery and description, the MEP pathway is widely distributed in nature. Many bacteria (including pathogens) (Giner and Jaun, 1998), green algae, higher plants, and protozoa (including the malaria parasite Plasmodium falciparum) use the MEP pathway. Bacteria appear to use either the MVA or the MEP pathways but not both. Mosses and liverworts, marine diatoms and higher plants use both pathways. Some parasitic micro-organisms appear to lack isoprenoid biosynthetic capabilities and may use terpenoids from their host (Eisenreich et al., 2001). Over 5000 sesquiterpene lactones (STLs) have been identified and a large number of these are biologically active against bacteria, fungi, and/or viruses. Many of the STLs interact with protein or DNA to elicit physiological responses. In higher plants 113 it is evident that monoterpenes and diterpenes are synthesized primarily by the MEP pathway but few studies have been conducted on the role of the MEP pathway in sesquiterpene biosynthesis. Recent reports show both the MVA and MEP pathways contribute to the biosynthesis of sesquiterpenes (Adam and Zapp, 1998). The purpose of experiments described in this chapter was to elucidate the biosynthetic origins of the sesquiterpene lactone parthenolide. This was accomplished by feeding feverfew shoots 14C and 13C enriched substrates (glucose, acetate, pyruvate and mevalonate) and quantifying 14C incorporation and the position of 13C enrichment in parthenolide. 5.2. Materials and Methods The feeding experiments were carried out in a modified fume-hood with full spectrum vita lights programmed for 16 hours light / 8 hours dark. The average temperature was 24 °C with the lights on and 20 °C with the lights off. Solutions were made with distilled water filtered through a milli-Q (Millipore) filtration system. All chemicals were purchase from Sigma unless otherwise stated. In both the 13C and 14C experiments, labeled feeding solutions were administered until the entire volume was taken up by the shoots after which water or glucose solution was supplied to allow the plants a longer period for label incorporation. The 14C-glucose experiments were conducted prior to the 13C experiments. The 14C-pyruvate, 14C-acetate, and 14C-mevalonolactone experiments were conducted simultaneously at the same location and under the same conditions. All shoots were harvested fresh and recut underwater before treatments were applied. 114 5.2.1. 14C feeding experiments UL-14C-glucose feeding 11.31 (j,Ci UL-14C-[D]-glucose (purchased from Cambridge Isotope Laboratories, CIL) was added to 100 ml of cold 1% D-glucose for a final activity of 0.1131 u.Ci / ml. Twelve young shoots with 4 to 7 leaves each were placed in four vials, each containing 25 ml UL-14C-D-glucose solution. The shoots were grown in an environmentally controlled fume hood under 16 hours light (24 °C) / 8 hours dark (20 °C) for seven days. The UL-14C-D-glucose solution was taken up in three days and replenished with cold 1% D-glucose for an additional four days. The control treatment was grown under the same conditions with the exception of cold 1% D-glucose instead of UL-14C-D-glucose as the substrate. 1-14C-glucose feeding One hundred and sixty ml of cold 1% D-glucose was mixed with 40 u.Ci of 1-14C- D-glucose (purchased from CIL) for a final activity of 0.25 u,Ci/ml. Twelve young shoots with 5 to 8 leaves each were placed in four vials containing the radio-labeled glucose solution. The shoots were incubated in a fume hood under 16 hours of light (24 °C) / 8 hours dark (20 °C) for nine days. The 1-14C-D-glucose solution was taken up in six days and then replenished with cold 1% D-glucose for three days. A control treatment was conducted at the same time using cold 1% D-glucose as a substrate. All other conditions were as described above. 1-14C-pyruvate feeding Cold 1 mM sodium pyruvate solution (40 ml) was prepared and mixed with 9.33 u.Ci sodium-1-14C-pyruvate for an activity of 0.23 u.Ci/ml. Two vials were filled with 20 ml of the feeding solution and three young shoots were placed in each vial. The shoots 115 were incubated in a fume hood under 16 hours of light (24 °C) / 8 hours dark (20 °C) for seven days. The labeled solution was used up within four days and the containers were refilled with distilled water for the remaining three days. The same number of shoots and conditions were used for the control treatment with the exception of 12C-pyruvate instead of 14C-pyruvate. 2-14C-acetate feeding A 1 mM Na+-acetate solution (40 ml) was prepared and mixed with 10.2 u.Ci Na+2-14C-acetate for a specific activity of 0.25 u.Ci/ml. Two vials were filled with 20ml of the feeding solution. Each vial contained three young shoots. The shoots were incubated in a fume hood under 16 hours of light (24 °C) / 8 hours dark (20 °C) for seven days. The labeled solution was taken up within four days and the containers were refilled with distilled water for the remaining four days. The same number of shoots and conditions were used for the control treatment except 12C-acetate was used instead of 14C-acetate. 2-14C-mevalonolactone feeding A 1mM mevalonolactone solution (40 ml) was prepared and mixed with 10.5 uCi 2-14C-mevalonolactone for a specific activity of 0.26 u.Ci/ml. Two vials were filled with 20 ml ofthe feeding solution. Each vial containing three young shoots was incubated in a fume hood under 16 hours of light (24 °C) / 8 hours dark (20 °C) for seven days. The labeled solution was taken up within four days and the containers refilled with distilled water for the remaining four days. The same number of shoots and conditions were used for the control treatment except 12C-mevalonolactone was used instead of 14C- mevalonolactone. 116 5.2.2. 13C feeding experiments 13C-Enriched substrates (pyruvate, acetate, mevalonolactone, and D-glucose) were fed to feverfew cuttings (all 13C experiments were conducted at the same time under the same conditions) grown in a modified fume hood equipped with full spectrum vita-lights. The cuttings were inserted in vials filled with the enriched solution for a total of seven days under 16 hours of light at 24 °C and 8 hours dark at 20 °C. The control treatments of unlabeled substrate was applied for three days followed by distilled water for four days. The control treatment was applied to five vials with three shoots in each. Vials from all treatments were arranged randomly together under one set of vita-lights. 