On the distribution of phytoecdysteroids in plants
L. Dinan*, T. Savchenko and P. Whiting
Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS (United Kingdom), Fax: +44 1392 263 700, e-mail: [email protected]
Received 2 April 2001; received after revision 4 May 2001; accepted 22 May 2001
Abstract. The occurrence and levels of phytoecdysteroidsin the seeds and other parts of plants grown from the seedsof 180 randomly selected plant species were assessed andcompared. Ecdysteroids are frequently detectable inleaves and flowers, but less so in stems, roots and seeds.The seeds of 290 species were assessed for the presence ofhydrolysable ecdysteroid conjugates. Low levels of conju-gates were detected in a significant number of species,large amounts being present only when levels of freeecdysteroids were high. Individual plants of Arabidopsisthaliana were assessed for the presence of phytoecdy-steroids. While plants of this species are generally ecdy-
ficial pharmacological and medicinal applications [5, 6].Consequently, elevation of ecdysteroid levels in cropspecies may provide an effective control method for manyinvertebrate predators without increasing risks for humanor other vertebrate consumers. Most crop species do notcontain detectable levels of ecdysteroids, spinach (Spina-cia oleracea) and quinoa (Chenopodium quinoa) beingnotable exceptions [7–10]. Elevation of ecdysteroidlevels in crop species could be brought about by geneticengineering of the ecdysteroid biosynthetic pathway orits regulation to provide adequate levels of particular phy-toecdysteroid analogues which are not readily detoxifiedeven by polyphagous predators. Some evidence suggeststhat most, if not all, plant species retain the genetic ca-pacity to produce ecdysteroids: the almost erratic distri-bution of ecdysteroid-positive species in the plant worldand the geographical variation in ecdysteroid levels in
steroid negative, individual plants in the population accu-mulate low levels of ecdysteroids. Extracts of seeds of 50“ecdysteroid-negative” species were concentrated andpartially purified to determine if they possess ecdy-steroids at levels below the usual detection levels. Ecdy-steroids were detectable by radioimmunoassay in almostall of these concentrated samples. Thus, all lines of evi-dence point to the conclusion that all species of plantshave the capacity to produce at least low levels of phy-toecdysteroids. This has important implications for theprotection of crop species through enhancing ecdysteroidlevels by breeding/genetic modification strategies.
Ecdysteroids are the steroid hormones of arthropods andprobably of many other invertebrate phyla. In insects,they regulate moulting and metamorphosis and may alsohave roles in reproduction, embryogenesis and diapause[1]. Phytoecdysteroids are analogues of these invertebratesteroid hormones (zooecdysteroids) that occur in a widevariety of plant species [2]. More than 200 analogueshave been isolated to date [3]. Their presumed function isto contribute to the deterrence of invertebrate predators;evidence is gradually accumulating that they are effectivetoxins or antifeedants towards non-adapted insectspecies. However, some polyphagous species are remark-ably tolerant towards ingested phytoecdysteroids [re-viewed in ref. 4]. Phytoecdysteroids are apparently non-toxic to mammals and may even have a number of bene-
* Corresponding author.
some species. Consequently, at a time of considerablepublic sensitivity and resistance towards genetic engi-neering, identifying varieties with elevated ecdysteroidlevels may be possible through traditional selection pro-cedures. Progress in this area is hampered by our limitedknowledge of the biochemistry and biology of phy-toecdysteroids, with only a restricted understanding ofthe biosynthetic pathway(s) in plants and no knowledgeof the regulation of the activity of these pathways [6, 11].A number of facets of the distribution of phytoecdys-teroids within the plant world are enigmatic. Five to sixpercent of all higher plant species accumulate detectablelevels of ecdysteroids [12, 13], but levels vary enor-mously between species and the distribution of ecdys-teroid-containing species is only partly understood [14,15]. When levels in ecdysteroid-containing species arecompared, distributions within the plants vary in species-and developmentally dependent ways. Ecdysteroid pro-files also vary considerably, although 20-hydroxy-ecdysone (fig. 1) and polypodine B (5b, 20-dihydroxy-ecdysone) are the most common major ecdysteroids. Theother analogues (usually occurring as minor components)differ from 20-hydroxyecdysone mainly in the numberand position of oxygen-containing functional groups orin the nature or positions of attachment of conjugatingmoieties (e.g. glycosides, acyl esters) [16]. Phytoecdys-teroids thus provide a rich diversity of analogues suitablefor quantitative structure-activity studies in insect sys-tems [e.g. ref. 17]. Most methods which have been ap-plied to the analysis of phytoecdysteroids are effective indetecting a subset of the plethora of possible analogues,but may completely miss others. Generally, approacheshave either used physicochemical properties of the mole-cule (the characteristic UV absorbance at 242 nm in al-cohol which is common to most, but not all, ecdysteroids)or the biological activity based on a bioassay (e.g. theChilo dipping test [12]). We have developed an approachwhich combines assessment of both structural similarityto ecdysone [using radioimmunoassay RIA] and biologi-cal activity (BII bioassay for ecdysteroid agonist activity)[13, 18]. These methods are sensitive and robust, allow-ing assessment of phytoecdysteroid levels in small sam-ples of plant material after simple solvent extraction andwork-up. In this report, we present data from four exper-
iments designed to shed light on the occurrence and dis-tribution of phytoecdysteroids. In the first experiment,the phytoecdysteroid levels in seeds of 180 randomlychosen species were compared to the levels present in thevarious parts of plants grown from those seeds. In the sec-ond experiment, extracts of seeds of 290 species were ex-amined for the presence of ecdysteroid conjugates, and inthe third and fourth experiments, we considered the ques-tion of whether all species are capable of producingecdysteroids, the levels in most species, though, being be-low usual detection levels.
