Plant Cell Reports
Plant Cell Reports (1987) 6:410-413
© Springer-Verlag 1987
Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures H.-M. Schumacher 1, H. Gundlach 1, F. Fiedler 2, and M. H. Zenk 1 : Lehrstuhl far Pharmazeutische Biologie der Universit~it Mtinchen, Karlstrasse 29, D-8000 MOnchen 2, Federal Republic of Germany 2 Lehrstuhl far Mikrobiologie der Universitat Mtinchen, Maria-Ward-Strasse 1a, D-8000 Mtinchen 19, Federal Republic of Germany Received September 21, 1987 / Revised version received October 14, 1987 - Communicated by K. Hahlbrock
ABSTRACT Quaternary benzophenanthridine alkaloids (sanguinarine, c h e l e r y t h r i n e , c h e l i r u b i n e , c h e l i l u t i n e and macarpine) are s p e c i f i c a l l y induced by c e l l wall components of Penicillium and Saccharomyces in a colorless s t r a i n of Eschscholtzia californica c e l l suspension cultures. Classical e l i c i t o r s such as t h e Phytophthora megaspema e l i c i t o r are i n a c t i v e . The alkaloid synthesis i s , however, strongly induced by certain polypeptide a n t i b i o t i c s . Out of 190 tested plant species the yeast e l i c i t o r provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from ( S ) - r e t i c u l i n e , is strongly stimulated during the e l i c i t a t i o n process. These r e s u l t s c l e a r l y demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial e l i c i t o r s . ABBREVIATIONS ACC EDTA LS-medium Pmg
= = = =
1-Aminocyclopropane-l-carbonic acid, Ethylenediaminetetraacetic acid, Linsmaier and Skoog medium,
Phytophthora megasperma
INTRODUCTION In 1981 we observed t h a t a callus c u l t u r e p e t r i dish containing a colorless s t r a i n of E. c a l i f o r n i c a had been infected by a P e n i c i l l i u m colony. At the contact zone between the callus and fungus a strong red zone had developed. This color reaction was provoked not only by the l i v i n g fungus, but also by autoclaved mycelium of t h i s microorganism when applied to the callus. The red compound developing under the influence of the fungus was found to be a benzophenanthridine a l k a l o i d . Obviously the e l i c i t a t i o n of isoquinoline a l k a l o i d synthesis by a fungal component had taken place. R e l a t i v e l y few cases have been reported where fungal e l i c i t o r s stimulate alkaloid synthesis ( E i l e r t et a l . , 1984, 1985; Wolters and E i l e r t , 1982; Heinstein, 1985; Funk et a l . , 1987). E l i c i t a t i o n of the production of sanguinarine, a benzophenanthridine a l k a l o i d , by homogenates of the fungus B o t r y t i s have been previously studied by E i l e r t et a1. (1985) using Papaver somniferum c e l l cultures. In order to study the mode of regulation of secondary product biosynthesis in c e l l cultures, the Eschscholtzia system introduced here may prove
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useful, in t h a t most of the enzymes of the r e t i culine-benzophenanthridine pathway are e i t h e r already known (Zenk et a l . , 1985) or are presently under investigation. MATERIAL and METHODS Cell cultures of Eschscholtzia californica Cham. were established more than 10 years ago in t h i s laboratory. A c u l t u r e derived from a single seedling was by c e l l u l a r cloning using UV (Deus-Neumann and Zenk, 1984) and v i s i b l e selection s p l i t into 4 d i f f e r ent c e l l l i n e s . These c e l l lines were distinguished by t h e i r varying contents of benzophenanthridine a l k a l o i d s . Over a period of 6 years these chemically defined d i f f e r e n t c e l l lines proved to be stable. Only t h a t s t r a i n colorless in appearance in v i s i b l e l i g h t is used in t h i s study. The cultures were grown in Linsmaier and Skoog (1965) medium. Suspension cultures were transfered every 7th day and were c u l t i v a t e d at 100 rpm at 23°C under 600 lux incandescent l i g h t . Light had no e f f e c t on the biosynthetic capacity of the c e i l s . Ceils were also grown in 24 well Nunc multi c u l t u r e dishes containing I ml of medium under s t e r i l e conditions at 140 rpm. Ceils were harvested by suction f i l t r a t i o n or c e n t r i fugation, extracted with ethanol / 0 . 1 N HCl at 60°C f o r 2 h. An a l i q u o t of t h i s e x t r a c t was subjected to HPLC chromatography (Nucleosil C18 10 ~m, 4 x 25 mm; l i n e a r programmed gradient; from 0 to 34 min (0 - 47% B); from 34 to 36 min (47 - 65% B); from 36 to 50 min (65 - 76% B); solvent A: 95% H20 / 5% CH3CN / 0.01% H3P04; solvent B: 10% H20 / 90% CH3CN / 0.01% H3P04; flow rate 3 ml/min; detection at 280 nm). Alkaloids were isolated on a preparative scale using a semipreparative HPLC system (Nucleosil C18 10 ~m, 16 x 50 mm column) and the structures v e r i f i e d by MS (Elmode) using a Finnigan-MAT 44S instrument, as well as comparison (TLC toluene : acetone : ethylacetate = 7 : 2 : I ; cyclohexane : diethylamine = 9 : I, and HPLC) with authentic material. The Penicillium fungus was grown in LS-medium. The mycelium was harvested by f i l t r a t i o n , autoclaved and thoroughly washed with water to remove soluble components. For preparation of the yeast e l i c i t o r I kg bakers yeast was suspended in 1.5 1Na c i t r a t e buffer (20 mM; pH 7.5) and autoclaved f o r 60 min. This preparation was centrifuged 20 min at 10 000 x g. To the supernatant was added one volume of ethanol. A f t e r s t i r r i n g over n i g h t , the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n as described, and the p r e c i p i t a t i o n procedure was repeated. The solids
411 isolated in the second p r e c i p i t a t i o n step were freeze dried and subsequently taken up in water at a defined concentration. This crude e l i c i t o r preparation was used in a l l experiments reported here. Further p u r i f i c a t i o n was achieved by d i a l y s i s , gel chromatpgraphy on Sephadex G-75, and chromatography on DEAE c e l l u lose. A f t e r hydrolysis, t h i s b i o l o g i c a l l y active preparation yielded as the only sugar component mannose (by GC-analysis of the acetates). Subcellular vesicles of suspension cultured c e i l s containing the berberine bridge enzyme were isolated according to Amann et a l . (1986) and the enzyme a c t i v i t y measured using [ N - C T 3 ] - ( S ) - r e t i c u l i n e (7 Ci/mM) according to Steffens et a l . (1985). A l l biochemicals used were of highest commercial p u r i t y a v a i l a b l e . The Pmg and A l t e r n a r i a e l i c i t o r preparations were g i f t s of Dr. J. Ebel and Dr. U. Matern, Freiburg, and are g r a t e f u l l y acknowledged.
