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Occurrence and activity of ecdysterone (insect moulting hormone) in plants Dreier, Susan I. 1987

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OCCURRENCE AND ACTIVITY OF ECDYSTERONE (INSECT MOULTING HORMONE) IN PLANTS by SUSAN I. DREIER B.Sc, The University of Guelph, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Botany We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1987 c SUSAN I. DREIER, 1987 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 of B o t a n y The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 D t June 25, 1987 D E - 6 ( 3 / 8 1 ) ABSTRACT The occurrence and biological activity of ecdysterone (insect moulting hormone) was examined in a number of plant species. A simple method was developed for the semiquantitative analysis of ecdysterone in plant extracts. The procedure consists of repeated washing of an aqueous methanolic extract of the plant with petroleum ether, followed by gradient elution of the freeze-dried aqueous extract on a SepPak (CIS reverse-phase cartridge), and high-performance liquid chromatography. Crude extract was purified on Sephadex L H 2 0 for spectral analysis. Twelve species of ferns, three species of gymnosperms, and five species of angiosperms were examined for the presence of ecdysterone. Ecdysterone was found in a number of plant species, the chemistry of which has not previously been examined with respect to phytoecdysteroids. These include four species of ferns (Aspidotis densa (Brackenr.) Lellinger., Cryptogramma crispa (L.) R.Br., Blechnum spicant (L.) Roth, Polypodium glycyrrhiza D.C. Eat); two species of gymnosperms {Taxus brevifolia Nutt., Taxus canadensis Marsh.); and two species of angiosperms (Trillium cernuum L, Trillium ovatum Pursh.). Applied ecdysterone had no effect in a cytokinin bioassay, but elicited a slight increase in elongation of mung bean epicotyls (GA 3 bioassay) and elongation of excised dwarf pea hypocotyl hooks (auxin bioassay). ii TABLE OF CONTENTS A B S T R A C T ii A C K N O W L E G E M E N T S vii 1. INTRODUCTION 1 1.1. Discovery of Ecdysteroids 1 1.2. Chemistry of Ecdysteroids 3 1.3. Distribution and Concentration of Ecdysteroids in Plants 8 1.3.1. Distribution 8 1.3.2. Concentration 10 1.4. Biological Activity of Ecdysteroids 12 1.4.1. Insects and Other Invertebrates 12 . 1.4.2. Plants ; 15 1.5. Occurrence and Biological Activity of Steroids in Plants and Fungi 17 1.6. Outline of Research 26 2. M A T E R I A L S A N D M E T H O D S 27 2.1. Plant Material 27 2.2. Solvent Extraction 28 2.3. Chromatography 30 2.3.1. Thin Layer Chromatography (TLC) 30 2.3.2. Column Chromatography 30 2.3.3. High Performance Liquid Chromatography (HPLC) 31 2.3.4. Droplet Countercurrent Chromatography (DCC) 32 2.3.5. Gas Chromatography (GC) 32 2.4. Spectroscopy 33 2.5. Plant Growth Bioassays 33 2.5.1. Mung Bean Epicotyl Elongation 33 2.5.2. Elongation of Cucumber Hypocotyls 35 2.5.3. Promotion of Expansion of Cucumber Cotyledons 37 2.5.4. Expansion of Dwarf Pea Hooks 37 2.5.5. Thiarubrine Production in Tumour Cultures of Chaenactis douglasii 41 3. R E S U L T S A N D DISCUSSION 42 3.1. Methods of Extraction and Isolation of Ecdysterone from Plant Material 42 3.1.1. Solvent Extraction 44 3.1.2. Chromatography 45 3.2. Occurrence of Ecdysterone in Plants Surveyed 50 3.3. Biological Activity of Ecdysterone in Plant Tissues 58 3.3.1. Production of Thiarubrine in Callus Cultures 58 3.3.2. Plant Growth Bioassays 60 3.3.2.1. Gibberellin Bioassays 60 3.3.2.2. Auxin Bioassay 66 3.3.2.3. Cytokinin Bioassay 70 3.3.2.4. Summary 72 iii 4. C O N C L U S I O N 74 5. BIBLIOGRAPHY 75 6. A P P E N D I X 1 8 5 iv LIST OF TABLES Table I. Chemical and Physical Data for Ecdysterone 7 Table II. Plant Species Containing High Concentrations of Ecdysterone 11 Table III. Dates and Locations of Plant Collections 27 Table IV. Occurrence of Ecdysterone in Plants Surveyed 53 Table V. Production of Thiarubrine in Callus Cultures from Transformed Chaenactis douglasii 59 v LIST OF FIGURES Fig. 1. Common ecdysteroids 2 Fig. 2. Cholesterol 4 Fig. 3. Amasterol 5 Fig. 4. Brassinolide 18 Fig. 5. Conformational formulae of ecdysterone and brassinolide 20 Fig. 6. Sterols 22 Fig. 7. Antheridiol; oogoniol 24 Fig. 8. Extraction and isolation of ecdysterone from plants 29 Fig. 9. Material for Elongation of Mung Bean Epicotyls bioassay 34 Fig. 10. Material for Elongation of Cucumber Hypocotyls bioassay 36 Fig. 11. Material for Expansion of Cucumber Cotyledons bioassay 38 Fig. 12. Material for Expansion of Dwarf Pea Hypocotyl Hooks bioassay 39 Fig. 13. U V absorption spectrum of ecd3'sterone 43 Fig. 14. H P L C trace of a mixture of ecdysteroids 47 Fig. 15. H P L C trace of crude extract of A. densa following partial purification by SepPak 47 Fig. 16. DCC elution profile of 0.5 mg ecdysterone 50 Fig. 17. Major steps in the mevalonic acid pathway, showing branch point to G A 3 and ecdysterone 61 Fig. 18. 24-epibrassinolide 62 Fig. 19. Elongation of cucumber hypocotyls 64 Fig. 20. Elongation of mung bean epicotyls (response to ecdysterone) 65 Fig. 21. Elongation of mung bean epicotyls (response to 24-epibrassinolide) 65 Fig. 22. Elongation of dwarf pea hypocotyl hooks 68 Fig. 23. Fresh weight increase of dwarf pea hypocotyl hooks 69 Fig. 24. Expansion of cucumber cotyledons , 71 vi ACKNOWLEGEMENTS I thank Dr. Towers for introducing me to the topic of phytochemistr}7, and for his support and enthusiasm throughout this study. I appreciate and thank all those people in the lab and the department who offered help and advice, and Risa Smith who gave me valuable assistance with statistical analyses, support, and an island retreat. Special thanks to Nesrin Tanrisever for her advice and encouragement during the later stages of this work. Finally, I would like to thank my family for their endless support and encouragement. vii 1. INTRODUCTION 1.1. DISCOVERY OF ECDYSTEROIDS Ecdysis, the periodic shedding and regeneration of the cuticle during insect development, was suspected to be under hormonal control after Kopec (1922) demonstrated that secretions from the brain were necessary for pupation of caterpillars of the gypsy moth, Lymantria dispar. Later studies (Hachlow, 1931; Fraenkel, 1935; Wigglesworth, 1934) indicated that substances secreted by the brain stimulate production of a "moulting hormone" (MH) by the prothoracic glands. Becker (1941) obtained active fractions from the blowfly Calliphora erythrocephala but the active compound was not isolated until Butenandt and Karlson (1954) managed to obtain 25 mg of pure crystalline hormone, which they named ecdysone, from 500 kg of silkworm (Bombyx mori) pupae. Karlson (1956) isolated a second active compound from B. mori and named it p'-ecdysone to distinguish it from the a-ecdysone isolated earlier. The structure of a-ecdysone was finally determined by Huber and Hoppe (1965) and it was first synthesized in 1966 simultaneously by two groups (Siddall et al, 1966; Kerb et al, 1966). Structures and nomenclature of a- and j3-ecdysone are shown in Fig. 1, and further information on the chemistry and structure of ecdysteroids (the generic name for this group of compounds) is given in Section 1.2. Owing to the difficult}7 of synthesis of a- and /3-ecdysone and the trace 1 INTRODUCTION / 2 Fig. 1. Common phytoecdysteroids. A: ecdysterone; 20-OHecdysone; /3-ecdysone; crustecdysone B: ecdysone; a-ecdysone C: ponasterone B D: ponasterone B E: polypodine B F: inokosterone INTRODUCTION / 3 amounts present in insects, research on moulting hormones was impeded by extreme scarcity of the compounds. It was with great interest therefore, that ecdysteroids were unexpected^ found in plant sources at concentrations several orders of magnitude greater than in insects. Nakanishi et al (1966) reported the isolation of ponasterone A (Fig. 1-C) from the conifer Podocarpus nakaii at the same time that Galbraith and Horn (1966) reported j5 -ecdysone in leaves of Podocarpus elata. Takemoto et al (1967a) isolated inokosterone (Fig. 1-F) and /3-ecdysone from Achyranthes radix. Many plants have since been screened for M H activity. Over 70 ecdysteroids have been isolated from plants (phytoecdysteroids) and 20 are known, in animals (zooecdysteroids) (Horn and Bergamasco, 1985). 1.2. C H E M I S T R Y O F E C D Y S T E R O I D S Ecdysteroids are polyhydroxylated steroids containing 27 to 29 carbon atoms, with a structure similar to that of cholesterol. The structure of cholesterol and conventional system of numbering carbon atoms and rings of steroid molecules is shown in Fig. 2. INTRODUCTION / 4 Fig. 2. Cholesterol Al l ecdysteroids have the following features in common: (1) steroidal structure. (2) 6-keto-7-ene moiety, (3) cis-fused A/B ring junction, and (4) full sterol side chain. These four features are necessary for M H activity (Bergamasco and Horn, 1983). The presence and position of hydroxyl groups have varied effects on M H activity. Presence of a 14a-hydroxyl group is important for high activity, OH groups at C3 and C22 are less important, and OH groups at C2, C20 and C25 have a marginal effect on M H activity' (Bergamasco and Horn, 1980). The most widespread and abundant phytoecdysteroid is ecdysterone (20-OH ecdysone). The nomenclature of this compound has been rather confusing in the past but it is now usually referred to as ecdysterone or 20-OH ecdysone. Structures of ecdysterone and other common phytoecdysteroids are shown in Fig. 1. INTRODUCTION / 5 Ecdysteroids are derived from cholesterol, but the steps of their biosynthesis in plants and animals are not well understood. The probable order of events in the biosynthesis of ecdysone in insects is: formation of the cis arrangement of A and B rings, followed by hydroxylations of the sidechain and a hydroxylation at C2, based on combined results of studies with a number of insect species (Rees and Isaac, 1985). Very little is known about ecdysteroid biosynthesis in plants. Labelled cholesterol is incorporated into ecdysterone and mevalonic acid is converted to ecdysterone in seedlings of various plants (Heftmann et al, 1968; Rees, 1971; Lloyd-Jones et al, 1973; Rees and Goodwin, 1974; Morgan and Poole, 1977). Amasterol (Fig. 3) was isolated from roots of Amaranthus viridus (Roy et al, 1982). This compound, containing a A5,7-diene, has been proposed as an intermediate of ecdysone biosynthesis in insects. Since Amaranthus spp. also contain ecdysterone, amasterol may well be a precursor of ecdj'sterone in these plants. Few studies