Materials and methods
Plant materialPlants of Arabidopsis thaliana (ecotype Col-0) weregrown in a compost mix of equal parts of vermiculite,perlite, John Innes No. 2 and silver sand. Individualrosette leaves were harvested when the plants were 6weeks old. Seeds of other species were obtained from theSeed Guild (Lanark, UK). Plants were grown in theglasshouses of the University of Exeter. After harvesting,plants were cut into the relevant sections, fresh weightswere determined and the material was then freeze-driedfor 4 days prior to extraction.
Extraction procedureSeeds and freeze-dried plant samples were crushed in apestle and mortar and a portion (25 mg) was extracted asdescribed elsewhere [13]. Briefly, the sample was ex-tracted with methanol (3 ¥ 1 ml, each for 3 h at 55 °C).The pooled extracts were mixed with water (1.3 ml) andpartitioned against hexane (2 ml). The aqueous methanolphase (4.3 ml) was used for ecdysteroid analysis.
Phytoecdysteroid analysis and quantificationDetails of the ecdysteroid-specific radioimmunoassaysand the BII bioassay are given elsewhere [13, 18–20]. Thetwo antisera used differ in their specificities for ecdys-teroids. The relative binding affinities for ecdysone, 20-hydroxyecdysone and polypodine B for the DBL-1 andBlack antisera are 1:1.3:10.8 and 1:68.2:124, respectively[13]. Portions of the aqueous methanol extracts were as-sessed by RIA with the DBL-1 antiserum (2 ¥ 5 ml ex-tract) and Black antiserum (2 ¥ 10 ml extract) and in theagonist and antagonist versions of the BII bioassay (4 ¥20 ml). When a positive response was obtained in initialRIA, the level of RIA-positive material (in mg ecdysoneequivalents/g) was quantified by repeating the RIA withfive known volumes (in duplicate) of the extract, plottingRIA response against volume and determining the gradi-ent of the regression line. Ecdysteroid-containing ex-tracts will give well-fitted straight lines and this proce-dure allows the recognition of extracts giving non-linear
1122 L. Dinan, T. Savchenko and P. Whiting Phytoecdysteroids
Figure 1. Structure of 20-hydroxyecdysone.
responses and deviations arising from interference atlarger sample sizes. In the case of the bioassay, a response(agonistic, antagonistic or cytotoxic) in the initial bioas-say was followed up by reassessing the extract in a seriesof ten-fold dilutions until no further activity was seen. Thethreshold for detection is 344 ng ecdysone equivalents/gwith the DBL-1 antiserum, 688 ng ecdysone equiva-lents/g with the Black antiserum and 1 mg 20-hydroxy-ecdysone equivalents/g in the agonist version of the BII
bioassay. To test for the presence of hydrolysable ecdy-steroid conjugates [18, 21], portions (2 ¥ 100 ml) of eachaqueous methanol phase were evaporated in Eppendorfvials. To one portion (control), 100 ml 0.1 M sodiumacetate buffer (pH 5.4) was added. To the other portion(hydrolysate), Helix pomatia hydrolases (Sigma, TypeH1; 10 mg/ml sodium acetate buffer) were added. After48 h at 37 °C, 0.8 ml ethanol was added and, after storingat 6 °C for several hours, the precipitated protein wasremoved by centrifugation (13,000 ¥ g). The supernatantwas used to quantify ecdysteroids by RIA (using theDBL-1 antiserum).
