Plant Cell Reports

Plant Cell Reports (1986) 5 : 2 3 - 2 6

© Springer-Verlag 1986

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Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media J. M. M~rillon ~, P. Doireau ~, A. Guiilot ~, J. C. Ch6nieux ~, and M. Rideau ~ ~ Laboratoire de Biotechnologie V~g~tale, Facult~ de Pharmacie, and ~ Laboratoire de Physiologie V~g~tale, Facult~ des Sciences, Universitfi de Tours, F-37032 Tours, France Received April 16, 1985 / Revised version received November 18, 1985 - Communicated by W. Barz

ABSTRACT ceils were cultured in t h r e e types of media. These media were : a low sucrose subculture medium and two high sucrose media, each o~ which d i l f e r e d in their mineral and hormonal c o n t e n t s . The kinetics ol t r y p t o p h a n decarboxyJase a c t i v i t y and the a c c u m u l a t i o n s oI tryptophan, t r y p t a mine, ajmalicine and serpentine were d i f f e r e n t in each series but no c o r r e l a t i o n b e t w e e n maximum e n z y m e a c t i v i t y and alkaloid c o n t e n t s was observed. Ajmalicine and serpentine productions were u n a f f e c t e d by addition of Trp to the media, whereas addition of secologanin e n h a n c e d alkaloid production. The results seem to imply t h a t the terpenoid pathway is the limiting f a c t o r in alkaloid production in C. r o s e u s cells. Catharanthus

roseus

INTRODUCTION The production of secondary metabolites by plant cells in v i t r o is, at least in part, dependent on the culture conditions employed (e.g. Deus and Zenk, 1982). In C a t h a r a n t h u s r o s e u s cells, stimulation of indole alkaloid production can be achieved by culturing the cells in "production media" (Zenk e t al., 1977) or in "induction media" (Knobloch and Berlin, 1981). However, the limiting factor in alkaloid a c c u m u lation has not been defined and may involve one or both of the indole and terpenoid pathways. In this study we c o m p a r e d t i m e courses of t r y p t o p h a n decarboxylase a c t i v i t y in C. roseus cells cultured in t h r e e media which p r o m o t e d d i f l e r e n t levels ol alkaloid production. We also studied the e f f e c t s ol supplementing these media with t r y p t o p h a n (indole pathway) and/or secologanin (terpenoid pathway). MATERIAL AND METHODS Cells~ culture conditions and media. The Catharanthus roseus G. Don cell culture (strain C20) was maintained under the conditions described by M&rillon et al., ([983). For the purpose of the experiments, 5 ml samples o£ the cell suspension (i.e. 107 cells) were transferred to 250 ml Erlenmeyer flasks which contained 145 m! of one of the following three media : SM, a medium used for subcultures , B5 medium (Gamborg el al., 1968)~ sucrose, 58 raM, 2,14D, 14.5 pM, Kin, 0.28 ~/VI ; PM, a medium identical to the SM, except in t h a t it had a sucrose c o n t e n t of 1714 mM ; IM___,an aqueous solution of sucrose, 1714 raM. This last medium r e s e m bled the "induction medium" used by Knobloch and Berlin (1981). Growth curves and d i n e courses