1-13 C-glucose feeding One hundred mg of 99% enriched 1-13C-D-glucose was added to 300 ml of a 1% D-glucose solution. The solution was divided into 10 vials, each containing three young shoots with 4 to 7 leaves. The solution was taken up by the plants in three days and replenished with 1 % glucose for another four days. 2-13 C-pyruvate, 2-13C-acetate and 2-13C-mevalonolactone feeding Three hundred ml each of 3 mM 2-13C-pyruvate, 3mM 2-13C-acetate and 2.5 mM 2-13C-mevalonolactone solutions (99% enriched) were each divided into ten vials (30 vials total). Three shoots with 4-7 leaves each were put into each of the vials. The 13C- enriched solutions were taken up in three days. Vials were replenished with water which was taken up for an additional four days. 117 5.2.3. Extraction methods, parthenolide isolation, and NMR analysis After feeding radio-labeled substrates to feverfew shoots, the surface of the fresh shoots were extracted twice using a rapid dichloromethane rinse. The extract was vacuum filtered with Whatman No. 1 filter paper and dried under vacuum at 30 °C using a rotary evaporator. The dry extract was weighed and resuspended in 1 ml methanol. An aliquot of 100 pi was put into a 7 ml scintillation vial with 5 ml of scintillation fluid to measure 14C activity using a scintillation counter. Activity in the extract was calculated from this sample. 14C-Parthenolide isolation Aluminum backed silica TLC plates with UV254 fluorescence indicator were used for the initial UL-14C-glucose experiment. For the rest of the 14C experiments, glass backed 0.25 mm preparative silica TLC plates with UV254 fluorescence indicator were used. The extracts were loaded in bands at the origin 1cm from the base and placed in the 80% dichloromethane : 20% acetone solvent system in a glass TLC chamber. This system resolved many bands including parthenolide (Rf=0.81). Bands were visualized under UV254 and with vanillin-sulfuric acid spray reagent. Each band on the TLC plate was scraped off and transferred to a 7 ml plastic scintillation vial with 5ml scintillation fluid and activity recorded with the scintillation counter. Purity of the resolved parthenolide band was confirmed with HPLC using methods described in Chapter 2. 13C-Parthenolide isolation Extracts were loaded in bands on 0.25mm silica TLC plates with UV254 indicator. The plates were developed with 80% DCM : 20% acetone. Bands were visualized under UV254 and with vanillin-sulfuric acid spray reagent. Parthenolide (Rf = 0.81) quenched the UV254 indicator but was not fluorescent. Developing the plate with vanillin sulfuric acid and heating resulted in a dark blue color for the parthenolide band. The 118 parthenolide band was scraped and the parthenolide eluted from silica with methanol. The extracted band was vacuum filtered with Whatman No. 1 filter paper and evaporated to dryness using a rotovap under vacuum at 30 °C. The dried extract was resuspended, transferred in 4 ml methanol to a vial, and concentrated under nitrogen at 40 °C. Parthenolide was purified further using preparative HPLC with a Waters NovaPak 2.5 X 10 cm (5u.m) Ci8 radial compression column. The mobile phase was acetonitrile:water (70:30) at 25 ml/min with detection at 210 nm. Injection volumes were 200 uJ and elution of the parthenolide peak was at 6 minutes. The fraction was dried under vacuum leaving a white residue (parthenolide). Purity of the isolated parthenolide was checked with analytical HPLC. NMR analysis The 13C-NMR spectra of parthenolide were recorded in deuterated chloroform (CDCI3) on a Bruker AMX-500 spectrometer. The 1H-NMR spectrum of parthenolide was recorded in CDCI3 on a Bruker AC-200E spectrometer. The residual chloroform CHCI3 (5 7.24 ppm) signal was used as reference. Enriched and non-enriched parthenolide samples were run sequentially under the same conditions. Enrichments were obtained through subtraction of the peak integrations and peak intensities of non- enriched samples from enriched samples. 119 5.3. Results 5.3.1. 14C labeling of parthenolide The 14C feeding experiments were carried out to determine feeding parameters and incorporation rates for use with the subsequent 13C experiments. A range of shoot sizes were tested with water or glucose. Shoots performed best when they were very young with 4 to 8 leaves and a glucose solution was better than water for shoot survival. 14C was incorporated into parthenolide after feeding the five labeled substrates to feverfew shoots. The 14C incorporation rates (Table 5.1), reported as relative specific activity (RSA), were 5.3 times greater when UL-14C-glucose was fed to shoots compared with 1-14C-glucose fed shoots. The highest RSAs after feeding the single labeled substrates were in shoots fed with either 1-14C-pyruvate or 1-14C-glucose. The substrates predicted to move solely through the mevalonic acid pathway, 2-14C- mevalonolactone and 2-14C-acetate, had the lowest RSAs. Table 5.1. Incorporation of 14C labeled substrates into parthenolide. Activity Activity in Specific taken up by surface activity Radiolabeled substrate the shoots extract (nCi) (nCi) InCii UL-14C-glucose 3430 188 0.624 0.0182 1-14C-glucose 37560 4860 1.28 0.00341 1- 14C-pyruvate 9325 1122 0.387 0.00415 2- 14C-acetate 10153 726 0.189 0.00186 2-14C-mevalonolactone 9455 689 0.276 0.00292 120 5.3.2. 13C enriched parthenolide The 1H-NMR spectrum of parthenolide (Figure 5.2 a) shows a characteristic chemical shift of two doublets around 5.7 and 6.3 ppm representing the two protons of the exocyclic methylene on the lactone ring (protons attached to carbon 13 of parthenolide in Figure 5.3). The 400 MHz 1H-NMR (Figure 5.2 b) shows one of these doublets with the corresponding satellite peaks. The satellite peaks are a result of 1H- 13C spin-spin coupling and can be used to quantify the 13C incorporation at that position. In Figure 5.2 b the large doublet (doublet 1) is the 1H attached to 13-12C-parthenolide and the small doublet (Figure 5.2 b, doublet 2) is the 1H attached to 13-13C- parthenolide. The ratio of integrals for the parent and satellite peaks gave the 13C abundance for carbon position 13 in parthenolide. After feeding the substrates, 13C abundance in carbon 13 of parthenolide calculated from 1H-NMR was 4% from 1-13C- glucose, 2% from 2-13C-acetate, 1.3% from 2-13C-pyruvate, and 1.2% from 2-13C- mevalonolactone fed shoots. 