Control and biological implications of alkaloid synthesis inCinchona seedlings

Phytochemistry,VoL 30, No. 11, pp. 3571-3577,1991 Printed in Great Britain.

0031-9422/91 $3.00+0.00 © 1991PergamonPress pie

CONTROL AND BIOLOGICAL IMPLICATIONS OF ALKALOID SYNTHESIS IN CINCHONA SEEDLINGS ROB J. AERTS,* WIM SNOEIJER, OLGA AERTS-TEERLINK,EDDY VAN DER MEIJDEN'~ and ROB VERPOORTE Department of Pharmacognosy, Center for Bio-Pharmaceutical Sciences, University of Leiden, P.O. Box 9502, 2300 RA Leiden; tDepartment of Population Biology, Research Group Ecology of Plants and Herbivores, University of Leiden, P.O. Box 9516, 2300 RA Leiden, The Netherlands (Received 19 September 1990)

Key Word Index--Cinchona ledgeriana; Rubiaceae; seedlings; alkaloids; autotoxicity; slug-repellency. Abstract--During germination of seeds of Cinchona ledgeriana, a rapid increase in alkaloid content occurs, which stops ca 4 days after the onset of germination. Cinchona alkaloids are derived from the precursor strictosidine, which itself is synthesized by enzymatic condensation of tryptamine and secologanin. When the increase in alkaloid content of the seedlings stops, both types of substrates and the necessary enzymatic activity for strictosidine synthesis are still present in the seedlings, suggesting that different intracellular compartmentation of these compounds prohibits further alkaloid biosynthesis. Indeed, upon homogenization and extraction in buffer of the seedlings, a pronounced increase in the amount of strictosidine was observed. External application of the alkaloids at concentrations higher than present in the seedlings showed autotoxic effects, which may explain why all substrate is not converted into alkaloid in the seedlings. At the concentration actually present in the seedlings, the alkaloids showed strong deterrence of feeding by slugs, indicating that this concentration is sufficient for ecological interactions. Moreover, the metabolic cost of carbon for alkaloid synthesis was estimated to be low.

INTRODUCTION The species from the genus Cinchona (family Rubiaceae) are tropical trees well-known for their production of pharmaceutically important alkaloids, including quinine [1]. The biosynthesis of Cinchona alkaloids is shown in Scheme 1. The first step of the pathway, the enzymatic condensation of tryptamine and secologanin to strictosidine, is common to the synthesis of monoterpenoid indole (and derived) alkaloids, and this step is estimated to play a role in the production of at least 2000 different alkaloid structures from numerous plant species mainly from three plant families: Apocynaceae, Loganiaceae and Rubiaceae. The enzyme catalysing this key step in alkaloid synthesis was named strictosidine synthase (EC 4.3.3.2), and the existence of strictosidine synthase activity has been shown in various plant species, including Cinchona [ 2 4 ] . Tryptamine, which provides the indole moiety of strictosidine and its derived alkaloids, is formed from tryptophan by action of the enzyme tryptophan decarboxylase (EC 4.1.1.28; [5]). Secologanin, which provides the monoterpenoid moiety, is derived from the terpenoid pathway. The steps after strictosidine in the synthesis of Cinchona alkaloids have not all been proven yet; corynantheal, however, is a putative intermediate [6]. Germinating Cinchona seeds proved to be a good system to study the biosynthesis of Cinchona alkaloids [4]. Soon after emergence of the radicles, a rapid increase in alkaloid content of the seedlings starts, reaching a

*Author to whom correspondence should be addressed.

plateau at the moment that the cotyledons emerge out of the seed coats, ca four to five days after the onset of germination [4]. In germinating seeds, the main alkaloids synthesized are the quinoline alkaloids cinchonine and its dihydro derivative. In addition, a small amount of the indole alkaloid quinamine is produced (Scheme 1). Strictosidine synthase activity rises gradually during germination, also reaching a plateau after ca four to five days. At the onset of germination, the first events leading to alkaloid biosynthesis are rapid, transient increases in both the tryptophan content and tryptophan decarboxylase activity of the seedlings, resulting in a rise in the level of tryptamine [4]. Not all tryptamine produced is subsequently converted into alkaloid; rather, when the increase in alkaloid content stops after four to five days of germination, a high tryptamine level is left in the seedlings. Because strictosidine synthase activity is also present, this led to the suggestion that, at this point, the monoterpenoid pathway might be lacking, thereby prohibiting further alkaloid synthesis [4]. We investigated this possibility. Furthermore, we tried, in a first attempt, to gain an insight into the possible ecological function of alkaloid production in Cinchona seedlings. RESULTSAND DISCUSSION Precursor pools for alkaloid biosynthesis in Cinchona seedlings