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RESULTS We have been using c e l l suspension cultures of the genus Eschscholtzia in the past f o r the i s o l a t i o n of s p e c i f i c enzymes involved in benzylisoquinoline biosynthesis (e.g. Rfiffer et a l . , 1981, 1983; Schumacher et a l . , 1983). The a l k a l o i d pattern of E. c a l i f o r n i c a c e l l cultures had been, however, previously investigated ( B e r l i n et a l . , 1983, and l i t e r a t u r e cited therein) and was found to consist of only the dihydro forms of the benzophenanthridine alkaloids, a l l of which are known to be constituents of the Eschs c h o l t z i a plant (Slavik et a l . , 1955; Slavikova et a l . , 1966). Quaternary Eschscholtzia alkaloids were not found to occur in t h e i r c e l l c u l t u r e . The chance observation that the i n f e c t i o n of our colorless s t r a i n of E. c a l i f o r n i c a by P e n i c i l l i u m resulted in the development of a strong red color in the contact zone between the fungus and c a l l u s led to the f i n d i n g t h a t under these conditions a quaternary benzophenanthridine a l k a l o i d is formed. Subsequently i d e n t i f i e d as macarpine in our laboratory (T. Tanahashi, unpublished r e s u l t s ) , t h i s a l k a l o i d was i d e n t i f i e d in Eschscholtzia (Slavikova et a l . , 1966) and i t s structure was l a t e r revised (Takao et a l . , 1981). The a l k a l o i d pattern of the colorless s t r a i n of E. c a l i f o r n i c a showed almost no UV absorbing material except f o r a small peak of macarpine representing a concentration of 5 mg/l or 0.29% dry weight (Fig. IA). This c u l t u r e , however, was e x t r a o r d i n a r i l y strongly induced to produce benzophenanthridine alkaloids by the addition of an e l i c i t o r (Fig. IB). As can be seen in Fig. I , there is a d r a s t i c increase in the concentration of the quaternary a l k a l o i d macarpine and i t s d i h y d r o d e r i v a t i v e under e l i c i t a t i o n . Furthermore, the synthesis of four other quaternary alkaloids: sanguinarine, c h e l e r y t h r i n e , c h e l i r u b i n e and c h e l i l u t i n e , none of which could be observed in the u n e l i c i t e d Eschscholtzia s t r a i n , in e i t h e r the dihydro or oxidized forms, is also induced. In a search f o r a more convenient e l i c i t o r than the c e l l wall of P e n i c i l l i u m , several preparations of b i o t i c e l i c i t o r s ( f o r review see: Brooks and Watson, 1985; Kauss, 1987) were tested in the Eschscholtzia system. The effects of these preparations are shown in Table I. The yeast mannan, termed here as yeast e l i c i t o r , was equally e f f e c t i v e in provoking a l k a l o i d synthesis as was the o r i g i n a l P e n i c i l l i u m c e l l wall preparation; i t stimulated the benzophenanthridine accumulation maximally. S u r p r i s i n g l y none of the classical e l i c i t o r s tested such as the Pmg or c h i t o sane were e f f e c t i v e in the Eschscholtzia system. On the other hand the cyclopolypeptide a n t i b i o t i c s c o l i s t i n and polymyxin B were even more e f f e c t i v e in the induction of a l k a l o i d synthesis than the c e l l wall
,b
io
io
Retention
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so
time
Fig. 1 Changes in the HPLC elution profile of an e x t r a c t of Eschscholtzia c a l i f o r n i c a cells a f t e r addition of a yeast elicitor to t h e culture medium (0.25 mg/ml). A. Elution profile of an e x t r a c t of cells grown in normal LS-medium. B. Elution profile of an e x t r a c t of cells 48 h a f t e r the addition of yeast elicitor to the culture medium, a. sanguinarine, b. c h e l e r y t h r i n e , c. chelirubine, d. c h e l i lutine, e. macarpine, f. dihydromacarpine.
Preparation
Concentration (mg/ml)*
E l i c i t a t i o n of synthesis of b e n z o p h e n a n t h r i d i n e alkaloids** in % of t h a t of t h e yeast elicitor
Penicillium cell walls
0.5
100
Yeast elicitor
0.2
100
Amphotericin B
0.001
Bacit racin
2.0
115
Colistin
0.2
100
Gramicidin S
0.03
25
Nyst atin
0.001
45
Pmg elicitor
0.25
45
0
Poly-L-Lysine
0.5
Poly-L-Ornithine
0.08
35 25
Polymyxin B
0.04
130
Ribonuelease
0.5
35
Skleroglucan elicitor 0.25 50 * C o n c e n t r a t i o n for maximum e l i c i t a t i o n **100% = 92 mg t o t a l alkaloid per litre medium T a b l e 1 E l i c i t a t i o n of b e n z o p h e n a n t h r i d i n e synthesis by d i f f e r e n t biotic and abiotic elicitors in cell c u l t u r e s of E. californiea. The cell suspensions were grown in Nune 24-well multi culture dishes (1 ml suspension per well). The dissolved or suspended elicitor was added in a 200 !al volume. Two days a f t e r t h e addition of elicitor t h e cells were h a r v e s t e d and the alkaloid c o n t e n t was measured.