121 Figure 5.2. 1H-NMR spectra of parthenolide. A) Full spectrum at 90 mHz and an expanded region B) at 5.4 to 5.7 ppm of a 400 mhz 1H-NMR spectrum showing the parent (1) and satellite peaks (2) of the attached proton of carbon 13. Figure 5.3. Carbon numbering of parthenolide and the predicted conformation of the three isoprene units. 122 m R S f? iS * :; s s 5 7 9 82 13 6 | a) 15/14 10 11 12 cn m n OJ mm ni ni IO ID ID r- —< IO O ^ MB R ru in iQ as R N s ID to ^ n n ni « - b) C-9 C-3 (4') (4) Figure 5.4. 13C NMR of parthenolide isolated from feverfew shoots a) fed with 2-12C- mevalonolactone and b) fed with 2-13C-mevalonolactone. 13C enrichment in carbon 9 and carbon 3 is greater in spectrum b than a. 123 The natural abundance of 13C is 1.1%. Patterns of 13C enrichment in excess of the natural abundance were found in parthenolide isolated from shoots from each of the four 13C feeding experiments (Table 5.2). 13C Abundance calculated from 1H-NMR (Figure 5.2) was consistent with the 13C incorporations calculated from 13C-NMR (Table 5.2 and Figure 5.5). Carbon 13 (see Fig. 5.3 for numbering scheme) of parthenolide was enriched after feeding the shoots 1-13C-glucose but not significantly enriched after feeding 2-13C-mevalonolactone or 2-13C-pyruvate. Parthenolide isolated from shoots after feeding 2-13C-mevalonolactone was enriched at Carbon 3 and Carbon 9, consistent with C-4 and C-4' of the isoprene subunits (Figure 5.3) whereas C-4" of the third isoprene subunit (equivalent to C-12) was not enriched. Parthenolide isolated from plants fed with 2-13C-pyruvate was enriched in carbons 4, 10, and 11 corresponding to C-3, C-3', and C-3" of the isoprene subunits. Carbons 6, 2, and 8 had intermediate enrichments which corresponded to C-1, C-1', and C-1" of the isoprene subunits. Many carbons in parthenolide were enriched after the 1-13C-glucose feeding experiment with the exception of carbons 4, 10, 11, and 12. Three of these non-enriched carbons (4, 10, and 11) corresponded to C-3, C-3', and C-3" of the isoprene subunits whereas the fourth non-enriched carbon corresponded to C-4" of the isoprene subunit. CD •4—' CD O 00CD00C0T-<0CDl0Or^00l0' C CD D) O CM CO Tt lO CN CO XT LO CM oo ^ in c —C Q. > 0 0 o '5 J3 W FT E ^ croc c £ "o CO lO 00 LO CN o Q- E CO w ^ .E 0 O to •— B * 125 Pathways Actual Enrichment Predicted Mevalonate Predicted MEP Figure 5.5. 13C enrichment patterns in parthenolide after feeding enriched substrates. Observed enrichments from table 5.2 are in the first column and predicted enrichment patterns are in the next two columns. Filled circles are carbons with strong enrichment and open squares indicate carbons with intermediate enrichment. 126 5.4. Discussion The biosynthetic origins of parthenolide were examined using 13C isotopic enrichment. The 13C feeding experiments showed parthenolide biosynthesis in feverfew utilized isoprene units from both the MVA and MEP pathways. Eichinger et al. (1999) and Eisenreich et al. (1996) conducted feeding studies using 13C-enriched substrates including 1-13C-glucose and found different labeling patterns in IPP dependent on the biosynthetic route utilized. In these studies the positions of enriched carbons in the isoprene units after feeding 1-13C-glucose were in carbons 1 and 5 when the MEP pathway was utilized and carbons 2, 4 and 5 when the MVA pathway was used. After feeding 1-13C-glucose to feverfew shoots, two isoprene units in parthenolide were enriched in C-1, C-2, C-4 and C-5 consistent with contributions from both the MVA and MEP pathways (numbering scheme in Figure 5.3). The third isoprene unit was enriched in C-1", C-2" and C-5" but not enriched in C-4". This pattern is difficult to explain but is more consistent with isoprene from the MEP pathway due to the strong enrichments in C-1" and C-5". The enrichment in C-2" may have resulted from metabolic turnover of the 1-13C-glucose. Alternatively, the enrichment in C-4" may have been masked by the three oxygen atoms in its vicinity which shifts the 13C peak downfield. In combination with the quaternary carbon, this caused reduced signal intensity. As expected, the C-3 positions of the isoprene units were not labeled. The enrichment in C-3, C-3' and C-3" is close to natural abundance and is in agreement with both biogenetic schemes (Figures 5.6 and 5.7). Acetate is an immediate precursor of the MVA pathway. Three molecules of acetyl-CoA condense to form IPP via mevalonic acid. There is no direct pathway for acetate to form G-3-P or pyruvate, and therefore it was not expected to move through 127 a) H OH a-D-glucose D-fructose-1,6-bisphosphate b) :0 Glyceraldehyde-3-phosphate -OH < r -OP =0 »—OP HO- -H H— -OH V-OH H- -OH 0=^—OP DHAP 1,3-bisphosphoglycerate ^OP F-1,6-BiP -OH H3Q ) OH O^OH O-^OH 0=^ OP 3-13C-Pyruvate Phosphoenolpyruvate 3-phosphoglycerate Figure 5.6. Glucose catabolism. a) The conversion of D-glucose to D-fructose-1,6- bisphosphate (F-1,6-BiP) and b) the production of glyceraldehyde-3-phosphate (G-3-P) and pyruvate from (F-1,6-BiP). The black circle shows the carbon position as glucose is catabolized. 128 Methvlerythritol phosphate pathway Mevalonate pathway C02 o Pyruvate o OH CoASH 2-13C-acetyl-SCoA G-3-P CoASH PO •i O o SCoA OH 2-13C-acetyl-SCoA-^ 0 PO^Y^V ^CoASH DXP OH CH3 OH O HOOC SCoA HO HMG-SCoA 3 OH HOOC OPP PPO. [>C02 IPP I CH9 CH2 OPP IPP FPP CH3 | OPP Observed 13C incorporation Predicted MEP pathway Predicted Mevalonate Pathway Figure 5.7. 1-13C-D-glucose feeding experiment. Observed and predicted patterns of 13C enrichment. 