of ecdysteroid synthesis in plants have been carried out. HO Fig. 3. Amasterol INTRODUCTION / 6 Physical and chemical data for ecdysterone are summarized in Table I. Ecdysteroids are water-soluble and are among the most polar naturally occurring steroids known (Bergamasco and Horn, 1 9 8 3 ) . Their polarity presents special problems in the extraction and isolation of ecdysteroids from plants. INTRODUCTION / 7 Table 1. Physical and Chemical Data for Ecdysterone MW = 480.6 m.p. = 237-239°C UV: Xmax 240nm, e = 12,000 IR: O-H 3440 cm" 1, C = 0 1650 cm" 1, C = C 1612 cm" 1 MS: m/z 462 (M-H 20), 444 (M-2H 20), 426 (M-3H 20), 408 (M-4H 20), 99, 81 Morgan and Wilson (1980) Banerji et al (1971) INTRODUCTION / 8 1.3. DISTRIBUTION A N D C O N C E N T R A T I O N O F E C D Y S T E R O I D S IN P L A N T S 1.3.1. Distribution Soon after the discovery of phytoecdysteroids, a large number of Japanese plants were screened for M H activity with the aim of finding new sources of moulting hormones and looking for chemotaxonomic relationships (Takemoto et al, 1967b; Imai et al, 1969a; Hikino et al, 1973). M H activity was determined by means of a bioassay in which ligated segments of insect larvae (Chilo suppressalis or Calliphora erythrocephala) were scored for pupation 24 h after injection of, or immersion in, a crude extract of the plant (Karlson and Hanser, 1953; Sato el al, 1968). Pupation was taken as an indication of M H activity, and plant extracts which resulted in pupation were assumed to contain phytoecdysteroids. Takemoto et al (1967b) obtained active extracts from 22/39 species of ferns and 27/74 species of higher plants surveyed. They found no evidence of M H activity in fungi (mushrooms) or algae. Phytoecdysteroids have, however, recently been isolated from the red alga, Laurencia pinnata Yamada (Fukuzawa et al, ' 1986). Imai et al (1969a) screened 1056 species of Japanese plants from 186 families of ferns, Gymnosperms and Angiosperms. Ferns and Gymnosperms had the highest incidence of M H activity, in particular the families Polypodiaceae, INTRODUCTION / 9 Taxaceae and Podocarpaceae. They also found M H activity in primitive vascular plants such as horsetails (Equisetaceae) and clubmosses (Selaginellaceae and Lycopodiaceae). In a later study, 283 species (76 genera from 20 families) of Japanese ferns were surveyed, 170 of which were MH-active (Hikino et al, 1973). It was noted that M H activity was most common in the more evolutionarily advanced Pteridophytes. Russell and Fenemore (1974) found M H activity in 24/64 New Zealand ferns, with most species of Blechnum, Todea, Rumohra and Adiantum being active. MH-active species are found in most families of Gymnosperms, and the probability of activity in species of Angiosperms is much lower than for Pteridophytes and Gymnosperms. Of 410 families of Angiosperms tested, 74 have species with M H activity. Phytoecdysteroids • are widespread in Angiosperms but the probability of activity in more than one species of the same genus is much higher than the probability of activity in more than one genus per active family (Bergamasco and Horn, 1983). To date, over 110 plant families are known to be MH-active or to contain phytoecdysteroids. The compounds have been found in all plant parts and concentrations within a single plant may vary from part to part. The five ecdysteroids most commonly isolated (in order of decreasing frequency) are ecdysterone, ponasterone A, polypodine B, ecdysone and pterosterone (Horn and Bergamasco, 1985). Comprehensive lists of plant families, genera and species from which ecdysteroids have been isolated can be found in INTRODUCTION / 10 reviews by Bergamasco and Horn (1983) and Horn and Bergamasco (1985). 1.3.2. Concentration Phytoecdysteroids commonly occur at concentrations between 0.001 and 0.1% of the dry weight of the plant (Jones and Firn, 1978), but have been reported in amounts as high as 2.9% of the dry weight of roots of Cyanotis arachnoidae C B . Clarke (Chou and Lou, 1980). Zooecdy steroids, on the other hand, occur at concentrations of 10" 9 to 1 0 " 5 % in insects (Bergamasco and Horn, 1983). The highest concentrations of ecdysteroids have been found in plants from a wide range of families. These are listed in Table II. Some authors have noted that M H activity tended to be greater in autumn than in the spring, in extracts of the same species (Hikino et al, 1973; Yen et al, 1974). In bracken fern (Pteridium aquilinum (L.) Kuhn), concentrations of ecdysone and ecdysterone were ver3' low throughout the growing season (<0.3 Mg/kg fresh weight) until October, when the concentration was at its peak (53 Mg/kg fresh weight) (Jones and Firn, 1978). Apparent increases in ecdysteroid concentrations in autumn may be due to a decrease in protein or other compounds concurrent with no change in actual ecdysteroid levels. T a b l e II P l a n t S p e c i e s C o n t a i n i n g High C o n c e n t r a t i o n s of E c d y s t e r o n e DIVISION SUBDIVISION FAMILY SPECIES % EC REFERENCE P t e r l d o p h y t a P o l y p o d l a c e a e Spermatophyta Gymnospermae Podocarpaceae Anglospermae Ranuncu1aceae CommelInaceae A l z o a c e a e Amaranthaceae L a b l a t e a e A s t e r a c e a e PoIypodIum vulgare L. Dacrydlum 1nt ermed!um K i r k . Podocarpus elatus R.Br. He I Ieborus mult IfIdus s s p . s e r v ( c u s (Adamovlc) Merzm. & P o d l . CyanotIs arachnoidae C B . C l a r k e Sesuv(um portuIacastrum L . Achyrant hes Japontca Nakal Ajuga Japontca MI que 1 Serratula xeranthemotdes 1.0 FW ( r h l z o m e s ) J l z b a ef al (1967) 1.0 DW ( b a r k ) R u s s e l l et al (1972) 0.45 DW ( b a r k ) G a l b r a l t h 8 Horn (1966) 0.36 DW ( a e r i a l Hardman S Benjamin p a r t s S f l o w e r s ) (1976) 2.9 DW ( r o o t s ) Chou 8 Lu (1980) 0.35 DW (whole p l a n t ) 0.20 DW ( l e a v e s ) 0.20 DW ( l e a v e s ) 0.33 DW ( I n f l o r e s c e n c e ) B a n e r j l et al (1971) Takemoto et al (196B) Imai et al (1969) Kholodova et a I (1979) S o u r c e : Horn and Bergamasco (19B5) 1.4. BIOLOGICAL ACTIVITY OF ECDYSTEROIDS INTRODUCTION / 12 1.4.1. Insects and Other Invertebrates Ecdysone and ecdysterone are the most commonly occurring ecdysteroids in invertebrates. Ecdysteroids have been found in insects as well as in other arthropods including the spider Pisaura mirabilis (Bonaric and DeReggi, 1977), the tick Amblyomma hebraeum (Delbecque et al, 1978) and the crustacean Limulus polyphemus (Winget and Herman, 1976). They have also been isolated from members of other invertebrate phyla. The parasitic nematode Ascaris lumbricoides (Horn et al, 1974), the mussel Mytilus edulis (Takemoto et al, 1967c), the snail Helix pomatia (Romer, 1979), the zoanthid Gerardia savaglia (Sturaro et al, 1'982) and the parasitic fluke Schistosoma mansonii (Torpier et al, 1982) have all been reported to contain one or more of these compounds. Ecdysteroids are present at all stages of insect and crustacean development. In insects, they are primarily synthesized by the prothoracic glands, but are also synthesized and accumulated in the ovaries of adult females, then transferred to eggs (Sail et al, 1983). Insect moulting involves a complex series of events and interaction of a number of hormones. Prothoracicotropic hormone (PTTH) from the brain, stimulates the prothoracic glands to release ecdysone. Changing titres of ecdysone and juvenile hormone interact to regulate cuticulogenesis and metamorphosis. There is a large body of literature concerning the hormonal regulation of insect moulting. For more information on this topic, see Kerkut and Gilbert (1985), Vol. 8. INTRODUCTION / 13 Studies on the activity of ecdysteroids at the gene level have had an important influence on ideas regarding the mode of action of vertebrate steroid hormones such as testosterone and estrogen. Clever and Karlson (1960) reported that puffing occurred in isolated salivary gland polytene chromosomes of Chironomus a few minutes after application of ecdysone. This was the first indication that steroid hormones may act directly on genes. Ashburner et al (1974) proposed a general model of steroid hormone action in which the steroid passively diffuses into the cell, where it binds to a receptor protein. The receptor-steroid complex then moves to the nucleus and binds to specific chromosomal sites, inducing transcription of mRNA at those loci. The presence of ecdysteroid receptor proteins was first demonstrated in 1978, in cells of Drosophila imaginal discs ("Yund and Fristrom, 1978) and Drosophila cell cultures (Maroy et al, 1978). These proteins, by definition, bind ecdysterone with high affinity and specificity. Gronemeyer and Pongs (1980) were able to visualize ecdysone bound to salivary gland polytene chromosomes of Drosophila. Ecdysteroids were photocrosslinked to their binding site and visualized by affinity labelling techniques. Brightly fluorescing regions on the chromosomes corresponded to the locations of ecdysteroid-induced puffs. The originally proposed model of steroid hormone action has been altered somewhat since O'Connor's group (Sage et al, 1982; O'Connor, 1983) demonstrated the presence of ecdysteroid receptors resident in the nucleus. See O'Connor (1983) for a discussion of ways in which ecdysteroids might influence transcription of specific proteins in insects. The biosynthetic precursor of ecdysteroids in insects is cholesterol. Since they cannot synthesize the sterol nucleus, most insects require cholesterol or INTRODUCTION / 14 phytosterols (primarily sitosterol and stigmasterol) in their diet. There is some debate regarding the fate of ingested ecdysteroids. If the compounds evolved in plants as a means of defense against insects (first suggested by Galbraith and Horn, 1966), one would expect them to have adverse effects when ingested. However, a number of insects have been shown to inactivate ecdysterone by conjugation, oxidation, epimerization and hydroxylation (at C26) (Hoffmann and Hetru, 1983). Robbins et al (1968) reared housefly (Musca domestica) larvae on an artificial diet containing either ecdysterone or ponasterone A. Ecdysterone was inactive at the highest dose tested (150 ppm) but ponasterone A (150 ppm) resulted in growth and metamorphosis abnormalities. The effects of ingestion of a mixture of ajugasterone A and ecdysterone by silkworm larvae (B. mori) has been used by the silk industry. Administration of M H at appropriate times shortens or prolongs the feeding period of last instar larvae, synchronizes coccoon spinning, and increases silk production (increase of approximately 19% in average coccoon weight compared to controls) (Chou and Lu , 1980). Kubo et al (1983a) fed B. mori larvae an artificial diet containing 25 to 100 ppm ecdysterone. Effects of dietary ecdysterone varied with concentration, developmental stage and duration of exposure and included death without moulting, death after prolonged moulting, and growth inhibition. In another study (Kubo and Klocke, 1983), larvae of the moth Heliothis sp. exhibited no developmental or morphological abnormalities following ingestion of greater than 3000 ppm phytoecdysteroids in an artificial diet. In the same study, phytoecdysteroids added to an aqueous diet were shown to deter feeding in the aphid Schizaphis graminim. Modde et al (1984) reported that ecdysterone ingested by fifth instar larvae of Locusta migratoria was rapidly converted into its 3-acetate and 3-acetate-2-phosphate INTRODUCTION / 15 derivatives. These metabolites were rapidly excreted and ecdysterone never entered the hemolymph. Locusts (Schistocerca gregaria) fed bracken fern (high in ecdysterone) showed no growth or developmental abnormalities (Carlisle and Ellis, 1968). In a study of the role of ecdysteroids in bracken fern (Pteridium aquilinum (L.) Kuhn.), Jones and Firn (1978) tested the effect of ecdysteroids on the feeding behaviour of seven species of insects. They concluded that the levels of ecdysterone in P. aquilinum were "2 x 10 6 to 9 x 10 3 times lower than the lowest levels which would be required to affect development, reproduction, or survivorship of insects feeding on the plant". They also point out that levels of ecdysterone are highest in the autumn, a time of year during which insect herbivory is declining. The theory of phytoecdysteroids as defensive compounds has been neither confirmed nor refuted. 1.4.2. Plants It is possible that ecdysteroids have a physiological function in plants. This has been investigated in very few studies. Carlisle et al (1963) reported that MH-active fractions of whole locust extracts were active in a gibberellin bioassay. The locust extract stimulated elongation of dwarf pea internodes significantly over that of controls and elongation was approximately 10% of elongation of GA 3 - t reated plants. It has since been pointed out that the extract was of unknown composition and may have contained gibberellin-like compounds (Hendrix and Jones, 1972). These latter investigators tested the activity of INTRODUCTION / 16 ecdysterone in four gibberellin bioassays. They found no change in: a-amylase production by barley half-seeds, dwarf pea internode length, leaf sheath length of dwarf corn, and dark germination of spores of the fern Anemia phyllitidis. Gibberellin-type activity has been reported for ponasterone A, ecdysterone and inokosterone in one study, however. These compounds stimulated the growth of the second leaf sheath and root of rice seedlings (Matsuoka et al, 1969). The effect of ecdysterone on flowering is unclear. Jacobs and Suthers (1971) observed no significant effect of ecdysterone on flowering of cultured shoot-tips of Xanthium pennsylvanicum. The same compound inhibited formation of female flowers on Cucurbita pepo (Felippe, 1979). Following the isolation of a number of ecdysteroids from culture medium of Pteridium aquilinum gametophytes, ponasterones A and C, and ecdysterone were tested for activity in antheridium induction bioassays. None of the compounds displayed any activity with gametophytes of Pteridium, Onoclea, and Anemia (McMorris and Voeller, 1971). The question of a physiological function (or lack of) for ecdysterone in plants has not been resolved and has not been investigated in a thorough manner. INTRODUCTION / 17 1.5. O C C U R R E N C E A N D BIOLOGICAL ACTIVITY OF STEROIDS IN PLANTS A N D FUNGI Ecdysteroids are members of a group of triterpenoid compounds, the steroids. These are C30 compounds with a characteristic four-ring nucleus, formed via the mevalonic acid pathway by condensation of six isoprene units. The major classes of phytosteroids are: 1) sterols, 2) progestagens, 3) estrogens and androgens, 4) cardiac glycosides, and 5) saponins. Activity in plant growth and development bioassays has been demonstrated for a number of steroids. This is interesting since the compounds are similar in structure to ecdysteroids. It is logical to consider that steroidal compounds may have a physiological role within the plants that produce them, in view of their strong biological activity in animal systems. Relevant studies of biological activity of plant steroids are reviewed in this section. There are a large number of known phytosterols, all with a long sidechain at C17. They are often classified on the basis of substitution at C3, as: 1) free sterols (OH at C3), 2) steryl esters (fatty acid esterified at C3), 3) steryl glycosides (carbohydrate at C3), and 4) acylsteryl glycosides (carbohydrate at C3, with a fatty acid esterified to C6 of the carbohydrate). The sterol most often isolated from higher plants is 0-sitosterol (Fig. 6-A). The function of phytosterols is largety unknown, although some have exhibited activity in plant growth bioassays. The most notable of these is brassinolide (Fig. 4), a steroidal plant growth INTRODUCTION / 18 hormone isolated in extremely low concentrations from pollen of Brassica napus L. (Grove et al, 1979). Fig. 4. Brassinolide Brassinolide (BR) has a very strong growth-promoting effect on plants, which involves an increase in both cell elongation and cell division (Worley and Mitchell, 1971). BR exhibits auxin activity, in some cases acting synergistically with IAA (Yopp et al, 1979, 1981). It has effects similar to kinetin in some cytokinin bioassays and is active in gibberellin assays (no synergism with G A 3 ) (Mandava et al, 1981). The mode of action of brassinosteroids is being investigated. It appears to be distinct from the activity of other groups of plant growth regulators. Ancymidol, an inhibitor of gibberellin-mediated growth has no effect on responses to brassinolide (Yopp et al, 1979; Gregory and Mandava. 1982), and brassinosteroids are inactive in some assays which are very sensitive to gibberellins (Takeno and Pharis, 1982). Likewise, brassinolide is not active in all cytokinin bioassays (Mandava et al, 1981). It acts synergistically with auxin INTRODUCTION / 19 in some tests, but has an effect opposite to that of auxin in maize root segments, the growth of which is inhibited by auxin (Yokota and Takahashi, 1986). It is possible that endogenous auxin may be necessary for BR activity (Arteca et al, 1985). Brassinolide and ecdysterone bear a strong structural resemblance to each other, but there are certain important differences. The B ring of brassinolide is a seven-membered (vs six-membered in ecdysterone) lactone ring. Stereochemical]}', ecdysterone and brassinolide displa}' some important differences. Fusion of the A and B rings is trans in brassinolide and cis in ecdysterone, giving brassinolide a relatively planar configuration as opposed to ecdysterone, which is kinked (see structures in Fig. 5). The hydroxyl groups at C2 and C3 in the A ring are oriented differently (j3 in ecdysterone, a in brassinolide), and' the hydroxyl at C22 in the sidechain is oppositely oriented in the two compounds. These differences almost certainly have serious effects on the biological activity of these molecules. INTRODUCTION / 20 Fig. 5. Conformational structure of ecdysterone (A), and brassinolide (B). INTRODUCTION / 21 Activity in plant growth bioassaj's has been reported for other plant sterols. Steryl glucosides, particularly stigmasteryl-/3-D-glucoside (Fig. 6-B), have exhibited auxin activity, and acted synergistically with auxin in assays of elongation of Avena coleoptile segments (Kimura et al, 1975; Tietz et al, 1977; Smith and VanStaden, 1978). Various functions have been proposed for phytosterols. Some sterols may act to stabilize membranes by controlling their permeability. Grunwald (1971), comparing the effects of free sterols, steryl esters and steryl glycosides on membrane permeability, found that only free sterols were effective in reducing ethanol-induced leakage of electrolytes from barley roots. There are certain structural requirements for activity of sterols as membrane stabilizers. These are: 1) presence of a free C3 hydroxyl group (for interaction with membrane phospholipids), 2) a relatively flat configuration (ie trans-fused rings), and 3) a ring system with at least one double bond (Grunwald, 1980). It is important to consider these characteristics in comparison with the structure of ecdysterone. Ecdysterone is unlikely to be membrane-stabilizing because: 1) it is hydrophilic, and 2) its A and B rings are cis-fused. Steryl esters and steryl glycosides may be involved in the transport of sterols and glucose within plants. Steryl glycosides may also be a storage form of plant sterols (Grunwald, 1980). Estrogens and androgens (Fig. 6-C and 6-D) are C18 and C19 steroids with strong hormonal activity in mammals. There are a few reports of estrogens in plants at concentrations in the ng kg" 1 range (see Grunwald, 1980). Androgens, including testosterone, were found in pollen of Scotch pine Pinus INTRODUCTION / 22 Fig. 6. Sterols. A: /3-sitosterol B: stigmastery]-j3-D-glucoside C: estradiol D: testosterone E: C o r t i s o l F: corticosterone INTRODUCTION / 23 sylvestris L. (Saden-Krehula et al, 1971). Applied estrogens and androgens have been reported to influence sex expression of Ecballium elaterium L. (Cucurbitaceae) grown in natural field conditions (Kopcewicz, 1970). Estrogens significantly increased the ratio of female to male flowers, whereas androgens had the opposite effect. Cortisone, a corticosteroid, increased the total number of flowers but had no effect on sex expression. Geuns (1974; 1977) studied the effect of various corticosteroids on etiolated mung bean seedlings and found that applied C o r t i s o l and c o r t i c o s t e r o n e (Fig. 6-E and 6-F) doubled the number of l a t e r a l r o o t s formed. Cortisol stimulated elongation of root cells and hypocotyls. There has only been one report of isolation of a corticosteroid from a plant. 