412 e l i c i t o r s . The most effective of the three antibiotics was polymyxin B where the dose response curve showed 40 ~g/ml cell culture to be optimal. No effect on alkaloid synthesis of the cell suspension cultures was shown by a wide range of concentrations of following compounds: actinomycin, arachidonic acid, capreomycin, chloramphenicol, D-cycloserine, dibutyryl-cAMP, kanamycin, neomycin, palmitoy1-DLcarnitine, p e n i c i l l i n G, phosphatidylinositol, phosphomycin, ristocetin, tetracycline, vanadylsulfate, vancomycin. A11 further experiments were conducted with the yeast e l i c i t o r . Dose response curves using the yeast mannan showed that the alkaloid induction system is satura2 ted at 1.5 mg/ml, half maximal e l i c i t a t i o n being achieved at about 0.1 mg/ml culture medium. The e l i c i t o r was t o t a l l y inactivated bytreatment with sodium periodate which fact indicates that the biological a c t i v i t y is due to the carbohydrate component and not to a peptide impurity. The e l i c i t a t i o n effect both of the yeast e l i c i t o r and the polypeptide antibiotics was abolished by the chelating agent EDTA (4 mM) or by lanthanchloride (4 mM). The EDTA i n h i b i t i o n was reversed by the addition of calcium ions. Omission of calcium ions from the medium also leads to almost no alkaloid accumulation in the presence of e l i c i t o r s . Cell growth under these conditions was not affected. Careful analysis of the magnitude of the e l i c i t a t i o n phenomenon with respect to the growth curve of the cells showed that addition of the e l i c i t o r at day 6 gave a maximal response in alkaloid synthesis. A comparison of the kinetics of alkaloid accumulation of the Eschscholtzia strain with and without addition of yeast e l i c i t o r is shown in Fig.2.
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8
10
12
(days)
Fig. 2 Accumulation of benzophenanthridine alkaloids during ce|1 growth by a cell culture of Eschscho|tzia califolrdca after addition of a yeast elicitor to LSmedium (0.25 mg/ml). The cells were grown in 300 ml Erlenmeyer flasks containing 75 ml medium.
While growth of the c e i l s was not affected by the e l i c i t o r , a clear increase in t o t a l a l k a l o i d production was seen as soon as 6 h a f t e r addition of the e l i c i t o r . In contrast, the benzophenanthridine content of the untreated cultures remained unchanged.
Following a metabolic period of 5 days a f t e r e l i c i t o r addition, the a l k a l o i d content was increased 6 - f o l d as compared to the control. Under the condition of e l i c i t a t i o n , no enhanced formation of ethylene was observed, although s l i g h t l y elevated levels of ACC and malonyl-ACC were measured. In order to t e s t the range of e l i c i t o r e f f i c a c y on plant c e l l cultures, a t o t a l of 190 cultures comprising 25 plant f a m i l i e s were transfered to LS-medium containing agar into which 0.5 mg/ml medium yeast e l i c i t o r had been incorporated. The c a l l i were v i s u a l l y inspected a f t e r a metabolic period of 21 days. Of these cultures, a change in color was observed in 32 cases. (Care was taken not to take any necrotic changes as f a l s e p o s i t i v e s . ) The strongest reactions were observed in a l l 7 species and subspecies of the genus Eschscholtzia tested, as well as in Chelidonium majus, Glaucium rubrum, G. flavum, Papaver somniferum and Corydalis ophiocarpa. In a l l these cases only quaternary benzophenanthridine synthesis was strongly stimulated, which might be due to the screening system (pigmentation) used. In a11 species, there was a drastic increase in quaternary alkaloid production observed (up to |0-fold stimulation). The alkaloid pattern for the species: E. caespitosa, E. californica, E. douglasii, E. glauca, E. 1obbii, E. pulcheIla, and E. tenuifolia showed quite some v a r i a b i l i t y in quality and quantity among these cells. The variation in alkaloid content in these cultures w i l l provide a useful choice of systems with which to perform further biosynthetic studies. The alkaloid pattern of several Berberis species in culture as well as suspensions of Stephania japonica were tested by HPLC. No change in the alkaloid quality or quantity was observed. Benzophenanthridine alkaloids are biosynthetically derived from (S)-reticuline, the central intermediate in isoquinoline biosynthesis (for review see Corde11, 1981), which is transformed to (S)-scouierine by action of the berberine bridge enzyme (Rink and BOhm, 1975; Steffens et at., 1985). This intermediate is then converted to benzophenanthridine alkaloids (Takao et at., 1983). Since the berberine bridge enzyme opens the pathway for benzophenanthridine alkaloid biosynthesis, i t could be expected that this branch point enzyme is induced in E. californica cell cultures under the influence of e l i c i t o r s . This hypothesis was experimentally tested. I f e l i c i t o r was added to a logarithmically growing cell culture and the enzyme level tested, one could observe a drastic increase in berberine bridge enzyme a c t i v i t y 8 h after addition. Enzyme a c t i v i t y peaked 20 h after addition and then gradually decreased (Fig. 3A). No such enzyme induction was observed in the non e l i cited control tissue. The berberine bridge enzyme is not a cytosolic enzyme but rather is exclusively contained in a Golgi-derived vesicle with the density of p = 1.4 g/ml (Amann et at., 1986). I f , as in our case, the berberine bridge enzyme a c t i v i t y is strongly increased by e l i c i t a t i o n , we expected this increase also to be reflected in the increase in a c t i v i t y in the vesicle band upon sucrose density centrifugation of a crude isoosmotic extract. Comparing the berberine bridge a c t i v i t y in the vesicle band from e l i c i t e d and non stimulated E. californica cell cultures, the expected drastic increase in enzyme a c t i v i t y in this band was noted (Fig. 3B). Both of these experiments shown in Fig. 3 demonstrate that the berberine bridge enzyme as well as other enzymes of the benzophenanthridine pathway (data not shown) are specifically induced during the e l i c i t a t i o n process.