129 the MEP pathway. Feeding feverfew shoots with 2-13C-acetate resulted in enrichment in positions C-2, C-4, C-5, C-2', C-4', C-5', C-2", C-4", and C-5" in parthenolide as predicted from the MVA pathway (Figure 5.8). Interestingly the third isoprene equivalent which forms the lactone ring (C-2", C4" and C-5") had lower levels of enrichment than the other two isoprenes. This suggests another source or pool of IPP is utilized for this isoprene unit, possibly derived from the MEP pathway. This is consistent with the 2-13C-mevalonolactone feeding experiment where there was enrichment at C-4 and C-4' in the first two isoprene units but insignificant enrichment in C-4" of the third isoprene comprising the lactone ring of parthenolide (Figure 5.9). The 2-13C-pyruvate feeding experiment also indicated contributions from two separate IPP pools (Figure 5.10). Isotopic enrichment of six carbons (C-1 and C-3 of each isoprene unit) was predicted if pyruvate was catabolized to acetate and then condensed to form IPP through the MVA pathway whereas only three carbons (C-3 in each isoprene unit) were predicted to be enriched if pyruvate went through the MEP pathway. Enrichment was greater in C-3, C-3', and C-3" while lower levels of enrichment were seen in C-1, C-1' and C-1". The higher enrichments at C-3 relative to C-1 positions ofthe isoprene units were consistent with contributions of IPP from the MEP pathway which resulted in higher enrichments at the C-3 positions. Due to the overlap in carbon enrichment from the two pathways, the results from 2-13C-pyruvate feeding can only confirm a contribution of IPP from the MEP pathway when there is no IPP contribution from the MVA pathway. A problem in identifying the contributions to the MEP pathway from pyruvate or G-3-P enriched in carbons 2 or 3 arises because carbon 1 of pyruvate is lost as CO2 in the MEP pathway o Predicted Mevalonate Pathway Figure 5.8. 2-13C-acetate feeding experiment. Observed and predicted patterns of 13C enrichment. Closed circles are carbons with strong enrichment and open boxes are carbons with moderate enrichment 131 Methylerythritol phosphate pathway Mevalonate pathway Predicted Mevalonate Pathw Figure 5.9. 2-13C-mevalonolactone feeding experiment. Observed and predicted patterns of 13C enrichment. Closed circles are carbons with strong enrichment. Methylerythritol phosphate pathway Mevalonate pathway Predicted MEP pathway Predicted Mevalonate Pathway Figure 5.10. 2-13C-pyruvate feeding experiment. Observed and predicted patterns of 13C enrichment. Closed circles are carbons with strong enrichment and open boxes are carbons with moderate enrichment. 133 with it's conversion to acetate. Only the enrichment of carbon 1 of G-3-P would be specific for the MEP pathway without seeing contributions from the MVA pathway. 13C Enriched deoxyxylulose is an alternative substrate to study the MEP pathway but is not commercially available. Another approach is to use double-labeled substrate which gives the additional advantage of studying 13C-13C interactions. The two independent terpenoid biosynthetic pathways are physically separated by compartmentation, but there is interaction between the two pathways. There is evidence that IPP, GPP, and FPP are exchanged between the compartments (Eisenreich et al., 2001). The superposition of two different labeling patterns in terpenoid compounds and the chimera nature of some terpenoids suggest a transport mechanism between the chloroplast and cytoplasm. 13C-Deoxyxylulose phosphate fed to Catharanthus roseus resulted in significant 13C enrichment in phytol, p-carotene and lutein and low levels of enrichment in the phytosterols (Arigoni, 1997). It has been established that plant sterols are primarily synthesized through the MVA pathway (Eisenreich et al., 2001). Mixed biosynthesis was found in callus cultures of the hornwort Anthoceros punctatus. 2H and 13C labeled mevalonate were incorporated preferentially into an FPP derived portion of chlorophyll a, p-carotene and some diterpenes which are all primarily synthesized through the MEP pathway (Itoh, 2000). Experiments with chamomile sesquiterpenes showed mixed biosynthesis. Two isoprenes from the MEP pathway and a third from the MVA pathway condensed to form the sesquiterpene chamazulene (Adam et al., 1999; Adam and Zapp, 1998). It is not clear how the plastidic terpenoid transport mechanisms work, but in chamomile either MVA derived IPP was transported into the chloroplast, or MEP derived GPP was exported to the cytoplasm, to form FPP. Sesquiterpenes were thought to be synthesized primarily in the cytoplasm but there are reports of sesquiterpene synthesis 134 via the MEP pathway in plastids. The leaves of lima bean (Phaseolus lunatus) incorporated 2H labeled deoxyxylulose into volatile monoterpenes and sesquiterpenes in chloroplasts after jasmonic acid treatment or spider mite infestation (Rohmer, 1999). Inducible sesquiterpene synthesis may occur in the plastids because of the proximity to the source of carbon fixation. The two precursors, pyruvate and G-3-P, of the MEP pathway are derived from the products of photosynthesis and thus may be induced faster than the MVA pathway. Some Streptomyces species synthesize sesquiterpenes via MEP (Eisenrich et al., 1998), and the sesquiterpenoids from mychorrhyzal barley roots are also synthesized through the MEP pathway (Walter et al., 2000). It is possible that parthenolide is biosynthesized in both the plastids and cytoplasm via the two independent pathways. This is the case for ABA biosynthesis which can be direct via MVA in the cytoplasm or through the catabolism of carotenoids in plastids (Dewick, 1999). Since parthenolide may be a defense compound it is possible that synthesis via the MEP pathway occurs under stress conditions such as herbivory, infection, or physical damage, but under unstressed conditions synthesis is through the MVA pathway. This could explain the superposition of the 13C labeling patterns found in parthenolide. The compartmental separation of the two IPP biosynthetic pathways is not absolute. At least one metabolite can be exchanged between the compartments. The extent of the transport depends on the species as well as on the presence and concentration of exogenous precursors. The movements of terpenoids between compartments appear generally small in intact plants under physiological conditions. The nature of the metabolic exchange between the compartments and the regulation of the process remain to be established. The sharing of terpenoids between the two pathways is one of the reasons for the failure in history to recognize the MEP route. 