At the onset of germination of Cinchona seedlings, a rapid increase in alkaloid content commences. After ca four days, this increase reaches a plateau; the seedlings then have produced 800-1000 pmol alkaloid

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Biosynthesis of Cinchona alkaloids.

Scheme 1. Biosynthesis of Cinchona alkaloids.

( ~ 0.4 nmol m g - 1 fr. wt). In the next 72 hr, from day 4 to day 7, there is little further change in alkaloid content of the seedlings [cf. 4] (Fig. 1A and B, bars). Also, the amount of tryptamine, the indolic precursor for alkaloid synthesis, which was synthesized at the onset of germination [4], remains virtually unchanged from day 4 to day 7

(Fig. 1A and B, bars). The alkaloids are present in both green and non-green parts of the seedlings [7]. We examined the precursor pools for alkaloid biosynthesis in the seedlings in more detail (see Experimental). The estimated steady-state pool sizes per s~dling when four- to eight-day-old are shown in Fig. 2~ Besides

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Fig. 1. Alkaloid content in Cinchona seedlings and spontaneous strictosidine formation in seedling extracts. In A and B, both the total alkaloid and the tryptamine content is shown of Cinchona ledgeriana Moens seedlings after four and seven days of germination (Tam: tryptaminc, Alk: alkaloids). The inserts show the spontaneous increases in the amount of strictosidine in seedling homogenates after over-night incubations at 30°. In C, the results are shown of incubations of seedling homogenatcs for 24 and 48 hr at 27°. The increases in the level of strictosidine as compared to the control level in intact seedlings are shown (Inc: incubation, Contr: control).

reported [9]. Finally, the seedlings contain a small amount of tryptophan, from which tryptamine is pro-

alkoloid ( 950 pmol )

Fig. 2. Estimated steady-state pool sizes of precursors and endproducts of alkaloid biosynthesis for days 4-8 Cinchona seedlings. Contents per seedling are shown. The alkaloid pool consists of cinchonine, dihydrocinchonine and quinamine.

tryptamine, both strictosidinc and secologanin are also present. In addition, the seedlings contain the monoterpenoid loganin, which has been shown to b c a specific precursor for alkaloid biosynthesis in Cinchona [8]. Although it has not yet been studied in Cinchona, conversion of loganin into secologanin by plant cells has been

duced. It seems surprisingthat pools of both tryptamine and secologanin exist, since strictosidinesynthase activity, which condenses tryptamine and secologanin to strictosidine,is also present in both green and non-green parts of the seedlings[4, 7]. Moreover, assuming that the loganin pool can be converted into secologanin, even more stdctosidinccould be produced by the seedlings.The,fact that these precursor pools do existin Cinchona seedlings, despite the presence of stnctosidine synthase activity, may indicate that these diverse components of the biosynthetic pathway are differentlycompartmentalized in the cells.This isnot uncommon for alkaloidbiosynthesis: in Catharanthus and Berberis, for instance, an elaborate intracellular compartmentation of enzymes involved in alkaloid synthesis has been reported [10, 11]. To test whether compartmentation prohibits further alkaloid synthesis in germinating Cinchona seedlings, the seedlings were homogenized and extracted with buffer. Subsequently, the crude extracts were simply left overnight at 30 ° with some added gluconic acid-lactone to prevent any synthesized strictosidine from being further processed [12]. The extracted seedlings are indeed able to spontaneously generate a considerable amount of strictosidine: in extracts from both four-and seven-day-old seedlings an increase in the amount of strictosidine of ca 500pmol was found after 15 hr (Fig. 1A and B, inserts). Additionally, when the seedlings were extracted and left with some gluconic acid-lactone at 27 °, the temperature

3574

R.J. AERTSet al.