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of Trp decarboxylase activity and alkaloid contents were studied over a period of 20 days. Five flasks from each treatment were taken every two days and the cells filtered off using Whatmann GF/A filters. In experiments on the effects of supplementing the media with secologanin and/or tryptophan the cells were cultured in the three media for l0 days (10 flasks per treatment). Filtration sterilised (Millipore, 0.22 pro) tryptophan (1.22 raM) and/or secologanin (0.[214 raM) were then added to 5 flasks and the cells cultured for another lO days. An equivalent volume of distilled water (2 ml) was added to 5 control flasks. Secologanin was obtained from young leafy shoots of Lonicera xylosteum L. using the extraction technique described by Kinast and Tietze ([976), and was compared against a pure standard by mass spectrometry and GLC and TLC chromatographies. In all experiments, the cells were agitated on a gyratory shaker, at 214°C, in the dark. Alkaloid and growth measurements. The procedures used for the determination of cell numbers and Trp, tryptamine, ajmalicine, and serpentine contents both in cells and in media were as described by M~rilion et ai., ([993, [9814). Tryptophan decarboxylase activity. Cells were frozen in liquid nitrogen and stored at -20°C. Enzyme extractions and measurements of a c t i v i t y were performed according to the method of Gibson et al. ([972) with the following modifications : (i) The cells (3 g) were ground under liquid nitrogen with sand (2 g) in a mortar and were extracted with a phosphate buffer (0.[ M, pI-i g, [00 ml) in the presence of 6 g polyclar A.T. (General aniline, France). (Li) The proteins were precipitated with 35-75 % saturated (Nh4)2SO,~ and were sedimented by centrifugation. The residue was recovered in phosphate buffer (0.05 [vb pH g, 2 mi) and desalted on Sephadex G25. The protein fraction obtained was then reduced to 500 ~l by u l t r a f i l t r a t i o n through Amicon membranes (yM 10). (rid The enzyme assay (total volume, 20 IA) contained enzyme solution (10 pl ) ; L-( ]4C methylene) tryptophan (Amersham), specific a c t i v i t y 3 Mbq~nmole (100 nmoles) ~ pyridoxal phosphate (14 nmotes) and phosphate buffer (0,0~ M ~ pH g). The reaction was stopped, after 1 h at ~7°C, by addition of Na2CO3(10 % ~ 5 I~l). (iv) .5 IA o~ the reaction medium was submitted to T i C (Sigei 60, 0.25 mm ~ Merck). The plates were developed with M e 2 C O - C ~ H s C H a - M e O H - 2 8 % NHgOH, 140-30-10-2, v/v. The tryptamine spot visualised under UV light was scrapped into a scintillation vial containing l0 ml of a liquid scintillation counting solution (OCS~

24 strain has lost its ability to accumuiate ajmalicine when cultured in this medium. (This phenomenon does not appear to be particularly unusual, since Deus-Neumann and Zenk (1984), have d e m o n s t r a t e d that the ability of C. roseus cells to accumulate alkaloids is an unstable trait). The high sucrose c o n c e n t r a t i o n s present in the PM and IM restored the capacity of cells to accumulate ajmalicine (fig. ID) and enhanced serpentine production (fig, I E).

Amersham) and its radioactivity measured. The protein c o n t e n t of the enzyme solution was d e t e r m i n e d as described by Bradford (1976). RESULTS 1. Growth Figure IA shows the changes in cell numbers with time over a culture period of 20 days. As previously d e m o n s t r a t e d by M4rillon et al. (1984), the growth curves in SM and PM were similar during the first eight days of incubation. The stationary phase occurred on day 8 in SM and on day 12 in PM. Stationary phase cultures in PM contained 20 % more cells than stationary phase cultures in S/Vi. Although t h e r e was a significant increase in cell dry weight in IN, (results not presented) the increase in cell numbers in this medium was low (after 20 days of cultivation, total cell number had increased only by 25 % compared to 1200 % in SM and 1650 % in PM).

4. Trp decarboxylase activity In SM, Trp decarboxylase activity was high up to day 8 of incubation, but declined sharply during the stationary phase. Sasse et al. (1983) have reported a similar e f f e c t in another strain of C. roseus cultured in a low sucrose medium. On the contrary in PM Trp decarboxylase activity remained high through out t h e e x p e r i m e n t a l period and p a r t i c u l a r l y during the alkaloid accumulation phase (fig. IF). In IM, a f t e r an important decrease in activity during the first 12 h, activities peaked at the end of the second day. If t h e s e activities are r e l a t e d to protein content (specific activity), this p a t t e r n is in good a g r e e m e n t with that reported by Knobloch et al., (1981), with the exception of the 12 first hours (fig. 2).