135 The low incorporation rates of mevalonate and acetate in many plant terpenoids were not because of restricted uptake or restricted use of the labeled precursors but likely reflected the small contributions of the MVA pathway to the biosynthesis of terpenoids predominantly formed via MEP. Feeding experiments are tedious and may not give the exact picture, but they do shed light on the main terpenoid biosynthetic route and on possible exchanges between the two routes. The presence of two different biosynthetic routes leading to the same metabolite (IPP), which functions in two separate compartments, may be a mechanism for plants to regulate isoprene biosynthesis. Experiments using molecular techniques may uncover the nature of interactions between the two pathways. Contradictions between some recent isotopic feeding studies of the MEP and MVA pathways may be explained by some inherent problems with feeding studies. Excessive feeding and the subsequent accumulation of metabolites, which are not naturally accumulated by cells such as MVA or DXP, do not represent normal physiological conditions. High concentrations of a substrate may unnaturally induce or increase synthesis of a terpenoid and lead to an over-estimation of the pathway's contribution. Feeding of an earlier metabolite such as glucose may result in a more accurate estimation of the contribution of each pathway to isoprenoid biosynthesis. However, feeding earlier precursors such as glucose may lead to confusing results due to prolonged metabolism. For example, in phototrophic organisms the recycling of CO2 may result in confusing labeling patterns. Other factors to consider with intact organisms are the differences in uptake and metabolism dynamics for each substrate. These affect incorporation into terpenoids and may invalidate direct comparisons between incorporation rates of different substrates. In feverfew for example, labeled compounds must move through the plant from stem to trichome and into parthenolide. 136 Labeled pyruvate moves to the cytoplasm, is metabolized to acetate and used in the MVA pathway but must move into the chloroplast to be used by the MEP pathway. 5.5. CONCLUSIONS It is clear that parthenolide biosynthesis in feverfew uses the mevalonate pathway. It appears that the MEP pathway also contributes to parthenolide biosynthesis. The extent of terpenoid movement between the two pathways seemed substantial. Further experiments are required to obtain conclusive evidence showing the extent of MEP contribution to STLs and parthenolide. This may be achieved with the use of inhibitors specific for either pathway such as mevinolin which inhibits the MVA pathway and fosmidomycin which inhibits the MEP pathway (Fellermeier et al., 1999). Feeding substrates in the presence of an inhibitor for one pathway would show the potential involvement of the other pathway. In addition, labeled methylerythritol phosphate or deoxyxylulose phosphate would give a better indication of MEP pathway involvement in parthenolide biosynthesis. The increasing antibiotic drug resistance of human pathogens has created an urgent need for novel therapeutic approaches. The MEP pathway is used by many pathogenic microorganisms and by the protozoa P. falciparum but is not present in humans or animals. Therefore, the MEP pathway is an ideal target for the development of novel antibiotics and antimalarial agents (Rohmer, 1998). In plants the inhibition of the MEP pathway may lead to the development of new herbicides. Understanding the mechanisms and regulation of both the MVA and MEP pathways will likely benefit the biotechnological production of commercially or pharmaceutically interesting isoprenoids by engineering increased production or production from non-typical sources. 137 5.6. REFERENCES Adam, K., Thiel, R., and Zapp, J. (1999) Incorporation of 1-[1-13C]deoxy-d-xylulose in chamomile sesquiterpenes. Archives of Biochemistry and Biophysics 369:127- 132 Adam, K., and Zapp, J. (1998) Biosynthesis of the isoprene units of chamomile sesquiterpenes. Phytochemistry 48:953-959 Araki, N., Kusumi, K., Masamoto, K., Niwa, Y., and Iba, K. (2000) Temperature- sensitive arabidopsis mutant defective in 1-deoxy-D-xylulose 5-phosphate synthase within the plastid non-mevalonate pathway of isoprenoid biosynthesis. Physiologia Plantarum 108:19-24. Arigoni, D., Sagner, S., Latzel, C, Eisenreich, W., Bacher, A., and Zenk, M.H. (1997) Terpenoid biosynthesis from 1-deoxy-D-xylulose in higher plants by intramolecular skeletal rearrangement. Proceedings of the National Academy of Science 94:10600-5. Dewick, P.M. (1999) The biosynthesis of C5-C25 terpenoid compounds. Natural Product Reports 16:97-130 Eichinger, D., Bacher, A., Zenk, M. H., and Eisenreich, W. (1999) Analysis of metabolic pathways via quantitative prediction of isotope labeling patterns: a retrobiosynthetic C NMR study on the monoterpene loganin. Phytochemistry 51:223-236 Eisenreich, W., Menhard, B., Hylands, P. J., Zenk, M. H., and Bacher, A. (1996) Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin. Proceedings ofthe National Academy of Science 93:6431-6 Eisenreich, W., Schwarz, M., Cartayrade, A., Arigoni, D., Zenk, M.H., and Bacher, A. (1998) The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chemistry and Biology 5:R221-R233 Eisenreich, W., Rohdich, F., and Bacher, A. (2001) Deoxyxylulose phosphate pathway to terpenoids. Trends in Plant Science 6: 78-84 Fellermeier M., Kis K., Sagner S., Maier U., Bacher A., and Zenk M. H. (1999) Cell-free conversion of 1-deoxy-d-xylulose 5-phosphate and 2-c-methyl-d-erythritol 4- phosphate into beta-carotene in higher plants and its inhibition by fosmidomycin. Tetrahedron Letters 40:2743-2746 Giner, J.L. and Jaun, B. (1998) Biosynthesis of isoprenoids in Escherichia coli: Retention of the methyl H-atoms of 1-deoxy-D-xylulose. Tetrahedron Letters 39:8021-8022. Itoh, D., Karunagoda, R.P., Fushie, T., Katoh, K., and Nabeta, K. (2000) Nonequivalent labeling of the phytyl side chain of the chlorophyll a in callus of the hornwort Anthoceros punctatus. Journal of Natural Products 63:1090-1093 138 Rohmer, M. (1998) Isoprenoid biosynthesis via the mevalonate-independent route, a novel target for antibacterial drugs? Progress in Drug Research 50:135-54. Rohmer, M. (1999) The discovery of the mevalonate-independent pathway for isoprenoid biosynthesis in the bacteria, algae, and higher plants. Natural Product Reports 16:565-574 Walter, M.H., Fester, T., and Strack, D. (2000) Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the 'yellow pigment' and other apocarotenoids. Plant Journal 21:571-8. 