at which the seedlings grow in the climate room, an increase in the amount of strictosidine of ca 500 pmol was found when after 24 hr the strictosidine levels of the seedling-extracts were compared with those of intact seedlings (Fig. 1C). Upon longer incubation (48 hr), the increase in the amount of strictosidine was to be lower (Fig. 1C), probably because the gluconic acid-lactone was gradually inactivated in time. Concomitant with the increases in strictosidine, decreases in tryptamine were observed (not shown). Furthermore, because incubations of secoioganin with tryptamine in extraction-buffer did not show any formation of strictosidine, it was concluded that the synthesis of strictosidine in the seedling extracts was not just chemical. The data suggest that for the synthesis of strictosidine, loganin had to be converted into secologanin (of. Fig. 2). Thus, the above results show that in fact all constituents for strictosidine synthesis are present in the seedlings, and that upon homogenization and extraction, strictosidine production indeed proceeds. This may indicate a different compartmentation of the constituents for strictosidine synthesis in the cells, although other mechanisms of regulation are also possible. If all precursor pools (Fig. 2) were ultimately to be converted into alkaloid, we estimate that ca twice as much quinoline alkaloid could be produced by the seedlings. We have indications that besides the above mentioned precursors (Fig. 2), also a derivative of corynantheal, the putative intermediate in Cinchona alkaloid biosynthesis, is present in the seedlings (see Experimental). Autotoxic effects of the alkaloids

Evidence indicates that alkaloids, like many secondary metabolites, can interfere with the normal germination of plant seeds, thereby establishing a means for ecological interactions between plants from the same or different species [13,14"1. Cinchona alkaloids have been reported to exert inhibitory effects on the germination of other plant species [13]. We investigated whether the alkaloids which are synthesized during germination in Cinchona seedlings, at higher concentrations can influence the normal development of Cinchona seedlings themselves. To test for these autotoxic effects, Cinchona seeds were germinated in the presence of increasing concentrations of a mixture of alkaloids as are present in the seedlings (cinchonine, dihydrocinchonine and quinamine in the ratio 2: 7:1). The influence of the alkaloid mixture on the length of the radicles, the most sensitive growth parameter during germination [14], was monitored. At concentrations above 1 mM, the alkaloids become strongly toxic and prohibit normal germination (Fig. 3). The arrow in Fig. 3 indicates the alkaloid concentration actually found in the seedlings (~0.4 nmol m g - 1 fr. wt), which is remarkably near the toxic concentrations. In other experiments, Cinchona seeds were first germinated for two days (either on soil or on Vermiculite, see Experimental), and only thereafter, when they were synthesizing alkaloids themselves, placed in external contact with the alkaloids. In these experiments also, strong toxic effects at concentrations higher than 1 mM were observed (not shown). These effects are specific and not merely due to changes in ionic strength, because no sign of toxicity was found with sodium chloride at the same concentrations and pH (not shown). Although in the seedlings differentiation between individual cells in alkaloid tolerance can exist, the above

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Fig. 3. The influence of Cinchona alkaloids on germination of Cinchona seeds. Increasing concentrations of a mixture of alkaloids such as present in germinating Cinchona seedlings were externally applied in a weak phosphate buffer to Cinchonaseeds. The mixture of alkaloids consisted of cinchonine, dihydrocinchonine and quinamine in the ratio 2:7:1. After germination, the lengths of the radicles were measured. Means (circles) and standard deviations of about 35 determinations are shown. The arrow indicates the alkaloid concentration in the seedlings themselves. H20: control germination in water. O: control germination in weak phosphate buffer.

shown sensitivity of the embryonic tissues for the alkaloids may explain why the synthesis of these compounds is so carefully dosed. If, as estimated above, from all precursors available twice as much alkaloid would be synthesized by the seedlings, the data indicate that indeed the range of toxic concentrations would be reached (see Fig. 3). In addition to the alkaloids, the precursors tryptamine a n d loganin were also found to be toxic when externally applied at concentrations higher than present in the seedlings (toxic effects at concentrations higher than 1 mM were observed). This implies that limits to the sizes of the precursor pools exist too. The observation that alkaloids can have autotoxic effects during germination has been made for tea seeds as well; the coats of these seeds were found to have an important protective function against the alkaloids which are released by the seeds into the environment, and removal of the coats led to inhibition of germination [15"1. We hypothesize that in Cinchona seedlings autotoxic effects are avoided by careful balancing of alkaloid synthesis and compartmentation. Indeed, we found that in seedlings of ca three-week-old, both the amount of alkaloid and the fresh weight double, resulting in the same average concentration as in seedlings of only a few days old ( ~ 0.4 nmol m g - 1 fr. wt). The question remains why the seedlings produce these alkaloids when they have autotoxic properties and, in addition, cost valuable nitrogen. Slug-repellent properties of the alkaloids