11-deoxycorticosterone was isolated from rice {Oryza sativa) husk oil (Bahadur and Srivastava, 1971). Corticosteroids regulate carbohydrate metabolism and sodium-potassium ion concentrations in mammals (Grunwald, 1980). The implications of these studies with respect to a possible hormonal function of estrogens, androgens or corticosteroids in plants, are unclear, since occurrence of the compounds in plants has been reported so rarely. Another interesting instance of biological activity of sterols concerns two compounds, antheridiol and oogoniol (Fig. 7), isolated from various species of the aquatic fungus Achlya (Barksdale, 1969; McMorris, 1978). These compounds initiate and coordinate sexual reproduction in Achlya spp. in the following manner. Antheridial hyphae secrete oogoniol, which stimulates female mycelium to form oogonial initials. Antheridiol secreted by the oogonial initials stimulates chemotropic growth of antheridial hyphae to the oogonia. The oogonia are then enveloped by the antheridial hyphae prior to fertilization. INTRODUCTION / Fig. 7. A: antheridiol; B: oogoniol. INTRODUCTION / 25 It has been demonstrated that sterols of plants and fungi do have significant biological activity in some cases. Our knowledge of hormones is far more comprehensive for the animal kingdom than for plants. This was aptly expressed by Parthier (1985), as "looking down from the summits of animal hormone action to the plains of phytohormone effects". Further research on activities and functions of phytosterols is needed. It is quite likely that some sterols have a hormonal function in plants. INTRODUCTION / 26 1.6. OUTLINE OF R E S E A R C H The aims of this study were: (a) to develop a reliable method for extraction and isolation of ecdysterone, (b) to screen selected ferns of British Columbia, and relevant plant species from groups other than the Pteridophytes, for the presence of ecdysterone, and (c) to address the question of the role of phytoecdysteroids by examining the biological activity of ecdysterone in a number of plant growth bioassays. 2. MATERIALS AND METHODS 2.1. PLANT MATERIAL Plant material used for extraction and isolation of ecdysterone was collected, weighed (fresh weight) and stored at -80°C. Al l material was frozen within 24 h of collection. Table III shows the date and location of collection for each species tested. Plant material was collected by the author unless otherwise indicated. Plant material for plant growth bioassays is described in Section 2-5, this Chapter. Table III. Dates and Locations of Plant Collections Species Date Location Aspidotis densa 9/7/84 Nanoose Hi l l , V.I. Cryptogramma crispa 18/7/84 Yale, B.C. Pityrogramma triangularis 9/7/84 Nanoose Hi l l , V.I. Adiantum pedatum 10/6/84 Squamish, B.C. Thelypteris phegopteris 2/9/84 Mission, B.C.* Athyrium filix-femina 18/6/84 Bowen Is.,B.C. Cystopteris fragilis 18/7/84 Yale, B.C. Dryopteris arguta - 18/6/84 Bowen Is., B.C. Polystichum munitum 18/6/84 Bowen Is., B.C. Blechnum spicant 18/6/84 Bowen Is., B.C. Polypodium glycyrrhiza 18/6/84 Bowen Is., B.C. Taxus baccata 20/7/85 U B C E L 27 M A T E R I A L S A N D M E T H O D S / 28 Taxus brevifolia 29/7/85 Taxus canadensis 30/6/85 Trillium cernuum 28/5/86 Trillium ouatum 1/6/86 Zebrina pendula 2/1/85 Tradescantia virginiana 6/1/85 Murdannia scapiflora 9/1/86 * collected by Dr. W.B. Schofield * * collected by Dr. W. Maas * * * collected by Dr. G.H.N. Towers Voucher specimens were placed in the U.B.C. Herbarium. Bot. Gdns., U B C Cambridge, N.S.* Kemptown, N .S . * * Victoria, B.C. U B C Bot. Greenhouse UBC Bot. Greenhouse Thai land*** 2.2. S O L V E N T E X T R A C T I O N A number of solvent extraction procedures were tested (Sauer et al, 1968; Morgan and Poole, 1976a; Hikino, 1981; Kubo and Klocke, 1983). Fig. 8 outlines the procedure that was ultimately used for all extracts. Crude extracts obtained from solvent partitioning procedures were freeze-dried, weighed and stored at - 1 0 ° C . MATERIALS AND METHODS / 29 HOMOGENIZE IN MEOH I 60 % AQUEOUS MEOH PET, ETHER EVAPORATE FREEZE-DRY SEP-PAK •HPLC SEPHADEX -(NMR), MS Fig. 8. Extraction and isolation of ecdysterone from plants. M A T E R I A L S A N D METHODS / 30 2.3. CHROMATOGRAPHY 2.3.1. Thin Layer Chromatography (TLC) Silica gel 60 G254, 0.2 mm precoated TLC plates (Merck) were used, with chloroform:95%ethanol (4:1) as the solvent system. Ecdysterone was visualized as a dark spot under U V or by spraying with vanillin-H 2 SO « reagent (Stahl, 1962). Plates were sprayed with a mixture of 1.5 g vanillin and 1 g concentrated H 2 S O t t in 50 ml 95% ethanol, then heated to 110°C for 5 min. Preparative TLC was carried out using a Chromatotron (Harrison Research Associates). This is a spinning T L C plate coated with silica gel 60 GF254 (thickness 2 mm). Fractions were eluted in chloroform:95%ethanol (4:1) at a flow rate of 4 ml min" 1 . The sample (1 mg ecdysterone) was applied in 2 ml of the solvent system. In order to test recovery from T L C , a band containing a known amount of ecdysterone (Sigma) was scraped off and eluted by centrifugation in MeOH (3 times, 15 min each). The concentration of ecdysterone in the combined supernatants was determined by H P L C . 2.3.2. Column Chromatography Silica gel columns (Si gel G60; Merck) were eluted with C H C l 3 : M e O H (80:20) in 20 ml fractions. M A T E R I A L S A N D M E T H O D S / 3 1 X A D - 4 A m b e r l i t e r e s i n w a s p r e w a s h e d i n ho t w a t e r a n d ho t M e O H . F i f t y m g Taxus brevifolia f r eeze -d r i ed e x t r a c t w a s app l i ed to the c o l u m n ( c o l u m n v o l u m e = 1 5 m l ) a n d e lu ted i n a g r a d i e n t of i n c r e a s i n g c o n c e n t r a t i o n s o f a q u e o u s M e O H (45 m l 3 0 % ; 30 m l 5 0 % ; 3 0 m l 7 0 % ; 3 0 m l 100%) . E l u e n t w a s co l lec ted i n ten 15 m l f r ac t i ons w h i c h w e r e s u b s e q u e n t l y e v a p o r a t e d to d r y n e s s , r e d i s s o l v e d i n M e O H a n d s c r e e n e d on T L C . S e p h a d e x L H 2 0 ( P h a r m a c i a ) w a s p r e s o a k e d i n M e O H . T w o h u n d r e d m g Aspidotis densa f r eeze -d r i ed e x t r a c t w a s app l i ed to the c o l u m n ( length 4 5 c m ; in t . d i a . 2 c m ) . T h e c o l u m n w a s r u n a t a f l ow r a t e of 5 m l / h r and 5 m l f r a c t i o n s w e r e co l l ec ted . A l l f r ac t i ons w e r e e v a p o r a t e d , r e d i s s o l v e d i n 2 0 0 n\ M e O H a n d spo t ted on T L C . 2.3.3. High Performance Liquid Chromatography (HPLC) H P L C w a s p e r f o r m e d o n a V a r i a n M o d e l 5 0 0 0 H P L C w i t h V a r i a n S e r i e s 6 3 4 v a r i a b l e w a v e l e n g t h de tec to r , u s i n g a V a r i a n M C H 1 0 R e v e r s e P h a s e c o l u m n . E c d y s t e r o n e w a s de tec ted b y U V a b s o r b a n c e a t 2 4 3 n m , a n d w a s e lu ted i n 2 0 % a c e t o n i t r i l e / H 2 O ( i soc ra t i c ; f l o w r a t e 1 m l m i n " 1 ) . C r u d e p l a n t e x t r a c t s w e r e p a r t i a l l y p u r i f i e d p r i o r to in jec t ion on H P L C b y the u s e of C 1 8 r e v e r s e p h a s e c a r t r i d g e s ( S e p P a k , W a t e r s A s s o c i a t e s ) . T h e s a m p l e w a s a p p l i e d to a S e p P a k i n 1 0 0 / i l 1 0 % a q u e o u s m e t h a n o l ( m a x i m u m 1 m g s a m p l e p e r ca r t r i dge ) a n d s e r i a l l y e l u ted w i t h 2 m l 1 0 % aqueous m e t h a n o l , 4 m l 2 0 % a q u e o u s m e t h a n o l , a n d 6 m l 6 0 % a q u e o u s m e t h a n o l (Rees a n d I s a a c , 1979 ) . T h e 6 0 % e lua te w a s e v a p o r a t e d a n d r e d i s s o l v e d i n 2 0 0 p\ M e O H . 2 0 p\ s a m p l e s w e r e r u n o n H P L C w i t h a n d MATERIALS AND METHODS / 32 without ecdysterone (Sigma) added. For each plant species examined, the 20% MeOH fraction collected from the SepPak was also tested on HPLC to ensure that all ecdysterone had eluted in the 60% MeOH fraction. 2.3.4. Droplet Countercurrent Chromatography (DCC) DCC is a form of chromatography in which the sample is partitioned between two immiscible liquid phases. Samples were chromatographed by DCC (Tokyo Rikakai Co.) using a solvent system of chloroform:methanol:water (13:7:4 v/v) in the ascending mode (Kubo et al, 1983b). The sample (maximum 2 mg) was injected in 3 ml of a. 1:1 (v/v) mixture of the mobile and stationary phases and eluent was collected in ' 5 ml fractions (35 min/fraction). Use of a UV monitor proved unreliable due to bubble formation of the immiscible solvents in the detection cell, so each fraction was evaporated, redissolved in methanol and optical density (O.D.) measured at 243 nm. 2.3.5. Gas Chromatography (GC) Ecdysterone was derivatized for GC by formation of trimethylsilyl (TMS) ethers. These were prepared and purified on TLC by the method of Bielby et al (1986). 2.4. SPECTROSCOPY M A T E R I A L S A N D METHODS / 33 U V spectroscopy was performed on a Pye Unicam SP8-100 UV/V IS spectrophotometer. Mass spectroscopy was carried out by F. Balza on a Finnigan 1020 GC/MS. 2.5. PLANT GROWTH BIOASSAYS Compounds used in plant growth bioassays were obtained from Sigma Chemical Co. unless otherwise indicated. 24-epibrassinolide was prepared by Dr. B. Abeysakara in the laboratorj7 of Dr. G.H.N. Towers. 