2. Trp and t r y p t a m i n e t i m e - c o u r s e s In all culture media, intra-cellular Trp concentrations peaked at the end of the first day of cultivation and then declined sharply (fig. IB). In cells cultured in IM, Trp levels b e c a m e very low a f t e r the 3rd day of cultivation (In these cultures t h e r e were few cells divisions and it is possible t h a t all the Trp had been converted to tryptamine, since the highest levels of t r y p t a m i n e observed in this experiment, 55 nmoles per 106cells, occurred in these cultures on the 4 th day of incubation). However, when the cells were cultured in PM or SM, Trp levels increased a f t e r day 4 and peaked on days 8 and 16 respectively. In P/vl, some Trp may be required for the continuation of cell division, and this requirement may explain the d e c r e a s e in Trp levels observed a f t e r day 8. In P~V; there was a further increase in Trp levels just before the end of the experiment due perhaps to proteolysis in senescent cells. Changes in t r y p t a m i n e levels followed similar p a t t e r n s in SM and PM (fig. IC), although levels tended to be slightly lower in PM cells.

5. E f f e c t s of Trp and/or secologanin Addition of Trp to the culture media enhanced t r y p t a m i n e production, but did not stimulate ajmalicine and serpentine production. The addition of secologanin always increased alkaloid levels. For example, the production of ajmaiicine and serpentine in cells cultured in PM were 20 fold higher when secologanin was added to this medium. The simultaneous addition of secologanin and Trp to PM or IM increased tryptamine levels but decreased alkaloid production (Table I). DISCUSSION The results presented in this article indicate that the kinetics of tryptophan decarboxylase activity depend on the culture medium used. This e f f e c t is particularly pronounced during the stationary growth phase when enzyme activity falls to low levels in ceils cultured in SM but remains at high levels in cells cultured in PM. This d i f f e r e n c e in the behaviour

3. Alkaloid time-courses In previous studies (M~riilon et al., 1983 ;1984) we have studied ajmalicine and serpentine production in the C20 strain of C. roseus cultured in SM. In the period following these studies the C20

Culture medium

Addition at day 10 of

Alkaloids (nmol. flask"I)

Growth (mg dr. wt. flask "l) T

SM

Trp Sec Trp + Sec PM

Trp Sec Trp + Sec IM

Trp Sec Trp + Sec

263 265 261 260 1271 1092 1263 1104

234 307 230 302

A ND ND 67,g + 80,3 +

15 9 11 6

1654 1929 562 1125

+ 74 ± 50 + 95 + Ii0

+ 65 ± 127 + 55 + 71

2840 5748 i554 4874

+ ± + +

130 640 180 243

74,6 36,0 611 #70

+ + ± +

362 1117 237 318

+ + + +

20 84 17 15

57,0 32,1 263,3 230,4

+

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8 II 10 7

S

6,0 13,3

20,1 23,0 32,1 33,3

_+ + + +

14,6 6,2 70,5 48,0

145,8 101,9 249,4 206,1

+ _% + +

7,2 5,0 18,3 17,1

15,8 12,6 27,8 15,5

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3,2 3,7 3,4 2,9

+ 25,9 _+ 9,5 +__ 22,1 + 10,9 z + + +

1,5 1,0 2,2 2,0

Table I : Influence of adding tryptophan (Trp) and secologanin (Sec) to SM, PM or IM, on alkaloid production and cell growth, a f t e r 20 days of incubation. T : Tryptamine ; A : Ajmalicine ; S : Serpentine ; ND : Non d e t e c t a b l e (n = 5)