413
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Fig. 3 Stimulation of the berberine bridge enzyme activity in cell cultures of Eschscholtzia californica by the addition of yeast elicitor. A. Enzyme activity measured in a cell raw extract after addition of elicitor on the sixth day after inoculation. B. Enzyme activity measured in c y t o solic vesicles purified by a linear sucrose density gradient from 20 to 40% w/w. Fractions (1.6 ml) were collected and assayed for enzyme activity. Elicitor was added 6 days after inoculation; cells were harvested 4 days after the addition of elicitor (0.25 mg/ml).
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Fractions
DISCUSSION
ACKNOWLEDGEMENTS
There is only l i m i t e d information on the stimulation of a l k a l o i d synthesis by fungal e l i c i t o r s in plant c e l l c u l t u r e systems ( E i l e r t et a i . , 1984, 1985; Funk et a l . , 1987). The evidence, however, which has accumulated up to now indicates that fungal c e l l wall preparations stimulate higher plant c e i l s to produce alkaloid phytoalexins which serve t h e i r oecochemical function in c o n t r o l l i n g pathogen attack. These systems are obviously quite s i m i l a r to the well studied f u r a nocoumarin induction systems (Scheel et a I . , 1987). A chance observation, the i n f e c t i o n of a c a l l u s culture by a fungus which resulted in a l k a l o i d synthesis, led us into the study of the e l i c i t a t i o n of benzophenanthridine synthesis. Fungal c e l l wall components and also selected polypeptide a n t i b i o t i c s stimulated c e l l cultures of the genus Eschscholtzia as well as other members of the family Papaveraceae to produce considerable amounts of benzophenant h r i d i n e a l k a l o i d s . Benzophenanthridines, s p e c i f i c a l ly sanguinarine, have a d i s t i n c t a n t i b a c t e r i a l a c t i v i t y (Southard, 1984). Under the influence of the e l i c i t o r s these cultures turn b r i g h t red or brown, while the growth of the suspension c e i l s as determined by increase of d r y w e i g h t was not affected by the e l i c i t o r . The chemically defined preparations (Table I) acting as e l i c i t o r s in the E. c a l i f o r n i c a system were s u r p r i s i n g l y d i f f e r e n t from the e l i c i t o r s of other a l k a l o i d phytoalexins, the furanocoumarin or callose (Kauss, 1987) systems. We succeeded here f o r the f i r s t time in the study of a l k a l o i d phytoalexin synthesis to demonstrate the e l i c i t o r induced change in a c t i v i t y of an important branch point enzyme, the berberine bridge enzyme, which is contained in a s p e c i f i c vesicle e x i s t i n g in the cytoplasm of t e t r a hydroprotoberberine producing plants (Amann et a i . , 1986). Using parsley c e l l cultures and fungal e l i c i t o r s , the molecular biology of the furanocoumarin phytoalexins has been e x c e l l e n t l y studied (Scheel et a I . , 1987). We believe t h a t the £schscholtzia system introduced here could y i e l d a s i m i l a r system f o r the study of e l i c i t o r recognition and molecular biology of alkaloid synthesis. We do, however, not bel~eve, t h a t t h i s e l i c i t o r system could be used to increase the p r o d u c t i v i t y of c e l l cultures f o r i n d u s t r i a l purposes. Experience has shown us t h a t high a l k a l o i d y i e l d i n g s t r a i n s cannot be f u r t h e r stimulated by the addition of e l i c i t o r s . Nevertheless, the study of the e l i c i t a t i o n phenomenon could y i e l d important information as to the a c t i v ation of genes in plant c e l l cultures which normally do not produce secondary products and could eventually c o n t r i b u t e to the use of plant c e i l s or the enzymes thereof in biotechnology.
We thank Dr. T.M. Kutchan f o r her i i n g u i s t i c help in the preparation of t h i s manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 145), and Fonds der Chemischen I n d u s t r i e . REFERENCES Amann M, Wanner G, Zenk MH (1986) Planta 167:310-320 Berlin J, Forche E, Wray V, Hammer J, H6sel W (1983) Z. Naturforschung 38c: 346-352 Brooks RJ, Watson DG (1985) Nat. Prod. Reports 2: 427-461 Cordell GA (1981) Introduction to alkaloids - a biogenetic approach, John Wiley & Sons, New York Deus-Neumann B, Zenk MH (1984) Pianta med. 50: 427-431 E i l e r t U, Ehmke A, Wolters B (1984) PIanta med. 6: 459-532 E i l e r t U, Kurz WGW, Constabel F (1985) d. Plant Physiol. 119:65-76 Funk C, GOgler K, Brodelius P (1987) Phytochemistry 26:401-405 Heinstein P (1985) J. Nat. Products 4 8 : I - 9 Kauss H (1987) Ann. Rev. Plant Physiol. 38:100-127 Linsmaier H, Skoog F (1965) Physiol. Plant 18: 100-127 Rink E, BQhm H (1975) FEBS Lett. 49:396-399 ROffer M, Ei-Shagi H, Zenk MH (1981) FEBS Lett. 129:5-9 ROffer M, Nagakura N, Zenk MH (1983) Pianta med. 49: 131-137 Scheel D, Dangl d, Douglas C, Hauffe KD, Herrmann A, Hoffmann H, Lozoya E, Schulz W, Hahlbrock K (1987) In: von Wettstein D (ed) NATO ASl Series, Plenum Press, in press Schumacher HM, ROffer M, Nagakura N, Zenk MH (1983) Planta med. 48:212-220 Slavik J, Slavikova L (1955) C o l l . Czech. Chem. Commun. 20:27-31 Siavikova L, Slavik J (1966) Coil. Czech. Chem. Commun. 3 1 : 3 3 - 6 2 Southard GL, US patent 4,517,172 Steffens P, Nagakura N, Zenk MH (1985) Phytochemistry 24:2577-2583 Takao N, Kamigauchi M, Sugiura M, Ninomiya I, Miyata O, Naito T (1981) Heterocycles 16:221-225 Takao N, Kamigauchi M, Okada M (1983) Helv. Chim. Acta 66:473-484 Woiters B, E i l e r t U (1982) Z. Naturforschung 37c: 575-583 Zenk MH, ROffer M, Amann M, Deus-Neumann B, Nagakura N (1985) J. Nat. Products 48:725-738
Plant Cell Reports (1987) 6:410-413
© Springer-Verlag 1987
Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures H.-M. Schumacher 1, H. Gundlach 1, F. Fiedler 2, and M. H. Zenk 1 : Lehrstuhl far Pharmazeutische Biologie der Universit~it Mtinchen, Karlstrasse 29, D-8000 MOnchen 2, Federal Republic of Germany 2 Lehrstuhl far Mikrobiologie der Universitat Mtinchen, Maria-Ward-Strasse 1a, D-8000 Mtinchen 19, Federal Republic of Germany Received September 21, 1987 / Revised version received October 14, 1987 - Communicated by K. Hahlbrock