2.5.1. Mung Bean Epicotyl Elongation The procedure described by Gregory and Mandava (1982) was used. Mung beans (Phaseolus aureus) were grown in moist vermiculite in a growth chamber at 27°C on a 16 h photoperiod (120 ^Einsteins m~ 2 s e c " 1 ) . Nine- to ten-day-old seedlings with epicotyls 35 to 60 mm long were harvested, their cotyledons removed and hypocotyls cut to 3 cm. Cuttings were placed in vials (5 cuttings per vial) containing 2 ml of test solution or water (Fig. 9) Epicotyl length was measured for each seedling, and from one to five India ink dots were placed on the epicotyl to identify individual seedlings within each vial. Epicotyl length was taken as the distance from the most apical cotyledonary scar MATERIALS AND METHODS / 34 Fig. 9. Material for Elongation of Mung Bean Epicotyls bioassay. M A T E R I A L S A N D M E T H O D S / 35 to the point where the primary leaves join the epicotyl. Test vials contained 1.98 ml distilled water and 20jul ecdysterone (EC) or brassinolide (BR) in methanol. Controls contained distilled water only. E C and BR were tested at concentrations of l O ' 6 , 1 0 " 7 , 1 0 " 8 , 10" 9 , 10 " 1 0 , 1 0 ' 1 1 and 1 0 ' 1 2 M . There were two vials per concentration, each containing five seedlings. Vials were maintained in a growth chamber with a 16 h photoperiod, at 27°C. Distilled water was added to vials if they dried out. A second measurement of epicotyl length was taken after 48 h. 2.5.2. Elongation of Cucumber Hypocotyls This bioassay was based on the methods of Katsumi et al (1965) and Mandava et al (1981). Seeds of Cucumis sativus (Marketer) were aerated in distilled water for 2 h, surface sterilized in 5% hypochlorite for 5 min, rinsed and sewn on wet paper towels. The seedlings were grown in the dark at 21 °C and aerated once a day under green light. After 5 days, when the hypocotyl was approximately 4 cm long, it was cut 5 mm below the concave surface of the hypcotyl hook. The apical segments of cut seedlings were incubated in Petri dishes containing 3 ml of test solution or water, in groups of 5 seedlings per dish (Fig. 10). Petri dishes were kept in a growth chamber under continuous light for 24 h (110 /jEinsteins m" 2 s e c " 1 ) at 22°C *-2°C. Four test solutions were used: 10" 4 M gibberellic acid ( G A 3 ) , 10" " M IAA (indole-3-acetic acid), 10* 5 M ecdysterone and 10" 5 M 24-epibrassinolide. There were three Petri dishes per test solution. After 24 h in continuous light, hypocotyl length was measured and mean elongation calculated for each Petri dish. M A T E R I A L S A N D METHODS / 36 Fig. 10. Material for Elongation of Cucumber Hypocotyls bioassay. M A T E R I A L S A N D METHODS / 37 2.5.3. Promotion of Expansion of Cucumber Cotyledons The procedure of Green and Muir (1978) and Mandava et al (1981) was followed. Seedlings of Cucumis satiuus (Marketer) were dark-grown as described in Section 2.5.2. Under a green safety light, cotyledons of 10-day-old seedlings were excised so that all of the hypocotjd hook was removed. Cotyledon pairs were weighed to the nearest mg in groups of 5 pairs, and floated adaxial side down, on 5 ml of test solution in a Petri dish (5 cotyledon pairs per dish) (Fig. 11). Test compounds were dissolved in a solution of lOmM C a C l 2 and 40mM KC1, pH 6.0. 10" " M solutions of the following compounds were used: 24-epibrassinolide, kinetin, 6-benzylaminopurine (BAP) and ecdysterone. Controls contained buffer only. There were 5 Petri dishes per treatment. After 4 days in a dark growth chamber at 25 °C, cotyledons were drained and weighed as for initial weighing. 2.5.4. Expansion of Dwarf Pea Hooks Dwarf pea seeds (Pisum sativum Laxton's Progress) were soaked in 5% hypochlorite for 15 min, rinsed thoroughly, sown in moist, sterile vermiculite at a depth of 1 cm and grown in complete darkness at room temperature. Seven-day-old seedlings were harvested by the methods of Adamson et al (1968), Sasse et al (1972), and Yopp er al (1981). Hypocotyl hooks were cut as shown in Fig. 12, and measured (at their longest point) to the nearest 0.2 mm using a stage micrometer. Only seedlings in which the scale leaf was passing, or had just passed, over the hook were used. Hook segments were weighed to the MATERIALS AND METHODS / 38 Fig. 11. Materia] for Expansion of Cucumber Cotyledons bioassay. M A T E R I A L S A N D METHODS / 39 ig. 12. Material for Expansion of Dwarf Pea Hypocotyl Hooks bioassay. M A T E R I A L S A N D METHODS / 40 nearest mg in groups of 10, then transferred to a 35 mm Petri dish containing 5 ml of the test solution (Fig 12). Al l procedures were carried out under a green safe-light. Test solutions were made by serial dilution of 10" * M stock solutions of IAA and ecdysterone in unbuffered 2% sucrose (Sasse et al, 1972) (pH 5.9). In order to dissolve test compounds completely, they were first dissolved in a small volume (200 ul) of ethanol. A l l possible combinations of IAA (0, 10" 7 , 10" 6 , and 10" 5 M ) and EC (0, 10" 1 , 10" 6 , and 10 ' 5 M ) were tested. Ten hook segments were floated on each test solution and kept under continuous light (110 ^Einsteins m" 2 s e c - 1 ) at 20°C ( i l ° C ) for 24 h, at which time they were again measured individually, blotted dry and weighed in groups of 10. M A T E R I A L S A N D M E T H O D S / 41 2.5.5. Thiarubrine Production in Tumour Cultures of Chaenactis douglasii Callus cultures from a crown gall tumor line of Chaenactis douglasii (Hook.) H . & A . were transferred to agar plates of SH medium (Schenck and Hildebrandt, 1972) (Appendix 1) without hormones. Experimental plates had either ecdysterone (10.5 |zM) or IAA (50 JIM) added to the medium. Controls contained agar and SH medium only. There were three replicate plates per treatment. Cultures were grown in the dark at 27°C for 4 weeks. For each plate, callus was removed, ground and thoroughly extracted into methanol. The residue was dried 24 h at 75 ° C , then weighed. The methanolic fraction obtained from the callus was diluted to 60% aqueous methanol and extracted twice into petroleum ether. Thiarubrine concentration was determined by measuring O.D. at 490 nm (e o9o=3000; M W = 228) (Cosio et al, 1986). Callus cultures were provided by Dr. E. Cosio/Dr. G . H . N . Towers. 3. RESULTS AND DISCUSSION 3.1. METHODS OF EXTRACTION AND ISOLATION OF ECDYSTERONE FROM PLANT MATERIAL Numerous methods have been published for the extraction and isolation of ecdysteroids from both plants and insects. Many combinations of solvent extraction and chromatography have been used, but the nature of ecdysteroids continues to pose problems. Ecdysterone is not coloured, and does not fluoresce under UV light. It is not distinguishable from other compounds on the basis of its UV spectrum. Absorption by the cyclohexenone B ring produces a spectrum with a single peak at 243 nm (Fig. 13). Many other organic compounds absorb UV strongly at this wavelength. For these reasons, it is difficult to detect ecdysterone in chromatographic systems. In addition, ecdysteroids are strongly polar, water-soluble compounds with solubilities similar to other polar phytochemicals such as glycosides. This limits the efficiency of solvent partitioning in the partial purification of ecdysteroid-containing plant extracts. This section is a discussion of the results of various procedures used in this study, leading to the adoption of a simple technique for semi-quantitative isolation of ecdysterone from fresh or frozen plant material. 42 RESULTS AND DISCUSSION / 43 , . _ | . . :i.vi::.l: -Jks i r -_! — r i. / 1 1 -.- !— : i 1 i 1 1 1 !— ' ' — ! — / • ! j — \ — • • ! : i : r - H - : i = ~ • ; [ l . i 1 i — ••' : j i i ; '; - - : - .-; :-(•- :- r - - ; i , ; U Z Z C ~r~—c :5t T Fig. 13. UV absorption spectrum of ecdysterone. RESULTS AND DISCUSSION / 44 3.1.1. Solvent Extraction A number of solvent extraction procedures were tested (see Methods), with little success, until it became apparent that two groups used diametrically opposing methods in order to extract the same compound. In the final step of their extraction procedure, Morgan and Poole (1976a) partitioned the extract between water and ethyl acetate and discarded the ethyl acetate fraction. On the other hand, Kubo et al (1983a) partition samples between ethyl acetate and water and keep the ethyl acetate fraction. Following this discovery, the solubility properties of pure ecdysterone were tested for a number of solvents, by partitioning followed by TLC of the contents of both phases. All ecdysterone ended up in the aqueous phase when partitioned between equal volumes of water and petroleum ether. When ecdysterone dissolved in water is extracted with ethyl acetate once, most of the ecdysterone remains in the aqueous phase. After six washes with ethyl acetate, approximately equal amounts of the compound are present in both phases. This was repeated three times, with identical results each time. Water and n-butanol were also tested. After one extraction some ecdysterone remains in the water, but after two extractions, it has all moved into the butanol phase. Butanol is difficult to evaporate, and it was concluded that ethyl acetate is not useful in the extraction of ecdysterone. Both butanol and ethyl acetate were, therefore, eliminated from the solvent extraction procedure. Further information on the partitioning behaviour of ecdysterone in mixtures of various solvents can be found in an article by Mamatkhanov et al (1984). RESULTS AND DISCUSSION / 45 It is desirable to use as few extraction steps as possible, since some compound may be lost in each step. The procedure finally adopted was very simple and consists of repeated extraction of a 60% aqueous methanolic extract with petroleum ether only (until the ether is colourless). This crude extract is then freeze-dried. 3.1.2. Chromatography TLC is useful for preliminary detection of ecdysterone in crude plant extracts. Ecdysterone has an Rf value of 0.2 in CHC1 3:95% EtOH (80:20). The compound can be visualized in two ways: 1) absorbance of visible light on silica gel containing a fluorescent indicator (F254), and 2) colour reaction with vanillin-H 2 SOy spray reagent. Ecdysterone appears as a yellowish-brown spot which is distinguishable from other ecdysteroids on the basis of their colour reaction to vanillin-H 2 SO <j. The limit of detection of ecdysterone by these methods was determined as 0.5 jug, which is in agreement with published values (Morgan and Wilson, 1980). Preparative TLC was considered as a means of further purification of crude extracts. Recovery of ecdysterone from silica gel plates was tested using two replicates. 58% of the applied compound was recovered from one replicate, 68% from the other. Percent recovery was deemed too low and too variable for this method to be of use. A second method of preparative TLC was investigated, in which fractions are eluted from a spinning circular TLC plate. One mg of ecdysterone was applied but it eluted over such a large volume of solvent that R E S U L T S A N D DISCUSSION / 46 it was not detectable in any of the fractions collected. Many methods have been published for determination of ecdysteroids by H P L C (Gilgan, 1976; Lafont et al, 1980; Baltaev et al, 1984), including more involved techniques in which ecdysteroids are fluorescently labelled prior to injection on H P L C (Kubo and Komatsu, 1986). The method used in this study was adapted from Lafont et al (1976). Good separations of mixtures of ecdysteroids are obtained using this elution program, as shown in Fig. 14. The retention time for ecdysterone is 9 to 10 min and the detection limit is 60 ng. The best results are obtained when the crude extract is partially purified prior to H P L C , by means of a SepPak C18 cartridge (modified method of Rees and Isaac, 1979). SepPaks are a very convenient means of purification of mixtures of polar compounds. The sample is loaded in a small volume of solvent and eluted in a step-gradient of increasing concentrations of MeOH. Fig. 15 shows the H P L C trace obtained for Aspidotis densa crude extract, following partial purification using a SepPak. Isolation of larger amounts of ecdysterone for spectroscopic analysis (MS, NMR) can be carried out on a Sephadex LH20 column, which separates molecules on the basis of size and, to a lesser extent, polarity. Other chromatographic techniques tested included GC, reverse-phase adsorption chromatography on XAD-4 resin, silica gel column chromatography and droplet countercurrent chromatography. R E S U L T S A N D DISCUSSION / 47 Fig. 14. H P L C trace of a mixture of ecdysteroids. A: ecdysterone; B: ecdysone; C: ponasterone A. INJECT TIME b<e:Zi:Zi /h^:'dj.hs, dl~^«. CtO^io^-P firT-lfj, . 