Ecdysteroids in individual plants of A. thalianaSingle leaves were removed from the rosettes of 334 in-dividual (pre-flowering stage) plants of A. thaliana andeach was placed in a 2 ml screw-top Eppendorf vial andfreeze-dried for 4 days. Methanol (1 ml) was added toeach vial and the leaves were extracted for 3 ¥ 3 h at55 °C. Aliquots (10 ml) of each extract were assessed induplicate by RIA (with the DBL-1 antiserum). Extractsgiving a positive response were reassessed at five aliquotsizes in duplicate.
Further purification of ‘ecdysteroid-negative’extractsFor 50 of the seed extracts from species which wereecdysteroid-negative (listed in table 3), a portion (2.5 mlfrom 4.3 ml) of the 70% aqueous methanol phase was di-luted to 10% methanol in water and applied to a pre-equi-librated C18 SPE column (Waters/Millipore, Watford,UK). The column was washed with 25% methanol in wa-ter (5 ml) and any ecdysteroids eluted with 75%methanol in water (5 ml). The cartridge was finally elutedwith methanol (5 ml). The 75% methanol fraction wasdried down and redissolved in 250 ml methanol and 5, 10,25 and 50 ml aliquots were taken in duplicate for RIA.Thus, the extract was concentrated ten-fold and the ma-ximum volume taken for RIA (DBL-1 antiserum) was in-creased ten-fold over the usual assay, thus increasing thesensitivity 100-fold and reducing the detection limit to3.4 ng ecdysone equivalents/g. New glassware and plas-ticware were used throughout the above experimentalprocedure to obviate the possibility of contamination.The residues from solvents used in these procedures wereall RIA-negative.
Results and Discussion
Randomness of the plant speciesThe seeds from which the plants were grown were pro-vided from the catalogue of the seed supplier without se-lection. In total, the seeds of 408 species were sown, ofwhich 180 germinated within 1 year. This is described asa random selection of species, but we recognise that it issubject to some bias towards species which are horticul-turally favoured and to those with shorter germinationtimes in a glasshouse environment.
Distribution of ecdysteroids in plantsTable 1 presents the data obtained by RIA with the DBL-1 and Black antisera and the agonist version of the BII
bioassay. However, each sample was also assessed in theantagonist version of the BII bioassay. A few speciesshowed antagonistic activity, but since all the pure phy-toecdysteroids which have been tested in the BII bioassay(over 100) [17, 19, 22] are agonistic, antagonistic activi-ties will not be discussed further here (this information isavailable from the first-named author on request). Gener-ally, there is excellent agreement between the data ob-tained with the Black and DBL-1 antisera and the agonistbioassay, taking into account the lower sensitivities of theRIA with the Black antiserum and the BII bioassay.RIA-positive material was detected in 16 of the seed ex-tracts (8.9%), of which 4 (2.2%) had levels above 1 mgecdysone eq./g with either the DBL-1 or Black antisera.These percentages are similar to those found earlier for alarger number of randomly selected seed extracts usingthe same methodology [13]. Many more leaf extracts ofthe species were found to be RIA positive (48%), butonly leaf extracts of the 4 species for which the seedswere highly positive (Eryngium maritimum, Helleborusfoetidissima, Limonium ramosissimum, Silene pusilla)contained levels above 1 mg ecdysone eq/g. Thus, theleaves of many species appear to contain detectableecdysteroid levels. The proportions of species with RIA-positive material in the stems (16%), roots (17%) andflowers (33%) were intermediate between the seeds andleaves. Within ecdysteroid-containing species the highestlevels may be present in the roots (E. maritimum, Incar-villea forestii, L. ramosissimum, S. pusilla), stems (H.foetidissima, Pittosporum tenuifolium) or seeds (Hiera-cium murorum). We and others [14, 15] have used seedextracts to assess plant species for the occurrence ofecdysteroids. However, the levels in seeds may be low incomparison to other parts of the plant (E. maritimum, H.foetidissima, S. pusilla) or much higher than in otherparts of the plant (H. murorum). Thus, one must concludethat there is no consistent pattern in the distribution ofecdysteroids within plants, unlike, for example, the dis-tribution of withanolides in solanaceous species whichpredominantly occur in leaves [23]. However, it can be
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1124 L. Dinan, T. Savchenko and P. Whiting Phytoecdysteroids
Table 1. Distribution of ecdysteroids (determined by RIA and BII bioassay) in randomly selected plant species.