139 CHAPTER 6 General discussion 6.1. RESEARCH OVERVIEW Phytochemicals are synthesized from water, nutrients and CO2 using energy from light to drive the reactions. Variation in any of these components affects plant biochemistry and physiology including secondary metabolism. Fluctuation of secondary compounds in response to the supply of resources for growth suggests plasticity in secondary metabolism. The role of many secondary compounds is for defence. Chemical concentrations and the level of chemical or physiological protection from the environment may thus depend on the availability of resources. For humans, secondary compounds are the primary source of biological activity of most medicinal plants as well as in flavours and aromas of culinary plants. If we can understand how different environments affect the regulation of secondary metabolism, we can manipulate medicinal and culinary plants toward higher production of commercially valuable compounds while maintaining high crop yields. We could improve agriculture and resource management if we had the ability to predict how secondary metabolism changes with the availability of resources. This thesis examined the relationship between growing conditions and growth and development, physiology and biochemistry of feverfew shoots. In Chapter 2, feverfew plants were grown with different nitrogen and water application rates in the field and greenhouse. Analysis of shoot yields and leaf parthenolide concentrations showed that higher rates of nitrogen application resulted in higher leaf parthenolide concentrations and higher shoot yields. Plants under mild water stress had lower shoot yields but higher leaf and flower parthenolide concentrations than non-stressed plants. 140 Parthenolide concentration has been examined in feverfew tissue culture (Brown, 1993, Brown et al., 1996), feverfew grown in different regions and climates (Maries et al., 1992), and in feverfew plants and commercial preparations (Awang et al., 1991; Heptinstall et al., 1992). These reports showed high variability in parthenolide concentration when measured in commercial products and authenticated feverfew plants. The range of parthenolide concentrations found in the literature is 0 % to 2.8 % in leaves and between 0.8% and 2.3% in flowers by dry weight (Awang et al., 1991; Maries et al., 1992). Leaf and flower parthenolide concentrations found in the experiments described in Chapter 2 were within the reported range. Variability in parthenolide concentrations resulted from varying water and nitrogen supply. Thus these two resources in the environment affected sesquiterpene metabolism. Environmental cues are required for normal plant development. Aside from providing energy for photosynthesis, light plays an important role in regulating many physiological processes such as germination, flowering and senescence. For example, the transition from vegetative to reproductive growth in many plants is cued by photoperiod. Experiments in Chapter 3 examined shoot yield and parthenolide concentration during shoot development in vegetative and flowering stages. Feverfew is a long-day plant that requires a dark period shorter than 12 hours per day to flower. When grown under short days for extended periods it remained in a vegetative stage and accumulated high concentrations of parthenolide (over 4% by dry weight) in leaf glandular trichomes. During the transition from vegetative to reproductive growth, leaf parthenolide concentrations decreased while the concentration of other more complex STLs increased. The accumulation of parthenolide may be a result of restricted biosynthesis of STLs downstream from parthenolide synthesis during the vegetative stage. The restriction may be due to an inhibition, or an absence of the required 141 enzymes for conversion of parthenolide to other STLs. The biosynthesis of other STLs during reproductive growth may be triggered by the same mechanism that triggers flowering. There are direct applications of these findings to the commercial cultivation of feverfew. Production of leaves with a high concentration of parthenolide and low levels of other STLs can be achieved by growing plants under short days. Alternatively, if the other STLs or flowers are pharmacologically important, feverfew can be induced to flower resulting in lower leaf parthenolide concentration and higher concentrations of other STLs. Trichomes are the site of parthenolide biosynthesis in feverfew. The relationship between trichomes, leaf development and parthenolide concentration was examined in Chapter 4. The mechanisms regulating STL biosynthesis are unknown. Experiments in Chapters 2 and 3 showed nitrogen and water availability and developmental status of feverfew had effects on leaf parthenolide concentrations. It is unknown exactly how these factors affected parthenolide biosynthesis. Parthenolide concentrations may be regulated by manipulation of trichome size and spatial distribution. I developed a new method for observing trichomes using slide preparations of dried epidermal peals under the light microscope. Drying the epidermis left the lipophilic trichomes intact and clearly visible. Trichome density was found to be related to leaf parthenolide concentration and both varied with leaf age and developmental stage. The difference