ABSTRACT Quaternary benzophenanthridine alkaloids (sanguinarine, c h e l e r y t h r i n e , c h e l i r u b i n e , c h e l i l u t i n e and macarpine) are s p e c i f i c a l l y induced by c e l l wall components of Penicillium and Saccharomyces in a colorless s t r a i n of Eschscholtzia californica c e l l suspension cultures. Classical e l i c i t o r s such as t h e Phytophthora megaspema e l i c i t o r are i n a c t i v e . The alkaloid synthesis i s , however, strongly induced by certain polypeptide a n t i b i o t i c s . Out of 190 tested plant species the yeast e l i c i t o r provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from ( S ) - r e t i c u l i n e , is strongly stimulated during the e l i c i t a t i o n process. These r e s u l t s c l e a r l y demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial e l i c i t o r s . ABBREVIATIONS ACC EDTA LS-medium Pmg
= = = =
1-Aminocyclopropane-l-carbonic acid, Ethylenediaminetetraacetic acid, Linsmaier and Skoog medium,
Phytophthora megasperma
INTRODUCTION In 1981 we observed t h a t a callus c u l t u r e p e t r i dish containing a colorless s t r a i n of E. c a l i f o r n i c a had been infected by a P e n i c i l l i u m colony. At the contact zone between the callus and fungus a strong red zone had developed. This color reaction was provoked not only by the l i v i n g fungus, but also by autoclaved mycelium of t h i s microorganism when applied to the callus. The red compound developing under the influence of the fungus was found to be a benzophenanthridine a l k a l o i d . Obviously the e l i c i t a t i o n of isoquinoline a l k a l o i d synthesis by a fungal component had taken place. R e l a t i v e l y few cases have been reported where fungal e l i c i t o r s stimulate alkaloid synthesis ( E i l e r t et a l . , 1984, 1985; Wolters and E i l e r t , 1982; Heinstein, 1985; Funk et a l . , 1987). E l i c i t a t i o n of the production of sanguinarine, a benzophenanthridine a l k a l o i d , by homogenates of the fungus B o t r y t i s have been previously studied by E i l e r t et a1. (1985) using Papaver somniferum c e l l cultures. In order to study the mode of regulation of secondary product biosynthesis in c e l l cultures, the Eschscholtzia system introduced here may prove
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useful, in t h a t most of the enzymes of the r e t i culine-benzophenanthridine pathway are e i t h e r already known (Zenk et a l . , 1985) or are presently under investigation. MATERIAL and METHODS Cell cultures of Eschscholtzia californica Cham. were established more than 10 years ago in t h i s laboratory. A c u l t u r e derived from a single seedling was by c e l l u l a r cloning using UV (Deus-Neumann and Zenk, 1984) and v i s i b l e selection s p l i t into 4 d i f f e r ent c e l l l i n e s . These c e l l lines were distinguished by t h e i r varying contents of benzophenanthridine a l k a l o i d s . Over a period of 6 years these chemically defined d i f f e r e n t c e l l lines proved to be stable. Only t h a t s t r a i n colorless in appearance in v i s i b l e l i g h t is used in t h i s study. The cultures were grown in Linsmaier and Skoog (1965) medium. Suspension cultures were transfered every 7th day and were c u l t i v a t e d at 100 rpm at 23°C under 600 lux incandescent l i g h t . Light had no e f f e c t on the biosynthetic capacity of the c e i l s . Ceils were also grown in 24 well Nunc multi c u l t u r e dishes containing I ml of medium under s t e r i l e conditions at 140 rpm. Ceils were harvested by suction f i l t r a t i o n or c e n t r i fugation, extracted with ethanol / 0 . 1 N HCl at 60°C f o r 2 h. An a l i q u o t of t h i s e x t r a c t was subjected to HPLC chromatography (Nucleosil C18 10 ~m, 4 x 25 mm; l i n e a r programmed gradient; from 0 to 34 min (0 - 47% B); from 34 to 36 min (47 - 65% B); from 36 to 50 min (65 - 76% B); solvent A: 95% H20 / 5% CH3CN / 0.01% H3P04; solvent B: 10% H20 / 90% CH3CN / 0.01% H3P04; flow rate 3 ml/min; detection at 280 nm). Alkaloids were isolated on a preparative scale using a semipreparative HPLC system (Nucleosil C18 10 ~m, 16 x 50 mm column) and the structures v e r i f i e d by MS (Elmode) using a Finnigan-MAT 44S instrument, as well as comparison (TLC toluene : acetone : ethylacetate = 7 : 2 : I ; cyclohexane : diethylamine = 9 : I, and HPLC) with authentic material. The Penicillium fungus was grown in LS-medium. The mycelium was harvested by f i l t r a t i o n , autoclaved and thoroughly washed with water to remove soluble components. For preparation of the yeast e l i c i t o r I kg bakers yeast was suspended in 1.5 1Na c i t r a t e buffer (20 mM; pH 7.5) and autoclaved f o r 60 min. This preparation was centrifuged 20 min at 10 000 x g. To the supernatant was added one volume of ethanol. A f t e r s t i r r i n g over n i g h t , the p r e c i p i t a t e was removed by c e n t r i f u g a t i o n as described, and the p r e c i p i t a t i o n procedure was repeated. The solids
411 isolated in the second p r e c i p i t a t i o n step were freeze dried and subsequently taken up in water at a defined concentration. This crude e l i c i t o r preparation was used in a l l experiments reported here. Further p u r i f i c a t i o n was achieved by d i a l y s i s , gel chromatpgraphy on Sephadex G-75, and chromatography on DEAE c e l l u lose. A f t e r hydrolysis, t h i s b i o l o g i c a l l y active preparation yielded as the only sugar component mannose (by GC-analysis of the acetates). Subcellular vesicles of suspension cultured c e i l s containing the berberine bridge enzyme were isolated according to Amann et a l . (1986) and the enzyme a c t i v i t y measured using [ N - C T 3 ] - ( S ) - r e t i c u l i n e (7 Ci/mM) according to Steffens et a l . (1985). A l l biochemicals used were of highest commercial p u r i t y a v a i l a b l e . The Pmg and A l t e r n a r i a e l i c i t o r preparations were g i f t s of Dr. J. Ebel and Dr. U. Matern, Freiburg, and are g r a t e f u l l y acknowledged.