Evidence is rapidly increasing that alkaloids in general can play important roles in the chemical defence of plants against various types of organisms [11, 12]. Alkaloids

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8001 Fig. 4. Feeding of slugs on the first stages of germinating Cinchona seedlings. Both the content of the main alkaloids of germinating Cinchona seedlings and the amount of seedling eaten when offered to Deroceras panormytanum slugs were determined.

often deter predation and, at the most profound level, can interfere with such diverse processes as DNA replication and transcription, protein synthesis and function, and signal transduction [16, 17]. Cinchona alkaloids have been shown to have amoebicidal properties [18], and they can be detected by deterrent receptors in various insect larvae [19]. In their natural habitat, the (relatively small) Cinchona seedlings are probably most subject to predation by soildwelling slugs, and therefore we investigated whether the alkaloids which the seedlings produce might have a protective function against these organisms. For these studies we used Deroceras panormytanum (Lessona and Pollonera, 1882), a typical moist-adapted slug species (see Experimental). In a first series of experiments, we offered germinating Cinchona seedlings of consecutive days old to the slugs, and determined both the alkaloid content in the seedlings and the amount of seedling eaten by the slugs. The gradual increase in alkaloid content during germination is strikingly correlated with a gradual decrease in the amount of seedling eaten by the slugs (Fig. 4). To test whether deterrence of feeding was really due to the presence of the alkaloids, solutions of increasing alkaloid concentration were top-applied to pieces of plant material which served as food for the slugs in the laboratory. The major alkaloids in Cinchona seedlings, cinchonine and dihydrocinchonine, indeed show strong deterrence of feeding by the slugs (Fig. 5), with halfmaximal effective doses of ca 0.2 raM, which is lower than the concentration found in the seedlings after four to five days ( ~ 0.4 nmol m g - 1 fr. wt). The concentration present in the seedlings is sufficient to effectively repel feeding by the slugs (Figs 4 and 5). Although we tested only one slug species, these data suggest that alkaloid synthesis in Cinchona seedlings may indeed have an important ecological function. Antigastropodan properties have been reported for alkaloids from other plant species also [20]. It is not uncommon that, as in our test system, plant substances strongly interact with animals from another geographical origin; for instance, insects have been shown to be frequently sensitive to compounds which they will never meet in their own biotope, implying that general mechanisms of perception are involved [19]. Interestingly, at very low concentrations the alkaloids seemed to

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slightly stimulate instead of deter feeding by the slugs (Fig. 5), a phenomenon which was consistently seen in several experiments and which has also been reported for some other plant-animal interactions [11]. To gain an insight into the metabolic costs of alkaloid synthesis in the seedlings, we compared the estimated daily flux of carbon atoms into respiration with that into alkaloid formation in the seedlings (cf. [21]). The carbon

R. J. AERTS et al.

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Table 1. Comparison of daily carbon atom flux into respiration and into alkaloid synthesis in germinating Cinchona seedlings

Respiration (dark) Alkaloid synthesis

02 uptake (nmol seedling- 1 day - 1)

Carbon atom flux (nmol seedling- t day- t)

986

968*

--

4 C in alkaloids ratio C in respiration

0.4%

*Assuming respiration quotient = 1.

a t o m s needed for alkaloid f o r m a t i o n only a m o u n t to 0.4% of the c a r b o n a t o m flux into respiration (Table 1). W e have indications t h a t there is n o t u r n - o v e r of the alkaloids d u r i n g the first days of g e r m i n a t i o n [4], b u t even if this m i g h t occur to some degree, the direct cost of c a r b o n a t o m s for alkaloid p r o d u c t i o n would still be low. Summarizing, o u r d a t a show t h a t in Cinchona seedlings appreciable precursor pools for alkaloid synthesis exist, which are p r o b a b l y n o t exhausted at once because of the potential autotoxicity of the alkaloids. Nevertheless, the c o n c e n t r a t i o n of alkaloid present in the seedlings seems sufficient for ecological interactions at relatively low costs of carbon. EXPERIMENTAL