in size and density of trichomes on leaves of different ages or between leaves of vegetative and reproductive shoots suggests a mechanism of control allowing the plant to alter total terpene production by changing the spacing and size of trichomes initiated on the leaf epidermis. Microsampling techniques made it possible to perform chemical analysis of individual trichomes from leaf and flower surfaces. Glandular trichomes from flower petals were significantly larger and contained low concentrations of parthenolide 142 compared to glandular trichomes on the leaves. This result seemed to contradict the consensus of reports showing that flowers had higher parthenolide concentrations than leaves (Banthorpe et al., 1990; Fontanel et al., 1990; Awang et al., 1991; Heptinstall et al., 1992). My research showed that parthenolide in feverfew flowers is most concentrated in trichomes on the receptacle and least concentrated in trichomes on the flower petals. Receptacle trichomes have high densities and therefore high parthenolide concentrations. The receptacle trichomes are of similar shape and size as the leaf trichomes and individually contain similar amounts of parthenolide. The relationship of trichome density and size to parthenolide and STL concentration has applications in research and agriculture. Trichomes can be easily isolated and manipulated by scientists to study terpenoid biosynthesis and trichome development. Varieties of feverfew and other crops with glandular trichomes of commercial importance may be selected for trichome density and size. Varieties could be selected for flowers with large receptacles or for other organs with high parthenolide concentration. Molecular techniques may be used to manipulate trichome density and size which could affect chemical concentration in the plant. The biosynthesis of terpenoids is under renewed investigation due to the recent discovery of a second terpenoid pathway (Rohmer et al., 1993). In plants, the methylerythritol phosphate (MEP) pathway to terpenoid biosynthesis occurs in plastids while the classically understood mevalonate pathway occurs in the cytosol (Rohmer, 1999). There is little known about the interaction between the two pathways and their contributions to STL biosynthesis. There appears to be transport of terpenoids across the plastidic membrane but the mechanism and rate of flux is unknown (Eisenreich et al., 2001). Some terpenoids produced in high concentrations, like isoprene and monoterpenes, are synthesized exclusively by the MEP pathway. Both pathways can 143 contribute subunits to the same terpenoid while other types of terpenoids appear to be synthesized exclusively through a single pathway. For example, the sesquiterpene chamazulene from chamomile is of mixed biosynthesis from the two pathways (Adam and Zapp, 1998; Adam et al., 1999) while the sterols are synthesized exclusively through the mevalonate pathway (Lichtenthaler et al., 1997). The pathway to parthenolide biosynthesis was examined in Chapter 5. Like the biosynthesis of chamazulene from chamomile, both the mevalonate and MEP pathways contribute to parthenolide synthesis. The mevalonate pathway was clearly demonstrated as a significant contributor to the biosynthesis of parthenolide. The extent of involvement of the MEP pathway however remains unclear and requires further study. 6.2. FUTURE RESEARCH The broad scope of this thesis presents many potential areas for future research. One of the underlying and unanswered questions is what are the control mechanisms linking environmental stimuli and shoot development to sesquiterpene biosynthesis? The nitrogen and water effects on sesquiterpene metabolism may be a response to altered physiology rather than a result of direct regulation of a terpenoid pathway. Likewise, shoot development and the transition from vegetative to reproductive growth alters physiology, which in turn may affect terpenoid biosynthesis. The possible indirect nature of these effects will make it difficult to answer the question. The best approach may be to determine what the genetic regulatory elements are in genes involved in the terpenoid biosynthetic pathways and then from these identify which factors cause the changes in terpenoid metabolism. Perhaps one of the most important but poorly understood processes in plant terpenoid metabolism is the transport of terpenoids across organelle membranes. 144 Transport proteins likely exist but have not been found to date. The non-polar terpenoids may diffuse across membranes but they often contain phosphate groups that would inhibit diffusion. A passive mechanism is unlikely since there are classes of compounds such as monoterpenes and triterpenes that utilize just one pathway for synthesis. Therefore it is more likely that there is strict control over the movement of terpenoids between compartments. The regulation of terpenoid pools in different cellular compartments is currently a primary target of investigation. The MEP pathway is responsible for synthesizing many important plastidic compounds for photosynthesis, electron transport, and photoprotection. Localization of the MEP pathway in plastids suggests that photosynthetic processes may be tightly linked to MEP pathway regulation. Research should focus on the relationship between plastidic processes, such as photosynthesis, and MEP pathway regulation. More research is required to determine the involvement of the MEP pathway in parthenolide biosynthesis. The ideal isotope feeding experiment to would be with enriched methylerythritol phosphate or 1-deoxyxylulose-5-phosphate. Both of these compounds are substrates for the MEP pathway. Methylerythritol phosphate is a committed precursor in the MEP pathway. Another approach is to use the mevalonate pathway inhibitor mevinolin while feeding labelled pyruvate. This will allow only the MEP pathway to contribute to parthenolide biosynthesis. The complimentary experiment using the MEP pathway inhibitor fosmidomycin with labelled mevalonate substrates could also provide insight into parthenolide biosynthesis. The variability of pharmacologically active terpenoids in commercial