14. 8 *E? .« Fig. 15. H P L C trace of crude extract of A. densa following partial purification by SepPak. Arrow indicates ecdysterone peak. R E S U L T S A N D DISCUSSION / 48 Ecdysterone is non-volatile and must be derivatized for analysis by GC (Morgan and Poole, 1976b; Bielby et al, 1986). Trimethylsilyl ethers are formed at each hydroxyl group, but due to the large number of variously positioned hydroxyl groups (six) in ecdysterone, incomplete silylation often occurs, resulting in the formation of a number of different derivatives. Although this method is used by a number of workers in the field, it was attempted, but discarded for use in this study, for the abovementioned reason. Adsorption chromatography on XAD-2 resin is described by Schooley et al (1972) as "an extremely efficient and simple extraction procedure for ecdysones from plants". A slightly more adsorptive resin (XAD-4) was employed in this study, but ecdysterone was not retained on this material. The compound was present in all fractions collected. This technique may bear further investigation, using XAD-7 or XAD-9 resins. Although silica gel columns are often used in the isolation of ecdysterone, their value is questionable for this type of compound since: 1) its retention time is very long, requiring large volumes of solvent, 2) as demonstrated by the T L C recoverj' tests mentioned earlier, significant amounts of ecdysterone will irreversibly bind to the silica gel, and 3) ecdysterone cannot be visualized on the column nor can it be distinguished from other compounds on the basis of its U V spectrum. Droplet countercurrent chromatography (DCC) has a large sample capacity (up to 2.5 g crude extract per injection) and has been successfully used by Kubo RESULTS AND DISCUSSION / 49 and coworkers in the isolation of mg quantities of ecdysteroids from plant sources (Kubo et al, 1983b; Kubo et al, 1985). Recovery of pure ecdysterone (0.5 mg) injected onto DCC was tested in the present study. DCC is a slow procedure and the compound eluted over fifteen five ml fractions (Fig. 16), 17.5 to 26 h after sample injection. The band was very broad and not very reproducible. In addition, ecdysterone (UV max. 243 nm) could not really be detected spectrophotometrically because the solvent system contains chloroform, which absorbs UV up to 245 nm. Even at 254 nm, absorption traces were erratic due to the presence of bubbles of the mobile phase passing through the stationary phase in the detection cell. For these reasons, DCC was eventually abandoned as a method for purification of phytoecdysterone. 7 -10 20 30 40 SO 60 70 tO FRACTION NUMBER (5 ml / f r ac t ion ) Fig. 16. DCC elution profile of 0.5 mg ecdysterone R E S U L T S A N D DISCUSSION / 50 In summary, although a wide variety of purification methods exist in the literature, many of these are used in the extraction and isolation of ecdysteroids from insects, not plants. Plant extracts contain many' polar compounds which behave in a manner similar to ecdysterone in solvent and chromatographic systems. The purification scheme ultimately selected in this study is straightforward, reproducible and semiquantitative (ie. relative amounts of ecdysterone in different plants can be determined). Fig. 8 outlines this scheme, and relevant physical and chemical data for ecdysterone are shown in Table I. For more accurate analysis of trace quantities of ecdysterone, one should probably turn to radioimmunoassay (RIA) techniques (see Borst and O'Connor (1972; 1974) and O'Connor (1985). Another immunoassay using a chemiluminescent derivative of ecdysone has also been developed (Reum et al, 1984). 3.2. OCCURRENCE OF ECDYSTERONE IN PLANTS SURVEYED Twelve species of ferns, three species of gymnosperms, and five species of angiosperms were examined for the presence of ecdysterone. A detailed summary of the results of this part of the study is given in Table IV. For each species, the presence of ecdysterone was determined by TLC and H P L C . The behaviour of the compound was the same for all species in which ecdysterone is recorded as present, and co-chromatography with commercially obtained ecdysterone resulted in peak enhancement on H P L C . For one species of fern (Aspidotis densa), the ecdysterone peak was isolated on Sephadex LH20 and mass spectrometry Table IV: Occurrence of Ecdysterone In Plants Surveyed DIVISION SUBDIVISION ORDER FAMILY SPECIES PLANT PART EC PREVIOUS REPORTS PterIdophyta PterIda 1es S1nopter1daceae Cryptogrammaceae Gymnogrammaceae Adlantaceae Asp1d1ales Thelypter(daceae Athyrlaceae Aspidotis densa (Brackenr.) Lei 1 Inger. Crypt ogramma crispa(L.)R.Br. Pityrogramma triangnIari s (Kaulf.) Maxon. Adiantum pedatum L . The Iypteris phegopteris (L.) Sloss. Athyrium fi I Ix-femina (L . ) Roth. fronds (s+f ) fronds (s) fronds (s) f ronds fronds fronds fronds NO REPORTS no MH ac t i v i t y no MH act 1v1ty HlkIno et a'(1973) Hlk Ino et aH 1973 ) NO REPORTS slight MH a c t i v i t y Hlklno et a'(1973) (some collection da tes) no MH a c t i v i t y (another species) high MH act1v1ty (other species) H1k1no et a!(1973) Hlklno ef a'(1973) 3 CO r co > co o d CO co o z Cyst opteris frag(!is(L.) Bernh. fronds Aspidlaceae Dryopteris arguta (Kaulf.) Watt . fronds Polysti chum mm i turn (Kaulf.) Presl fronds Blechnales B1echnaceae 0 I e c h n u m spicant(L.) Roth. fronds (s+f) + Polypodla 1es Polypodlaceae PoIypodium gIycyrrhiza D.C.Eat . rhlzomes Spermatophyta Gymnospermae Taxa1es Taxaceae Taxus baccata L. 1 eaves Taxus leaves + + + b r e v I f o l I a Ncitt. EC isolated Htklno & Hlklno (1970) no MH ac t i v i t y Hlklno et *M 1973) slight MH a c t i v i t y Hlklno et a)( 1973) no MH a c t i v i t y Hlklno ef 3/(1973) (another species) EC isolated (other Takemoto et «M1973) spec 1es) EC Isolated from Bergamasco 8 Horn other species (1983) EC Isolated Hoffmelster cf aM 1967) EC Isolated from T. cuspi dat a Sieb 8 Zucc. Ima1 ef al(1967 ) T axus canadensI s Marsh. 1 eaves Anglospermae L i H a l e s LI 11aceae Trt nIum cernuum L. rhizomes E C Isolated from other species Imai et al(19G9) TriI I Ium ovatum Pursh. rh1 zones CommelInales Commelinaceae Zebrina pendula Sch lz l . 1 eaves NO REPORTS TradescantI a v i r g i n I ana L . 1 eaves roots NO REPORTS NO REPORTS E C ecdysterone E D ecdysterold(s) (s) s t e r i l e fronds (f ) f e r t 1 I G fronds + number of "+" no E C detected Murdannia s c a p i f l o r a tubers Indicates re lat ive concentration of EC EC Isolated from another species Hou et al(1980) Pterldophyta c l a s s i f i e d according to the scheme of Plcchl-Serm o111 (1958) H CO f CO > a CO O a CO CO o •z O i CO RESULTS AND DISCUSSION / 54 confirmed the identity of the peak as ecdysterone. Ecdysterone was found in fronds of Aspidotis densa (Brackenr.) Lellinger., Cryptogramma crispa (L.) R.Br., Dryopteris arguta (Kaulf.) Watt, and Blechnum spicant (L.) Roth., and in the rhizomes of Polypodium glycyrrhiza D.C. Eat. These five species belong to five separate families of Pteridophytes (Pichi-Sermolli, 1958). The concentration of ecdysterone in Aspidotis densa is approximately 0.01% FW. There are no previous reports of ecdysteroids or moulting hormone activity in this species of fern, nor has the genus Aspidotis been studied previously. Both sterile and fertile fronds of Cryptogramma crispa contain ecdysterone. This species was previously reported to have no moulting hormone activity in bioassays performed by Hikino et al (1973) using extracts of a large number of species of Japanese ferns. In the same study, extracts of Dryopteris arguta showed slight moulting hormone activity, which concurs with the results of the present study. A small amount of ecdysterone was detected in Dryopteris arguta. Sterile fronds of Blechnum spicant contain >0.001% FW ecdysterone, as determined in this study. Ecdysteroids have been isolated from three other species of Blechnum and moulting hormone activity was observed in a fourth. B. amabile Makino and B. niponicum Makino contain ponasterone A and ecdysterone (Takemoto et al, 1973) and Chong et al (1970) isolated 2-deoxy-20-hydroxyecdysterone (0.01% FW) from Blechnum minus (R.Br.) Ettingsh. Strong moulting hormone activity was observed in B. castaneum L. but extracts R E S U L T S A N D DISCUSSION / 55 of B. orientale L. showed no activity whatsoever (Hikino et al, 1973). The genus Blechnum has a high incidence of ecdysteroid-containing species, with moderate to low concentrations of ecdysteroids. Various members of the Polypodium complex, most notabfy Polypodium vulgare L. contain ecdysteroids, with reports of concentrations as high as 1% of the fresh weight of rhizomes (Jizba et al, 1967; Horn and Bergamasco, 1985). It was therefore expected that P. glycyrrhiza D.C. Eat. from British Columbia might contain high concentrations of the compound, but rhizomes of this species contained relatively low amounts of ecdysterone. For most of the species of ferns examined in which ecdysterone was undetectable, the results correspond with published information on those species or members of the same genus (see Table IV). No ecdysterone was detected in fronds of Pityrogramma triangularis (Kaulf.) Maxon. This genus has not previously been examined. There does not appear, from this study, to be a relationship between presence of ecdysterone and degree of evolutionary advancement in the ferns, but the number of species examined is too low for any meaningful conclusions to be drawn. When Hikino et al (1973) screened 283 families of Japanese ferns for M H activity, they noticed that M H activity was more common in the most evolutionarily advanced families. Ecdysterone may be of use in distinguishing between species of confusing groups such as the Polypodium complex, but further screening of all Polypodium species is required, using more quantitative, sensitive RESULTS AND DISCUSSION / 56 techniques such as RIA. To shed more light on the question of ecdysterone as an herbivore