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Fig. i. Growth of C. roseus ceils (A) and t i m e courses of Trp (B), t r y p t a m i n e (C), ajmalicine (D) and s e r p e n t i n e (E) levels and of Trp decarboxylase a c t i v i t y (EA : F), in cells cultured in SM (O--O), PM (O--O) and IM (13....rn) of the enzyme may explain the g r e a t e r a c c u m u l a t i o n of alkaloids exhibited by cells cultured in PM. It is p e r t i n e n t to point out here the difficulties involved in i n t e r p r e t i n g t h e s e physiological results as long as one does not or c a n n o t distinguish b e t w e e n a c t i v e and i n a c t i v e pools of i n t e r m e d i a r y m e t a b o l i t e s . Maximal values for t r y p t o p h a n decarboxylase a c t i v i t y (expressed as general e n z y m e a c t i v i t y in Fig. IF and as specific e n z y m e a c t i v i t y in Fig. 2) were of the same order of magnitude in all 3 of the media studied. However i n t r a - c e l l u l a r alkaloid a c c u m u lation and t o t a l alkaloid production differed greatly b e t w e e n the 3 media (Table [I). It t h e r e f o r e appears t h a t t h e r e is no clear relationship b e t w e e n maxima values of t r y p t o p h a n decarboxylase a c t i v i t y and the a c c u m u l a t i o n of ajmalicJne and serpentine• This conclusion differs from t h a t put forward by Knobloch e l al._~ (19gl) who found t h a t , when cells were diluted

in an 80 g/l sucrose solution, an increase in tryptophan decarboxylase a c t i v i t y was a c c o m p a n i e d by an increase in alkaloid accumulation. However the same authors have also reported t h a t strains of C. roseus s e l e c t e d for high t r y p t o p h a n decarboxylase a c t i v i t y do not necessarily a c c u m u l a t e large quantities of alkaloids (Sasse et al., 1993). F u r t h e r m o r e we have found t h a t the maximum value of tryptophan decarboxylase activity in an h a b i t u a t e d strain of C. roseus (selected from the strain C20) r e m a i n e d unchanged whereas alkaloid a c c u m u l a t i o n was increased (manuscript in preparation). These last two observations also indicate the absence of a c o r r e l a t i o n b e t w e e n t r y p t o p h a n decarboxylase a c t i v i t y and alkaloid production. The addition of secologanin (but not tryptophan) markedly s t i m u l a t e d the a c c u m u l a t i o n of ajmalicine and s e r p e n t i n e w h a t e v e r the culture medium used. This appears to i n d i c a t e t h a t , in strain C20 at least, one of limiting factors in alkaloid production is one

26 Total production

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PM

IM

3#00 ND

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550

20 ND

25 1,2

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115 9

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ABBREVIATIONS 2,4D = 2,4-Dichlorophenoxyacetic GLC = gas-liquid chromatography; Kin tin; TLC = thin-layer chromatography; S M = c e l l c u l t u r e m e d i a as d e s c r i b e d Material and Methods•

acid; = kineIM, PM, under

REFERENCES Bradford MM (1976) Anal. Biochem., 72 : 248-254 Deus B, Zenk MH (1982) Biotechn. Bioengin., 24 : 1965-1974 Deus-Neumann B, Zenk MH (1984) P l a n t a med.~ 50: 427-431

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ACKNOWLEDGEMENTS The authors wish to thank Dr Tietze (Organisch Chemisches Institut der Universit~t G6ttingen, G e r m any) for supplying pure secotoganin and Dr C. Viel (ERA-alZCh&tenay Malabry, France) for carrying out m a s s - s p e c t r o m e t r y of isolated secologanin. The t e c h nical assistance of Mrs F. Boucher (UA 596, CNRS), M. Thiersault, M. Brillard and F. Andreu is gratefully acknowledged. Dr A.D. Mills (SRA-INRA, Nouzilly, France) is thanked for help with p r e p a r a t i o n of the English manuscript.

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Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media.

Catharanthus roseus cells were cultured in three types of media. These media were: a low sucrose subculture medium and two high sucrose media, each of...
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