crops, particularly in processed medicinal herbs used for one compound of interest, raises concerns over safety and efficacy of these products. Manipulating terpenoid biosynthesis by cultivation methods is idealistic and may be impractical for many 145 producers in diverse climates and geographies. To ensure safety for consumers of medicinal and culinary crops with potentially harmful terpenoids, genetic and chemical analysis could be performed to authenticate species, determine levels of unsafe compounds, and ensure safe dosage. Determining chemical or genetic markers to authenticate a species is relatively simple and should be pursued by regulating bodies. On the other hand, determining correct dosage for medicinal plants is generally difficult and understudied. The principle pharmacological activities in most herbal medicines are unknown. This is an area that requires significantly more research. Feverfew is among the most studied medicinal herbs during the past 30 years. Even though its efficacy in relieving migraine has been demonstrated, compounds other than parthenolide are thought to contribute to feverfew's antimigraine medicinal properties. Thus although feverfew is a well-studied medicinal plant, dosage will be difficult to recommend unless the other antimigraine compounds are identified and their interaction with parthenolide identified. As with all medicinal plants, systematic research should be conducted to develop standard procedures for product authentication, and whenever possible standardization of dosage, to ensure safe products for consumers. 146 6.3 REFERENCES Adam, K.P., Thiel, R., and Zapp, J. (1999) Incorporation of 1-[1-C-13]deoxy-D-xylulose in chamomile sesquiterpenes. Archives of Biochemistry and Biophysics 369:127-132. Adam, K.P. and Zapp, J. (1998) Biosynthesis ofthe isoprene units of Chamomile sesquiterpenes. Phytochemistry 48:953-959. Awang, D.V.C., Dawson, B.A., Kindack, D.G., Crompton, C.W., and Heptinstall, S. (1991) Parthenolide content of feverfew (Tanacetum parthenium) assessed by HPLC and 1H-nmr spectroscopy. Journal of Natural Products 54:1516-1521. Banthorpe, D. V, Brown, G. D, Janes, J. F, and Marr, I. M. (1990) Parthenolide and other volatiles in the flowerheads of Tanacetum parthenium I. Schultz bip. Flavour & Fragrance Journal 5:183-186. Brown, G. D. (1993) Production of anti-malarial and anti-migraine drugs in tissue culture of Artemisia annua and Tanacetum parthenium. Acta Horticulturae 330:269-276. Brown, A.M.G., Lowe, K.C., Davey, M.R., and Power, J.B. (1996) Feverfew (Tanacetum parthenium): Tissue culture and parthenolide synthesis. Plant Science 116:223-232. Eisenreich, W., Rohdich, F., and Bacher, A., (2001) Deoxyxylulose pathway to terpenoids. Trends in Plant Science 6:78-84 Fontanel, D., Bizot, S., and Beaufils, P. (1990) HPLC determination ofthe parthenolide content of Chamomile Tanacetum parthenium (L.) Schulz-bip. Plantes Medicinales et Phytotherapie 24:231-237. Heptinstall, S., Awang, D.V., Dawson, B.A., Kindack, D., Knight, D.W., and May, J. (1992) Parthenolide content and bioactivity of feverfew (Tanacetum parthenium (L.) Schultz-Bip.). Estimation of commercial and authenticated feverfew products. Journal of Pharmacy and Pharmacology 44:391-395. Lichtenthaler, H.K., Rohmer, M., and Schwender, J. (1997) Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants. Physiologia Plantarum 101:643-652. Maries, R.J., Kaminski, J., Arnason, J.T., Pazos-Sanou, L., Heptinstall, S., Fischer, N.H., Crompton, C.W., Kindack, D.G., and Awang, D.V. (1992) A bioassayfor inhibition of serotonin release from bovine platelets. Journal of Natural Products 55:1044-1056. Rohmer, M. (1999) The discovery of a mevalonate-independent pathway for isoprenoid biosynthesis in bacteria, algae and higher plants. Natural Product Reports 16:565-574. 147 Rohmer, M., Knani, M., Simonin, P., Sutter, B., and Sahm, H. (1993) Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochemical Journal 295:517-524. Smith R.M. and Burford M.D. (1992) Supercritical fluid extraction and gas chromatographic determination of the sesquiterpene lactone parthenolide in the medicinal herb feverfew (Tanacetum parthenium). Journal of Chromatography 627:255-261. 148 APPENDIX 1 Table 1. Field irrigation and nitrogen application trial fertigation schedule. The 50 kg/ha feed schedule was used for the field irrigation frequency trial. Date Application Rate Fertilizer kq/ha qm/N treatment area 0 50 100 0 50 100 May 22 0-53-34 15 15 15 53 48 53 (transplanted 34-0-0 0 18.4 36.8 0 59 130 May 16) MgS04 20 20 20 71 64 71 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 June 5 0-53-34 15 15 15 53 48 53 34-0-0 0 18.4 36.8 0 59 130 MgS04 40 40 40 142 128 142 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 June 19 34-0-0 0 18.4 36.8 0 59 130 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 July 3 34-0-0 0 18.4 36.8 0 59 130 MgS04 30 30 30 106 97 106 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 July 17 34-0-0 0 18.4 36.8 0 59 130 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 July 31 34-0-0 0 18.4 36.8 0 59 130 MgS04 30 30 30 106 97 106 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 August 14 34-0-0 0 18.4 36.8 0 59 130 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 August 28 34-0-0 0 18.4 36.8 0 59 130 MgS04 30 30 30 106 97 106 Chelates 1.5 1.5 1.5 5.3 4.8 5.3 1) May 10, 400 kg/ha of gypsum was applied to the beds and raked in. 2) Dissolve nutrients in warm water and adjust pH between 5.5-6.5. Make up to 20 L with water. 3) Fertigate both high and low water treatments, of the same N rate, at the same time. / 149 Table 2. Field and greenhouse irrigation and medium trial nutrient feed formula. Field solution was made in 100 I tank and greenhouse solution was made in 500 I water. Nutrient solutions were replenished weekly. All greenhouse-grown plants received this nutrient feed. This feed formula was used for all greenhouse experiments. Nutrient Fertilizer Concentration weight delivered to (g) plants (g/l) NH4NO3 (34-0-0) 133 0.266 CaCI2 196 0.392 KH2PO4 (0-53-34) 100 0.2 KH2SO4 (0-0-50) 278 0.556 micronutrients 7.5 0.015 H2SO4 adjust pH to 6