defense agent, it would be interesting to compare insect herbivory on sympatric species of the same genus (eg. Blechnum) which contain a range of concentrations of ecdysteroids. The problem becomes increasingly complex however, when one considers the number of ecdysteroids and ecdysteroid-glycosides which may be present, each with different effects on insects, as well as the other classes of secondarj7 metabolites involved in plant-insect interactions. Several species of plants from groups other than the Pteridophyta were also examined in the course of the study. These were chosen from families in which certain species are known to contain relatively high concentrations of ecdysteroids. In the gymnosperms, members of the genus Taxus, are reported to contain up to 0.002% (fresh weight) ecdysterone in their leaves. The steroid has been isolated from T. baccata L. (Hoffmeister et al, 1967), and T. cuspidata Sieb. and Zucc. (Imai et al, 1967). The former species is native to Europe, N. Africa and W. Asia; the latter to Japan, Korea and Manchuria. T. baccata L. was collected from the UBC Endowment Lands and analyzed for ecdysterone mainly as a means of confirming the validity of the isolation technique. As expected, leaves of this species contained significant quantities of ecdysterone. Two species of North American yew were tested as well: one Western species (7\ brevifolia Nutt.) and one Eastern (T. canadensis Marsh.). These species have not previously been RESULTS AND DISCUSSION / 57 examined with respect to ecdysteroid content. The concentration of ecdysterone is approximately four to five times greater in T. brevifolia than in T. canadensis. T. baccata contains an amount intermediate between the other two species. The highest concentration of ecdysterone known in any plant species to date, occurs in Cyanotis arachnoidae CB. Clarke (Commelinaceae). The dry roots contain 2.9% ecdysterone (Chou and Lu, 1980) and the plant is grown for production of ecdysterone in China, where this compound is used in the silk industry. Another species of the same genus, C. vaga Schult., contains 0.7% (fresh weight) ecdysterone in the leaves (Santos et al, 1970; 1972). Three other genera from the Commelinaceae were examined, based on the possibility that other genera producing large amounts of ecdysterone may be found in this family. No ecdysterone was detected in leaves of Zebrina pendula Schizl., or in leaves or roots of Tradescantia virginiana L. These plants have not previouslj' been examined. One species of the genus Murdannia (Commelinaceae) is reported to contain an ecdysteroid. Polypodine B has been isolated from Murdannia triquetra (Wall.) Bruckn. (Wang et al, 1984). Tubers of Murdannia scapiflora, collected in Thailand, did not contain detectable amounts of ecdysterone in the present study. The number of species examined in this survey is too small for conclusions to be drawn regarding chemotaxonomical relationships with respect to ecdysterone. The compound is so widespread in plants, and present in such a range of concentrations, that it may be of chemotaxonomic significance in the future, when a greater number of species have been examined chemically. The RESULTS AND DISCUSSION / 58 results of this study confirm earlier observations (Bergamasco and Horn, 1983) that ecdysterone will not necessarily occur in more than one genus of an ecdysteroid-containing family, but is often present in varying concentrations in more than one species of the same genus. The findings of this survey are useful as an addition to the growing body of information concerning the occurrence of ecdysterone in plant species. 3.3. BIOLOGICAL ACTIVITY OF ECDYSTERONE IN PLANT TISSUES 3.3.1. Production of Thiarubrine i n Callus Cultures Callus cultures of crown gall tumours from Chaenactis douglasii (Hook.) H. & A. produce thiarubrines — sulfur-containing polyacetylenes with antimicrobial activity (Cosio et al, 1986). Production of secondary metabolites has, in some instances, been enhanced by administration of "elicitor" compounds to plant tissue and cell cultures (Bohm, 1980). The effect of ecdysterone on thiarubrine production in callus cultures of transformed Chaenactis douglasii was tested. The results are shown in Table V. Ecdysterone had no discernible effect on production of thiarubrine in this tissue culture system. RESULTS AND DISCUSSION / 59 Table V. Production of Thiarubrine in Callus Cultures from Transformed Chaenactis douglasii Treatment Control EC IAA Total Thiarubrine* (mg/g dry weight) 4.4 (1.6) 3.7 (0.94) 4.7 (2.3) *Values are means of three replicates. Values in brackets are standard errors. RESULTS AND DISCUSSION / 60 3.3.2. Plant Growth Bioassays Ecdysterone has a widespread distribution in plants, and its role as a defensive compound remains in the realm of speculation. Steroid molecules, as a group, tend to exhibit high biological activity in many organisms (see an article by Karlson (1983): "Why are so many hormones steroids?"), and plants contain a large number of sterols, the functions of which are still a mystery. Very few studies have investigated the physiological activity (if any) of ecdysteroids in plants (see Introduction). These facts, and the structural similarity between ecdysteroids and the potent plant growth substance, brassinolide, prompted the testing of ecdysterone for activity in several plant growth bioassays in which brassinolide exhibits high activity. Two gibberellin assays, one auxin, and one cytokinin assay were carried out. The results of these tests are discussed below. 3.3.2.1. Gibberellin Bioassays Gibberellins are a group of diterpenoid phytohormones which have a range of biological activities in excised tissue from species of the Cucurbitaceae, and in intact plants. Gibberellins and ecdysteroids share a common biosynthetic origin in the mevalonic acid pathway (see Fig. 17), with gibberellins arising from the C15 compound farnesylpyrophosphate, and ecdysteroids arising from addition of an isoprene unit to farnesylpyrophosphate, followed by cyclization and oxidations. Due to some structural similarities to gibberellins, and a common biosynthetic origin, it has often been suggested that the ecdysteroids may play RESULTS AND DISCUSSION / 6 1 MONOTERPENES ( C I O ) SESQUITERPENES ( C 1 5 ) ACETYL COA MEVALONIC ACID ((WA) ISOPRENE (C5) 2 1SOPRENES GERANYLPP FARNESYLPP IPP C20 2 FARNESYLPP DITERPE'IES ( C 2 0 ) I GIQBERELLINS TRITERPE'lES (C30) SQUALENE CHOLESTEROL ECDYSTERONE AND OTHER STEROIDS O H O H I Fig. 1 7 . Major steps in the mevalonic acid pathway, showing branch point to GA 3 and ecdysterone. RESULTS AND DISCUSSION / 62 some gibberellin-like role within plants. This has been tested only twice, with opposing results. Hendrix and Jones (1972) found no activity of ecdysterone in four gibberellin bioassays, whereas Matsuoka et al (1969) had observed stimulation of growth in rice. The effect of ecdysterone on elongation in gibberellin-sensitive plant tissues was re-examined in this study. Ecdysterone was applied to excised and semi-intact tissue in two gibberellin bioassays: 1) elongation of cucumber hypocotyls, and 2) elongation of mung bean epicotyls. Ecdysterone effects were compared with those of 24-epibrassinoIide (Fig. 18). Brassinolide is active in both bioassays (Mandava et al, 1981; Gregory and Mandava, 1982). Fig. 18. 24-epibrassinolide RESULTS AND DISCUSSION / 63 Ecdysterone had no effect on elongation of excised cucumber hypocotyls with cotjdedons attached (Fig. 19). IAA does not promote elongation in this system (Katsumi et al, 1965) but both GA 3 and 24-epibrassinolide do. Elongation of GA 3-treated hypocotyls was approximately seven times greater, and brassinolide-treated hypocotyls elongated four times more, than controls. There was no significant difference between elongation of the controls and the ecdysterone-treated hypocotyls. The results were not as clear-cut for the elongation of mung bean epicotyls. These were epicotyls of semi-intact seedlings (ie. the leaves and the tissue being measured (the epicotyl) were intact, but the hypocot3d was cut to a length of 3 cm as shown in Fig. 9). The initial trial of this experiment indicated that there was a significant effect of ecdysterone concentration on epicotyl elongation. The experiment was repeated a total of four times. In two of the trials, the activity of 24-epibrassinolide was tested as well. The results of the experiment are shown in the graphs for ecdysterone (Fig. 20) and 24-epibrassinolide (Fig. 21). Conclusions are difficult to draw from these results, because although analysis of variance showed a significant effect of ecdysterone concentration on epicotyl elongation (p = 0.05), there was also a significant difference between trials. Comparisons between means of initial length of epicotyls for each trial showed significant differences as well. It might, therefore, be useful to repeat the experiment, germinating a large number of seeds in order to obtain enough seedlings with equal epicotyl lengths (+- 1 mm). It is important to note that the effects of ecdysterone, while statistically significant, are very small and may have no biological significance. Jones and Matloff (1986) have pointed out RESULTS AND DISCUSSION / 64 10-U 3 5-EC 10 -4 BR GA3 IAA 10 -5 10 -5 CONCENTRATION ( M ) Fig. 19. Elongation of cucumber hypocotyls. Values are means of three replicates. Bars indicate standard deviations. Controls (C), ecdysterone (EC), 24-epibrassinolide (BR), gibberellic acid (GA 3), indole-3-acetic acid (IAA). RESULTS AND DISCUSSION / 65 7.0 6.0-E 5.0-5 « .o-3 ? 3.0-2.0-I.O rf, rh rh th rh rh 0 10"12 10* : 1 IO - 1 0 IO'3 IO"8 i o - 7 10"5 Ecnrs-so* :onciiTi«r:oii r n > Fig. 20. Elongation of mung bean epicotyls. Values are means of four trials. Bars represent standard deviations. E S 25.0-21.0-23.0-Z2.0-s 9.0-1.0-7.0-6.0-5.0-4.0-J.0-2.0-1.0-rh rh rh rh 0 10"12 10'1 J IO - 1 3 ID"3 IC"8 IO"7 ID-'-2»-i»ir.asi»:'.K tw.trvr.w, < r i Fig. 21. Elongation of mung bean epicotyls. Values are means of two trials. Bars represent standard deviations. RESULTS AND DISCUSSION / 66 the dangers of assigning biological significance to very slight, but statistically significant, effects. The ANOVA showed a significant, linear effect of ecdysterone concentration on epicotyl elongation, but with a regression coefficient of 0.07. Comparison of means for each concentration showed that only the 10" 6 M treatment had significantly greater elongation than the controls (Scheffe test, p = 0.01). Student-Newman-Keuls procedure, a less strict test (p = 0.05), indicated that epicotyls from the three highest concentrations (10~ 8, 1 0 - 7 , and 10" 6 M) and the lowest concentration (10" 1 2M) were all significantly longer than controls. Maximum elongation of ecdy steroid-treated (10' 6M) seedlings was 1.8 times greater than controls, and application of 24-epibrassinolide at the same concentration resulted in elongation 6.2 times that of controls. In another study (Gregory and Mandava, 1982), GA 3 elicited a 3.7-fold elongation relative to controls in this bioassaj'. It appears that ecdysterone may have some effect on elongation in this system, and it may be worthwhile to repeat the experiment with a more strict size limitation placed on initial length of epicotyls, as previously mentioned. 