Species a Familyb Seeds Ecdysteroid content (RIA [DBL-1]/RIA [BL]/agonist bioassay)c
said that the majority of plants which accumulate low le-vels of ecdysteroids will have the highest levels in theleaves and those which accumulate high levels of ecdy-steroids will contain significant levels in all organs.Since ecdysteroids are detected in species across a widerange of monocotyledonous and dicotyledonous families,the ability to synthesise phytoecdysteroids is apparently ageneral property of plants. This conclusion is also sup-ported by the observation that ‘ecdysteroid-negative’ Zeamays can convert [2-14C]mevalonic acid and [4-14C]cho-lesterol into radiolabelled 20-hydroxyecdysone andecdysone [24].
Occurrence of ecdysteroid conjugatesEcdysteroid conjugates are common in insect extracts,where their presence is usually qualitatively or quantita-tively assessed [25]. A number of ecdysteroid conjugateshave been isolated from plant sources [3], but their oc-currence has not been systematically assessed. Seed ex-tracts of 290 randomly selected species were assessed forthe presence of ecdysteroid conjugates by incubation ofthe extract with a mixture of hydrolases from the gutjuices of H. pomatia (table 2). The majority of species re-mained ecdysteroid-negative after incubation of the seedextracts with the hydrolases, demonstrating that the oc-currence of covert conjugates is not a common feature ofseed extracts. Further, where significant levels of conju-gates were detected, it is in those species (e.g. H. foe-tidissima, H. murorum, S. pusilla, Veronica urticifolia)where significant levels of ecdysteroids were detectedprior to hydrolysis. However, from the few studies whichhave specifically tested for phytoecdysteroid conjugates,it is known that they do not always accompany free ecdy-steroids [26]. In a number of extracts with no or low le-
vels of ecdysteroids when incubated without the hydro-lases, slightly higher levels of RIA-positive material weredetected after incubation with the hydrolases, indicatingthat low levels of conjugates are present in the extract.However, with other extracts, the RIA response was re-duced after incubation with the hydrolases. Neitherecdysone nor 20-hydroxyecdysone is affected by incuba-tion with Helix hydrolases [18], but whether other freeecdysteroids could be modified by these enzymes is notknown. Furthermore the high proportion of extracts giv-ing a RIA-response may be a consequence of interferencein the RIA deriving from the large aliquot size taken forassay.
Can all plants produce ecdysteroids?Previous initial assessments of seeds and plant materialof A. thaliana for the presence of phytoecdysteroids hadconcluded that this species, in common with many otherspecies in the Cruciferae, was ecdysteroid-negative [un-published data]. We have now assessed the presence ofecdysteroids in individual leaves of over 300 individualplants. While the vast majority of leaf extracts wereecdysteroid-negative, a few extracts proved ecdysteroid-positive. In the case of these extracts (fig. 2), a good li-near relationship could be demonstrated between the RIAresponse and the volume of extract assayed, indicatingthat the RIA-positive material is ecdysteroidal rather thaninterfering substances, since only compounds structurallyrelated to the antigen will give a linear response. Thus,while A. thaliana is generally ecdysteroid-negative, indi-vidual plants within a population do accumulate ecdy-steroids (ca 200–800 ng ecdysone eq./g fresh weight).Such levels are not regarded as being effective at deter-ring insect predators in themselves [4], but may act syner-
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Table 1. (continued)
Species a Familyb Seeds Ecdysteroid content (RIA [DBL-1]/RIA [BL]/agonist bioassay)c
a Species and families according to Brummitt [31]. Abbreviations C, M and P for species in the Leguminosae refer to the subfamiliesCaesalpinioideae, Mimosoideae and Papilionoideae, respectively.b D, dicotyledonous; M, monocotyledonous.c DBL-1; RIA with the DBL-1 antiserum (mg ecdysone equivalents/g); BL, RIA with the Black antiserum (mg ecdysone equivalents/g);agonist bioassay, activity in agonist version of BII bioassay; -, inactive; +-, active as neat extract; ++-, active as 10-fold dilution; +++, ac-tive at 100-fold dilution; C–, cytotoxic as neat extract.