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RESULTS We have been using c e l l suspension cultures of the genus Eschscholtzia in the past f o r the i s o l a t i o n of s p e c i f i c enzymes involved in benzylisoquinoline biosynthesis (e.g. Rfiffer et a l . , 1981, 1983; Schumacher et a l . , 1983). The a l k a l o i d pattern of E. c a l i f o r n i c a c e l l cultures had been, however, previously investigated ( B e r l i n et a l . , 1983, and l i t e r a t u r e cited therein) and was found to consist of only the dihydro forms of the benzophenanthridine alkaloids, a l l of which are known to be constituents of the Eschs c h o l t z i a plant (Slavik et a l . , 1955; Slavikova et a l . , 1966). Quaternary Eschscholtzia alkaloids were not found to occur in t h e i r c e l l c u l t u r e . The chance observation that the i n f e c t i o n of our colorless s t r a i n of E. c a l i f o r n i c a by P e n i c i l l i u m resulted in the development of a strong red color in the contact zone between the fungus and c a l l u s led to the f i n d i n g t h a t under these conditions a quaternary benzophenanthridine a l k a l o i d is formed. Subsequently i d e n t i f i e d as macarpine in our laboratory (T. Tanahashi, unpublished r e s u l t s ) , t h i s a l k a l o i d was i d e n t i f i e d in Eschscholtzia (Slavikova et a l . , 1966) and i t s structure was l a t e r revised (Takao et a l . , 1981). The a l k a l o i d pattern of the colorless s t r a i n of E. c a l i f o r n i c a showed almost no UV absorbing material except f o r a small peak of macarpine representing a concentration of 5 mg/l or 0.29% dry weight (Fig. IA). This c u l t u r e , however, was e x t r a o r d i n a r i l y strongly induced to produce benzophenanthridine alkaloids by the addition of an e l i c i t o r (Fig. IB). As can be seen in Fig. I , there is a d r a s t i c increase in the concentration of the quaternary a l k a l o i d macarpine and i t s d i h y d r o d e r i v a t i v e under e l i c i t a t i o n . Furthermore, the synthesis of four other quaternary alkaloids: sanguinarine, c h e l e r y t h r i n e , c h e l i r u b i n e and c h e l i l u t i n e , none of which could be observed in the u n e l i c i t e d Eschscholtzia s t r a i n , in e i t h e r the dihydro or oxidized forms, is also induced. In a search f o r a more convenient e l i c i t o r than the c e l l wall of P e n i c i l l i u m , several preparations of b i o t i c e l i c i t o r s ( f o r review see: Brooks and Watson, 1985; Kauss, 1987) were tested in the Eschscholtzia system. The effects of these preparations are shown in Table I. The yeast mannan, termed here as yeast e l i c i t o r , was equally e f f e c t i v e in provoking a l k a l o i d synthesis as was the o r i g i n a l P e n i c i l l i u m c e l l wall preparation; i t stimulated the benzophenanthridine accumulation maximally. S u r p r i s i n g l y none of the classical e l i c i t o r s tested such as the Pmg or c h i t o sane were e f f e c t i v e in the Eschscholtzia system. On the other hand the cyclopolypeptide a n t i b i o t i c s c o l i s t i n and polymyxin B were even more e f f e c t i v e in the induction of a l k a l o i d synthesis than the c e l l wall
,b
io
io
Retention
~o
so
time
Fig. 1 Changes in the HPLC elution profile of an e x t r a c t of Eschscholtzia c a l i f o r n i c a cells a f t e r addition of a yeast elicitor to t h e culture medium (0.25 mg/ml). A. Elution profile of an e x t r a c t of cells grown in normal LS-medium. B. Elution profile of an e x t r a c t of cells 48 h a f t e r the addition of yeast elicitor to the culture medium, a. sanguinarine, b. c h e l e r y t h r i n e , c. chelirubine, d. c h e l i lutine, e. macarpine, f. dihydromacarpine.
Preparation
Concentration (mg/ml)*
E l i c i t a t i o n of synthesis of b e n z o p h e n a n t h r i d i n e alkaloids** in % of t h a t of t h e yeast elicitor
Penicillium cell walls
0.5
100
Yeast elicitor
0.2
100
Amphotericin B
0.001
Bacit racin
2.0
115
Colistin
0.2
100
Gramicidin S
0.03
25
Nyst atin
0.001
45
Pmg elicitor
0.25
45
0
Poly-L-Lysine
0.5
Poly-L-Ornithine
0.08
35 25
Polymyxin B
0.04
130
Ribonuelease
0.5
35
Skleroglucan elicitor 0.25 50 * C o n c e n t r a t i o n for maximum e l i c i t a t i o n **100% = 92 mg t o t a l alkaloid per litre medium T a b l e 1 E l i c i t a t i o n of b e n z o p h e n a n t h r i d i n e synthesis by d i f f e r e n t biotic and abiotic elicitors in cell c u l t u r e s of E. californiea. The cell suspensions were grown in Nune 24-well multi culture dishes (1 ml suspension per well). The dissolved or suspended elicitor was added in a 200 !al volume. Two days a f t e r t h e addition of elicitor t h e cells were h a r v e s t e d and the alkaloid c o n t e n t was measured.