Growth of seedlings. Seedlings of Cinchona ledoeriana Moens were grown on sterilized soil in garden-frames in a climate room where the temperature was maintained at 27 ° and the humidity at 60% as described previously [4]. For these experiments a fresh batch of seeds, kindly provided by Multiplant Holding B.V. (Maarssen), was used. Determination of alkaloids and their precursors. Determination of alkaloids and precursors was principally as described previously [4]. In short, after freezing, grinding, and extraction of the seedlings (25-60) with buffer [0.2 M Bis-Tris-propane, pH 7.8, 1 mM EDTA; 2/3-1 vols (v/w ft. wt)], the homogenates were centrifuged for 40 min at 3 °. The pellets were extracted twice with EtOH and all extracts were freeze-dried or vacuum centrifuged. The frs were pooled and analysed on three different HPLC systems according to ref. [22] (secologanin), ref. [23] (strictosidine, tryptamine), and ref. [4] (cinchonine, dihydrocinchonine, quinamine, tryptophan, tryptamine, loganin, secologanin). In addition, a slight modification of the last mentioned system was used: the eluent was 80 mM KH2PO, and 50 mM hexylamine adjusted to pH 3.0 with orthophosphoric acid, and 15% (v/v) MeCN plus 0.15% (v/v) THF. Identification of all compounds was both by Rt and UV-spectrum. In addition to the compounds mentioned above, a derivative of corynantheal (either dihydrocorynantheal or corynantheol) was found in the seedlings. Analysis of secoloaanin and loffanin by TLC. Secologanin and loganin were, in addition to HPLC, also identified by TLC. Samples of extract (see above) and reference compound were applied to pre-coated TLC plates [Silicagel 60, F254 (Merck)]. For secologanin analysis, the plates were developed with EtOAc-MeOH-H20-HCO2 H (10:1:2:2) and subsequently sprayed with Schiffreagent. For loganin analysis, the plates were developed with the eluent described above or with CHCI3-MeOH (3:1) and subsequently sprayed with anisaldehyde reagent [24], and heated, during which colour changes followed.

Analysis of loganin by GLC. For qualitative detection of loganin by GLC, the seedling extracts and loganin reference were first treated with a reaction mixt. according to ref. [25] for silylation. After a short incubation at room temp., the samples were analysed by GLC on a capillary column, using a gas chromatograph Packard 436S equipped with FID and connected with a chromatographic data processor. The GLC conditions used were: fused silica column, 10 m x 0.22 mm i.d.; stationary phase, CP-Sil 5CB; film thickness 0.13 #m; oven temperature programmed, isothermal 100° (3 min), 100-290 ° (20°min-l), isothermal 290° (10min); carrier gas, N 2 (0.84mlmin-1); splitting ratio, 1 : 50; injector and detector, 275 °. In the extracts, a peak with the retention time of loganin was identified. Strictosidine formation in seedlinff extracts. Seedlings (day 4 and day 7) were frozen, ground and extracted with buffer [0.2 M Bis-Tris-propane, pH 7.8, 1 mM EDTA; 2/3-1 vols (v/w fr. wt)]. After centrifugation, aliquots of crude extract were incubated in 0.1 M NaH2PO,-NaOH, pH 6.8, 0.1 M o(+)-gluconic acid-6lactone, either overnight at 30° or for 24 or 48 hr at 27 °. The reactions were stopped with CClaCO2H (2.5%), and analysed on a HPLC system as described in ref. [23], with photodiode-array detection (see above). Controls were stopped immediately after extraction and prepared either at the beginning of the overnight incubations or at the end of the 24 and 48 hr, incubations. In addition, control mixt. consisting of comparable amounts of secologanin and tryptamine such as present in the extracts were incubated under the same conditions as described above. Germination inhibition experiments. The germination experiments were performed in the climate room as described above (see growth of seedlings). A mixt. of alkaloids, consisting of cinchonine, dihydrocinchonine and quinamine in the ratio 2:7:1, was dissolved in 3 mM Pi buffer, pH 6.5 in increasing concns. Seeds were placed on tissues soaked with these solns in small, sterile containers. Ca one week after germination, the lengths of the radicles were measured. Alternatively, seeds were first germinated, either on soil or on Agra-Vermiculite, and after 2 days placed on tissues soaked with the alkaloid solutions in sterile dishes; ca one week later the lengths of the radicles were measured. Similar experiments were also performed with tryptamine, loganin or NaC1 instead of the alkaloids. In all experiments, control germinations on H 2 0 and on Pi buffer only were monitored also. Sluff repellency experiments. Deroceras panormytanum (Lessona and Pollonera, 1882) was collected in the surroundings of Leiden. This species originates from Mediterranean territory, but becomes increasingly common in The Netherlands and Belgium. This is the second mention in the Dutch province ZuidHolland [26, 27]. Individual slugs (4-8 per condition) were offered either germinating Cinchona seedlings of consecutive days old or pieces of Taraxaeum officinale Weber with top applied cinchonine or dihydrocinchonine in increasing concentrations dissolved in 10 mM phosphate buffer, pH 6.0. Control of