3.3.2.2. Auxin Bioassay Auxins promote growth by cell extension in particular, types of excised plant tissues. Applied auxin stimulates growth in excised stems, but not intact ones, due to creation of a deficiency of endogenous auxin in excised segments, which does not exist in intact plants. Brassinolide shows strong synergism with auxin in certain auxin bioassays, such as inhibition of opening of etiolated bean RESULTS AND DISCUSSION / 67 hypocotyl hooks (Yopp et al, 1979); elongation of azuki bean epicotyl segments; and expansion of dwarf pea hooks (Yopp et al, 1981). In the expansion of dwarf pea hooks, brassinolide alone did not elicit elongation or fresh weight increase. IAA alone elicited little or no elongation, but did promote lateral expansion, as indicated by an increase in fresh weight of the hook segments (39% greater than controls). When applied simultaneous^, brassinolide and IAA acted in a synergistic manner. A combination of IAA (3 x 10" 6M) with brassinolide (10" 6 M) resulted in elongation 67% greater, and fresh weight increase 78% greater, than those of control segments (Yopp et al, 1981). The effect of brassinolide on the elongation response in this bioassay is not observed unless IAA is added to the system, under which conditions, significant elongation and fresh weight increases occur. Since ecdysterone had exhibited a slight effect on elongation in the mung bean epicotyl bioassay, it seemed possible that it might elicit significant elongation in the presence of IAA in a manner similar to brassinolide. Brassinolide enhances elongation in stem bioassays specific to either IAA or GA 3 (Mandava et al, 1981). In the present study, excised dwarf pea hooks were treated with combinations of ecdysterone and IAA at concentrations of .0, 10" 7, 10" 6, and 10" 5 M. Figs. 22 and 23 show the results for elongation and fresh weight increase respectively. Percent elongation and percent fresh weight increase were calculated as (increase/initial x 100). The data were log-transformed prior to statistical analysis by two-way analysis of variance, because the variance around means increased with the magnitude of the response, thus obscuring possible RESULTS AND DISCUSSION / 68 Fig. 22. Elongation of dwarf pea hypocotyl hooks. Values are means of three trials. RESULTS AND DISCUSSION / 69 . Fig. 23. Fresh weight increase of dwarf pea hypocotyl hooks. Values are means of three trials. RESULTS A N D DISCUSSION / 70 effects. As expected from the results of Yopp et al (1981), IAA had a significant effect on fresh weight increase, but not elongation (p = 0.01). Ecdysterone had a significant, but slight, effect on elongation, but not fresh weight increase (p = 0.05). There was no interaction between IAA and ecdysterone for elongation or fresh weight increase. It is difficult to numerically compare these results with those reported by Yopp et al (1981). They present elongation as the mean final length of hooks (mm), and fresh weight increase as mean final weight (mg). This is not a valid presentation of results, since initial lengths and weights of segments are not at all uniform when prepared according to the methods for this bioassay. It is interesting that G A 3 elicits a slight, but significant increase in elongation in this system, but no increase in fresh weight (Adamson et al, 1968). This response to G A 3 is comparable to the response to ecdysterone observed in the present study. Possible implications of this are discussed in the summary section of this chapter. 3.3.2.3. Cytokinin Bioassay Applied ecdysterone at a concentration of 10" " M had no activity in a cytokinin bioassay of expansion of cucumber (Cucumis sativus) cotyledons. The results of the experiment are shown in Fig. 24. Expansion was measured as percent increase in fresh weight. Ecdysterone-treated cotyledons did not have weight increases significantly different from controls, whereas 6-benzylaminopurine (10 - *M) enhanced expansion 3 times relative to controls and RESULTS AND DISCUSSION / 71 140-L U CO CO LU 120i 100-60-10-20-EC BR BAP KIN TEST SOLUTION ( lO"*1 M ) Fig. 24. Expansion of cucumber cotyledons. Values are means of five replicates. Bars represent standard deviations. Controls (C), ecdysterone (EC), 24-epibrassinolide (BR), 6-benzylaminopurine (BAP), kinetin (KIN). R E S U L T S A N D DISCUSSION / 72 kinetin(10~ 4 M)-treated cotyledons had fresh weight increases over 4 times greater than controls. 24-epibrassinolide (10" 4 M ) was also tested and had no effect on cotyledon expansion. In the same bioassay, Yopp et al (1981) found that this concentration of brassinolide resulted in a significant fresh weight increase 1.5 times greater than that of controls. 6-Benzylaminopurine and kinetin are synthetic c.ytokinins which probably do not occur in plants, but their structures are similar to naturally occurring (in plants) cytokinins such as zeatin. They are all adenine-derived compounds with a purine ring and their structure and biosynthetic origins are different from those of ecdysterone. This is probably the reason that ecdysteroids have not previously been tested in cytokinin bioassays, and may explain their inactivity. 3.3.2.4. Summary Ecdysterone showed no activity in a cytokinin bioassay and in one gibberellin bioassay. In both tests, the response of excised tissue from Cucumus sativus to applied ecdysterone was measured. Elongation and lateral expansion of dwarf pea hypocotyl hooks are elicited by G A 3 (Adamson et al, 1968) and IAA (Yopp et al, 1981) respectively. Ecdysterone had no effect on lateral expansion of hook segments, and no interaction with IAA, but it did elicit a slight increase in elongation (the gibberellin-responsive parameter of this system). Ecdysterone also elicited an increase in elongation in a gibberellin bioassay using semi-intact mung bean epicotyls. Although the effects of ecdysterone were not great, they were statistically RESULTS AND DISCUSSION / 73 significant and it is interesting to note that for the tests in which a response was seen: 1) both are responsive to GA 3, to approximately the same degree as observed for ecdysterone; 2) both are elongation responses; and 3) both involve non-Cucurbitaceous species. The results of this study do not exclude the possibility of a physiological role for ecdysteroids and there is some indication that ecdysterone could have biological activity in plant tissues. It would be interesting to pursue this area using a different approach. Ongoing searches for gibberellin and auxin receptors in plants have demonstrated the presence of high-affinity binding sites for both classes of plant growth regulators (Stoddart, 1986; Libbenga et al, 1986). As outlined in the Introduction, ecdysteroid receptors in Drosophila cells have been the subject of much research. Combining the technologies of these three areas, one could demonstrate the presence or absence of receptors, or at least high-affinity binding sites, for ecdysterone in plant tissues. If these sites exist, it would follow that ecdysterone does have a physiological role in plants which should be studied in greater detail. 4. CONCLUSION A simple, reliable method was developed for semi-quantitative analysis of ecdysterone in plant material. By this procedure, ecdysterone was found in a number of plant species which have not previous!}' been examined with respect to phytoecdysteroids. These include four species of ferns, two species of Taxus, and two species of Trillium. Physiological studies indicated that applied ecdysterone could have biological activity in plant tissues. Future work in this area would best be directed along the following lines. More plant species should be screened for ecdysteroid content, using more sensitive analytical techniques such as immunoassay. Phytoecdysteroids may prove to be ubiquitous in plants at low concentrations. The question of a physiological role for ecdysterone in plants would probably best be answered by searching for ecdysteroid receptors in plant tissues. Much more could be learned regarding the biosynthesis of phytoecdysteroids, and the localization of these compounds in plant tissues and cells. So little is known about this group of compounds, that for now they are still largely regarded as a curiosity of plant biochemistry, and this area of research is full of interesting questions with elusive answers. This study provides some suggestions regarding the types of research on phytoecdysteroids which should be most fruitful. 74 5. BIBLIOGRAPHY Achrem, A.A., LS. Levina and Y.A. Titov. 1973. "Ecdysones - Steroidal Hormones of Insects". Nauka i Technika, Minsk. Adamson, D., V.H.K. Low and H. Adamson. 1968. Transitions between different phases of growth in cells from etiolated pea stems, Jerusalem artichoke tubers and wheat coleoptiles. In: "Biochemistry and Physiology of Growth Substances" (F. Wightman & G. Setterfield, eds.). The Runger Press, Ottawa, pp. 505-520. Arteca, R.N., J.M. Bachmann, J.H. Yopp and N.B. Mandava. 1985. 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USA 75: 6039-6043. 6. APPENDIX 1 S H Growth Medium Stock Solutions (per 100 ml): #1 M g S o , . 7 H 2 0 20.0 g #2 C a C l 2 . 2 H 2 0 20.0 g #3 FeSO„ .7H 2 0 1.5 g + N a 2 E D T A . 2 H 2 0 2.0 Vitamins: Nicotinic acid Thiamine.HCl Pyridoxine.HCl 500 mg 500 mg 50 mg Micronutr ients: M n S O „ . 4 H 2 0 1320 mg H 3 B O 3 600 mg KI 100 mg ZnSO„ .7H 2 0 100 mg C u S O f l . 5 H 2 0 20 mg C o C l 2 . 6 H 2 0 10 mg N a 2 M o O a . 2 H 2 0 10 mg B. Medium: Stock #1 2 ml/1 Stock #2 1 ml/1 Stock #3 1 ml/1 Vitamins 1 ml/1 Micronutrients 1 ml/1 inositol 1.0 g/1 K N O 3 2.5 g/1 N H , H 2 P 0 , 0.3 g/1 sucrose 30 g/1 agar 7.0 g/1 * Adjust pH to 5.7 From: Schenck and Hildebrandt (1972). 85 

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