412 e l i c i t o r s . The most effective of the three antibiotics was polymyxin B where the dose response curve showed 40 ~g/ml cell culture to be optimal. No effect on alkaloid synthesis of the cell suspension cultures was shown by a wide range of concentrations of following compounds: actinomycin, arachidonic acid, capreomycin, chloramphenicol, D-cycloserine, dibutyryl-cAMP, kanamycin, neomycin, palmitoy1-DLcarnitine, p e n i c i l l i n G, phosphatidylinositol, phosphomycin, ristocetin, tetracycline, vanadylsulfate, vancomycin. A11 further experiments were conducted with the yeast e l i c i t o r . Dose response curves using the yeast mannan showed that the alkaloid induction system is satura2 ted at 1.5 mg/ml, half maximal e l i c i t a t i o n being achieved at about 0.1 mg/ml culture medium. The e l i c i t o r was t o t a l l y inactivated bytreatment with sodium periodate which fact indicates that the biological a c t i v i t y is due to the carbohydrate component and not to a peptide impurity. The e l i c i t a t i o n effect both of the yeast e l i c i t o r and the polypeptide antibiotics was abolished by the chelating agent EDTA (4 mM) or by lanthanchloride (4 mM). The EDTA i n h i b i t i o n was reversed by the addition of calcium ions. Omission of calcium ions from the medium also leads to almost no alkaloid accumulation in the presence of e l i c i t o r s . Cell growth under these conditions was not affected. Careful analysis of the magnitude of the e l i c i t a t i o n phenomenon with respect to the growth curve of the cells showed that addition of the e l i c i t o r at day 6 gave a maximal response in alkaloid synthesis. A comparison of the kinetics of alkaloid accumulation of the Eschscholtzia strain with and without addition of yeast e l i c i t o r is shown in Fig.2.
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8
10
12
(days)
Fig. 2 Accumulation of benzophenanthridine alkaloids during ce|1 growth by a cell culture of Eschscho|tzia califolrdca after addition of a yeast elicitor to LSmedium (0.25 mg/ml). The cells were grown in 300 ml Erlenmeyer flasks containing 75 ml medium.
While growth of the c e i l s was not affected by the e l i c i t o r , a clear increase in t o t a l a l k a l o i d production was seen as soon as 6 h a f t e r addition of the e l i c i t o r . In contrast, the benzophenanthridine content of the untreated cultures remained unchanged.
Following a metabolic period of 5 days a f t e r e l i c i t o r addition, the a l k a l o i d content was increased 6 - f o l d as compared to the control. Under the condition of e l i c i t a t i o n , no enhanced formation of ethylene was observed, although s l i g h t l y elevated levels of ACC and malonyl-ACC were measured. In order to t e s t the range of e l i c i t o r e f f i c a c y on plant c e l l cultures, a t o t a l of 190 cultures comprising 25 plant f a m i l i e s were transfered to LS-medium containing agar into which 0.5 mg/ml medium yeast e l i c i t o r had been incorporated. The c a l l i were v i s u a l l y inspected a f t e r a metabolic period of 21 days. Of these cultures, a change in color was observed in 32 cases. (Care was taken not to take any necrotic changes as f a l s e p o s i t i v e s . ) The strongest reactions were observed in a l l 7 species and subspecies of the genus Eschscholtzia tested, as well as in Chelidonium majus, Glaucium rubrum, G. flavum, Papaver somniferum and Corydalis ophiocarpa. In a l l these cases only quaternary benzophenanthridine synthesis was strongly stimulated, which might be due to the screening system (pigmentation) used. In a11 species, there was a drastic increase in quaternary alkaloid production observed (up to |0-fold stimulation). The alkaloid pattern for the species: E. caespitosa, E. californica, E. douglasii, E. glauca, E. 1obbii, E. pulcheIla, and E. tenuifolia showed quite some v a r i a b i l i t y in quality and quantity among these cells. The variation in alkaloid content in these cultures w i l l provide a useful choice of systems with which to perform further biosynthetic studies. The alkaloid pattern of several Berberis species in culture as well as suspensions of Stephania japonica were tested by HPLC. No change in the alkaloid quality or quantity was observed. Benzophenanthridine alkaloids are biosynthetically derived from (S)-reticuline, the central intermediate in isoquinoline biosynthesis (for review see Corde11, 1981), which is transformed to (S)-scouierine by action of the berberine bridge enzyme (Rink and BOhm, 1975; Steffens et at., 1985). This intermediate is then converted to benzophenanthridine alkaloids (Takao et at., 1983). Since the berberine bridge enzyme opens the pathway for benzophenanthridine alkaloid biosynthesis, i t could be expected that this branch point enzyme is induced in E. californica cell cultures under the influence of e l i c i t o r s . This hypothesis was experimentally tested. I f e l i c i t o r was added to a logarithmically growing cell culture and the enzyme level tested, one could observe a drastic increase in berberine bridge enzyme a c t i v i t y 8 h after addition. Enzyme a c t i v i t y peaked 20 h after addition and then gradually decreased (Fig. 3A). No such enzyme induction was observed in the non e l i cited control tissue. The berberine bridge enzyme is not a cytosolic enzyme but rather is exclusively contained in a Golgi-derived vesicle with the density of p = 1.4 g/ml (Amann et at., 1986). I f , as in our case, the berberine bridge enzyme a c t i v i t y is strongly increased by e l i c i t a t i o n , we expected this increase also to be reflected in the increase in a c t i v i t y in the vesicle band upon sucrose density centrifugation of a crude isoosmotic extract. Comparing the berberine bridge a c t i v i t y in the vesicle band from e l i c i t e d and non stimulated E. californica cell cultures, the expected drastic increase in enzyme a c t i v i t y in this band was noted (Fig. 3B). Both of these experiments shown in Fig. 3 demonstrate that the berberine bridge enzyme as well as other enzymes of the benzophenanthridine pathway (data not shown) are specifically induced during the e l i c i t a t i o n process.