Alkaloid synthesis in Cinchona seedlings buffer only were also monitored. After 1 day, the amounts eaten were determined. Very small or large specimens were excluded from the experiments. Respiration measurements. The respiration of the seedlings in 20 mM Hepes, pH 6.5, 0.2 mM CaC12 was monitored with an O2-electrode at 25 °. The seedlings were adapted to dark prior to the measurements. Acknowledgements--We wish to thank Dr P. M. Brakefield for suggesting the slug-repellency experiments, and Dr E. Gittenberger for identification of Deroceras panormytanum (Lessona and Pollonera, 1882). Cinchona ledgeriana Moens seeds were kindly provided by Multiplant Holding B.V. (Maarssen). Financial support by Multiplant Holding B.V. (Maarssen) is gratefully acknowledged. REFERENCES

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12. Treimer, J. F. and Zenk, M. H. (1978) Phytochemistry 17, 227. 13. Harborne, J. B. (1988) Introduction to Ecological Biochemistry. Academic Press, London. 14. Wink, M. (1987) in Allelochemicals: Role in Agriculture and Forestry (Waller, G. R., ed.), pp. 524-533. Am. Chem. Soc. Symposium Series no. 330, Washington. 15. Evenari, M. (1949) Bot. Rev. 15, 153. 16. Shilling, D. G. and Yoshikawa, F. (1987) in Allelochemicals: Role in Agriculture and Forestry (Waller, G. R., ed.), pp. 334-342. Am. Chem. Soc. Symposium Series no. 330, Washington. 17. Suzuki, R. and Waller, G. R. (1987) in Allelochemicals: Role in Agriculture and Forestry (Waller, G. R., ed.), pp. 289-294. Am. Chem. Soc. Symposium Series no. 330, Washington. 18. Robinson, T. (1974) Science 184, 430. 19. Robinson, T. (1979) in Herbivores (Rosenthal, G. A. and Janzen, D. H., eds), pp. 413-448. Academic Press, London. 20. Keene, A. T., Harris, A., Phillipson, J. D. and Warhurst, D. C. (1986) Planta Med. 52, 278. 21. Schoonhoven, L. M. (1982) Ent. Exp. Appl. 31, 57. 22. Wink, M. (1984) Z. Naturforsch. 39C, 553. 23. Frischknecht, P. M., Ulmer-Dufek, J. and Baumann, T. W. (1986) Phytochemistry 25, 613. 24. Van der Heijden, R., Lamping, P. J., Out, P. P., Wijnsma, R. and Verpoorte, R. (1987) J. Chromatogr. 396, 287. 25. Pennings, E. J. M., Van den Bosch, R. A., Van der Heijden, R., Stevens, L. H., Duine, J. A. and Verpoorte, R. (1989) Anal. Biochem. 176, 412. 26. Stahl, E. (1967) Diinnschicht-Chromatographie. Ein Laboratoriumshandbuch, p. 817 (no. 15). Springer, Berlin. 27. Inouye, H., Uobe, K., Hirai, M., Masada, Y. and Hashimoto, K. (1976) J. Chromatogr. 118, 201. 28. Gittenberger, E., Bachhuis, W. and Ripken, Th. E. (1970) De landslakken van Nederland. Bibl. van de Kon. Ned. Nat. Hist. Vereniging no. 17, Amsterdam. 29. De Winter, A. J. (1984) Corr. blad van de Ned. Malacologische Vet. 219, 1547.