1128 L. Dinan, T. Savchenko and P. Whiting Phytoecdysteroids
Table 2. Effect of incubation with Helix pomatia hydrolases on thelevels of RIA-positive material in seed extracts. RIA was performedwith the DBL-1 antiserum. Species are allocated to Families ac-cording to [31]: D = dicot, M = monocot.
gistically with other defence chemicals to provide protec-tion.To test further ‘ecdysteroid-negative’ species, extracts of50 species were partially purified and concentrated. RIAwas used to determine whether these samples containeddetectable levels of ecdysteroids. Almost all the samples(48/50) were RIA-positive (table 3) and, further, the re-sponse was linearly related to sample size (fig. 3), indi-cating strongly that the response was a consequence ofthe presence of ecdysteroids. The other two samples ge-nerated non-linear positive responses, which may indi-cate the presence of interfering substances in addition toecdysteroids. The levels present in the majority of theseconcentrated samples were generally in the region of 20–150 ng ecdysone equivalents/g dry weight. Why the ex-
1130 L. Dinan, T. Savchenko and P. Whiting Phytoecdysteroids
RIA was performed with the DBL-1 antiserum. Species are allo-cated to families according to [31]: D = dicot, M = monocot, NL =non-linear response.
Figure 3. Relationship between RIA response and aliquot size forconcentrated extracts of seeds of representative “ecdysteroid-nega-tive” species. Seed extracts (2.5 mL; equivalent to ca. 14.5 mgseeds) were partially purified by reversed-phase solid-phase extrac-tion cartridges and the 75% methanol fraction concentrated to 250mL. Aliquots of this concentrate were assessed by RIA (DBL-1 an-tiserum): (✳ ) Allium albidum, (�) Aconitum vulparia, (¥) Arge-mone mexicana, (�) Bellevalia romana, (+) Agastache foenicu-lum, (�) Alemanchier florida, (✩ ) Chaenorhinum minus and (�)Arabis arenosa.
tract of Anthemis arvensis (1436 ng ecdysone eq./g) wasnot revealed as ecdysteroid-positive in the initial screen-ing and Helix hydrolysis experiment (table 2) is not clear,as the level found after concentration is above the origi-nal detection limit, but presumably partial purificationhas removed substances which interfere in the RIA.
ImplicationsThe finding that individual plants of A. thaliana (Cru-ciferae) may accumulate low levels of ecdysteroids indi-cates that even species within genera, tribes or familieswhich are not known to contain ecdysteroid-containingspecies retain the capacity to synthesise and accumulateecdysteroids (albeit at a low level). Thus, one must con-clude that most (if not all) plants retain this capacity,even if in 95% of species, this pathway is very poorlyactivated. The data presented here raise the ques-tion whether this individual variation in A. thaliana is de-pendent on genetic or environmental factors. However,the well-characterised genome of this species [27, 28]offers distinct advantages to elucidate this question andto study phytoecdysteroid biosynthesis and its regula-tion.The universality of phytoecdysteroid presence in plants issupported by the detection of low levels of ecdysteroidimmunoassay-positive material in concentrated extracts
of ‘ecdysteroid-negative’ species. Ecdysteroids have con-siderable potential for the protection of crop speciesagainst invertebrate predators, or, alternatively, plantsmight be efficient sources of these compounds forpharmaceutical/medical applications [6]. Thus, elevationof phytoecdysteroid levels in ecdysteroid-poor specieswill not require the introduction of genes correspondingto the enzymes of the biosynthetic pathway. Rather, levelscan be altered by altering the regulation of expression ofexisting genes. This is a much more feasible goal, whichcould be achieved by traditional breeding strategies or ge-netic modification.Ecdysteroid levels in S. oleracea are inducible by me-chanical or insect damage of roots [29, 30]. This appearsto be mediated by jasmonate signalling pathways [29].Thus, the application of appropriate elicitors may be afurther way to elevate ecdysteroid levels in crop species.The same situation probably applies to other classes ofsecondary products, including those with direct medici-nal applications, such that elucidation of the regulation ofthe biosynthetic pathways may enable strategies to ele-vate levels in convenient crop species and thus facilitatethe use of plants as ‘cell-factories’ for the many pharma-ceuticals of plant origin.
Acknowledgements. This research was supported by the Biotech-nology and Biological Sciences Research Council of the U.K. andby EU-INTAS (Contract No. 96-1261). Antisera were generouslydonated by Prof. Dr. Jan Koolman (University of Marburg, Ger-many). We thank James Kingdon for the growth and care of theplants.
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