413
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Time
u
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(hours)
50
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Fig. 3 Stimulation of the berberine bridge enzyme activity in cell cultures of Eschscholtzia californica by the addition of yeast elicitor. A. Enzyme activity measured in a cell raw extract after addition of elicitor on the sixth day after inoculation. B. Enzyme activity measured in c y t o solic vesicles purified by a linear sucrose density gradient from 20 to 40% w/w. Fractions (1.6 ml) were collected and assayed for enzyme activity. Elicitor was added 6 days after inoculation; cells were harvested 4 days after the addition of elicitor (0.25 mg/ml).
N>'
Fractions
DISCUSSION
ACKNOWLEDGEMENTS
There is only l i m i t e d information on the stimulation of a l k a l o i d synthesis by fungal e l i c i t o r s in plant c e l l c u l t u r e systems ( E i l e r t et a i . , 1984, 1985; Funk et a l . , 1987). The evidence, however, which has accumulated up to now indicates that fungal c e l l wall preparations stimulate higher plant c e i l s to produce alkaloid phytoalexins which serve t h e i r oecochemical function in c o n t r o l l i n g pathogen attack. These systems are obviously quite s i m i l a r to the well studied f u r a nocoumarin induction systems (Scheel et a I . , 1987). A chance observation, the i n f e c t i o n of a c a l l u s culture by a fungus which resulted in a l k a l o i d synthesis, led us into the study of the e l i c i t a t i o n of benzophenanthridine synthesis. Fungal c e l l wall components and also selected polypeptide a n t i b i o t i c s stimulated c e l l cultures of the genus Eschscholtzia as well as other members of the family Papaveraceae to produce considerable amounts of benzophenant h r i d i n e a l k a l o i d s . Benzophenanthridines, s p e c i f i c a l ly sanguinarine, have a d i s t i n c t a n t i b a c t e r i a l a c t i v i t y (Southard, 1984). Under the influence of the e l i c i t o r s these cultures turn b r i g h t red or brown, while the growth of the suspension c e i l s as determined by increase of d r y w e i g h t was not affected by the e l i c i t o r . The chemically defined preparations (Table I) acting as e l i c i t o r s in the E. c a l i f o r n i c a system were s u r p r i s i n g l y d i f f e r e n t from the e l i c i t o r s of other a l k a l o i d phytoalexins, the furanocoumarin or callose (Kauss, 1987) systems. We succeeded here f o r the f i r s t time in the study of a l k a l o i d phytoalexin synthesis to demonstrate the e l i c i t o r induced change in a c t i v i t y of an important branch point enzyme, the berberine bridge enzyme, which is contained in a s p e c i f i c vesicle e x i s t i n g in the cytoplasm of t e t r a hydroprotoberberine producing plants (Amann et a i . , 1986). Using parsley c e l l cultures and fungal e l i c i t o r s , the molecular biology of the furanocoumarin phytoalexins has been e x c e l l e n t l y studied (Scheel et a I . , 1987). We believe t h a t the £schscholtzia system introduced here could y i e l d a s i m i l a r system f o r the study of e l i c i t o r recognition and molecular biology of alkaloid synthesis. We do, however, not bel~eve, t h a t t h i s e l i c i t o r system could be used to increase the p r o d u c t i v i t y of c e l l cultures f o r i n d u s t r i a l purposes. Experience has shown us t h a t high a l k a l o i d y i e l d i n g s t r a i n s cannot be f u r t h e r stimulated by the addition of e l i c i t o r s . Nevertheless, the study of the e l i c i t a t i o n phenomenon could y i e l d important information as to the a c t i v ation of genes in plant c e l l cultures which normally do not produce secondary products and could eventually c o n t r i b u t e to the use of plant c e i l s or the enzymes thereof in biotechnology.
We thank Dr. T.M. Kutchan f o r her i i n g u i s t i c help in the preparation of t h i s manuscript. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 145), and Fonds der Chemischen I n d u s t r i e . REFERENCES Amann M, Wanner G, Zenk MH (1986) Planta 167:310-320 Berlin J, Forche E, Wray V, Hammer J, H6sel W (1983) Z. Naturforschung 38c: 346-352 Brooks RJ, Watson DG (1985) Nat. Prod. Reports 2: 427-461 Cordell GA (1981) Introduction to alkaloids - a biogenetic approach, John Wiley & Sons, New York Deus-Neumann B, Zenk MH (1984) Pianta med. 50: 427-431 E i l e r t U, Ehmke A, Wolters B (1984) PIanta med. 6: 459-532 E i l e r t U, Kurz WGW, Constabel F (1985) d. Plant Physiol. 119:65-76 Funk C, GOgler K, Brodelius P (1987) Phytochemistry 26:401-405 Heinstein P (1985) J. Nat. Products 4 8 : I - 9 Kauss H (1987) Ann. Rev. Plant Physiol. 38:100-127 Linsmaier H, Skoog F (1965) Physiol. Plant 18: 100-127 Rink E, BQhm H (1975) FEBS Lett. 49:396-399 ROffer M, Ei-Shagi H, Zenk MH (1981) FEBS Lett. 129:5-9 ROffer M, Nagakura N, Zenk MH (1983) Pianta med. 49: 131-137 Scheel D, Dangl d, Douglas C, Hauffe KD, Herrmann A, Hoffmann H, Lozoya E, Schulz W, Hahlbrock K (1987) In: von Wettstein D (ed) NATO ASl Series, Plenum Press, in press Schumacher HM, ROffer M, Nagakura N, Zenk MH (1983) Planta med. 48:212-220 Slavik J, Slavikova L (1955) C o l l . Czech. Chem. Commun. 20:27-31 Siavikova L, Slavik J (1966) Coil. Czech. Chem. Commun. 3 1 : 3 3 - 6 2 Southard GL, US patent 4,517,172 Steffens P, Nagakura N, Zenk MH (1985) Phytochemistry 24:2577-2583 Takao N, Kamigauchi M, Sugiura M, Ninomiya I, Miyata O, Naito T (1981) Heterocycles 16:221-225 Takao N, Kamigauchi M, Okada M (1983) Helv. Chim. Acta 66:473-484 Woiters B, E i l e r t U (1982) Z. Naturforschung 37c: 575-583 Zenk MH, ROffer M, Amann M, Deus-Neumann B, Nagakura N (1985) J. Nat. Products 48:725-738
