Plant Cell Reports

Plant Cell Reports (1987) 6:.142-145

© Springer-Verlag 1987

Increased accumulation of indole alkaloids by some cell lines of C a t h a r a n t h u s roseus in response to addition of vanadyl sulphate J. I. Smith 1, N. J. Smart 1, M. M i s a w a 1, W. G. W. Kurz 2, S. G. Tallevi 3, and F. D i C o s m o 3, 4 1 2 3 4

Allelix Inc., 6850 Goreway Drive, Mississauga, Ontario L4V 1PI, Canada Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan S7N OW9, Canada Centre for Plant Biotechnology, Department of Botany, University of Toronto, Ontario M5S 1A1, Canada Institute for Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 1A1, Canada

Received November 13, 1986 / Revised version received December 12, 1986- Communicated by F. Constabel

ABSTRACT Vanadyl sulphate (10-500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular a c c u m u ~ of catharanthine and ajmalicine. T h i s response was demonstrated in both flask and fermenter (30 l i t r e ) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. T h i s process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.

ABBREVIATIONS ~m, ajmalicine; Cath, catharanthine; CAS, ceric ammonium sulphate; VOS04, vanadyl sulphate; FW, fresh weight; n.d., not detected. INTRODUCTION It is well recognised that f o r any biotechnological process aimed at producing useful secondary metabolites from cultured plant cells, i t would be cost-effective to trigger an increase in product biosynthesis and accumulation early in the culture cycle. A variety of parameters, namely nutritional, hormonal and environmental are known to have a major effect on the physiology and metabolism of cultured plant cells (DiCosmo and Towers, 1984 and references therein; Morris, 1986), but i t is s t i l l the general experience that such cells usually f a i l to accumulate significant quantities of secondary products. The stimulation of secondary metabolism with microbially derived e l i c i t o r s which enhance the accumulation of secondary products in plant cell cultures is one method now being considered (DiCosmo and Misawa, 1985; E i l e r t et a l . , 1985). Such secondary products may eitE~-r-h-ave specific anti-microbial a c t i v i t y (Rokem et a l . , 1985), or be stress metabolites (Ayabe ~ aT., 1986, E i l e r t et a l . , 1984) the precise--~u~tions of which ~ t ~ n the plant system are unclear. In addition, chemically defined compounds have been found to stimulate secondary metabolism in cultured plant cells (Lee Offprint requests to: J. I. Smith

et a l . , 1981; Arfmann et a l . , 1985). Hattor] ~d-O-hta (1985) report t~t~odium orthovanadate stimulated the accumulation of isoflavone glucosides in suspension cultures of Vigna angularis. We have discovered that cell c u l ~ of Catharanthus roseus which normally produce low levels of indolea--~T!T~loids respond to a pulse of vanadyl sulphate with a rapid increase in alkaloid accumulation, and propose that the use of this agent could be exploited in a commercial process. MATERIALS AND METHODS Maintenance of Plant Cell Cultures Cell suspensions of Catharanthus roseus G. Don, derived from anther callus culturesl--Tn-1-979, were maintained in a Murashige and Skoog medium (Murashige and Skoog, 1962) containing I mg/l ~-naphthaleneacetic acid (NAA), 0.1 mg/l kinetin and 3% (w/v) sucrose. For small scale studies, cells were grown as 60 ml cultures in 250 ml Erlenmeyer flasks under continuous diffuse light on a rotary shaker (120 rpm; 25°C). Stock cultures were subcultured weekly using a one to five dilution of cells to medium. One cell line (FD/ST-84/07) was propagated in the dark on a rotary shaker (120 rpm; 27°C) in 500 ml Erlenmeyer flasks, in 100 ml of Schenk and Hildebrandt medium (Schenk and Hildebrandt, 1972), containing 4 mg/l NAA as the sole growth regulator. Cells were subcultured into fresh medium every 14 days using a one to four dilution of cells to medium. Preparation of Inocula for Fermenter Experiments Inoculum cultures w e r e grown in 2 l i t r e Erlenmeyer flasks containing i l i t r e of medium and 120 mls of cell inoculum. After 11-14 days of growth, three of these cultures were transferred to a p r e - s t e r i l i s e d 4 l i t r e Erlenmeyer flask, modified with a 2.5 cm orifice on the base, to which was attached 60 cm of s t e r i l i s e d silicon rubber tubing (0.9 cm bore). Transfer of the culture to the fermenter was f a c i l i t a t e d by making an aseptic connection between the open end of the s t e r i l e rubber tubing and a s t e r i l e isolator valve on the fermenter head plate.

143

Treatment with Vanadyl Sulphate (VOSO4) For small-scale experiments, vanadyl sulphate ( A l d r i c h Chemical Co., Milwaukee, Wis.) was dissolved in double d i s t i l l e d water and filter-sterilised using a 0.22 ~m M i l l i p o r e HA filter. A 1 ml stock solution was then added to lO-day-old C. roseus c e l l suspensions to give the desired f i ~ a l ~ n t r a t i o n . Control cultures were set up using an equivalent volume of d i s t i l l e d water. Cells were harvested and t h e i r a l k a l o i d content determined a f t e r a f u r t h e r 3 days. All experiments were performed i n duplicate. For 30 l i t r e studies, the vanadyl sulphate was added as a I l i t r e s o l u t i o n , made up at such a concentration as to give a f i n a l c e l l broth concentration of 50 mg/l. The solution was filter-sterilised using a O.2/~m Nalgene f i l t e r u n i t and transferred a s e p t i c a l l y to the fermenter from a p r e - s t e r i l i s e d b o t t l e , via a s i l i c o n tube (4 mm bore). Fermentation Equipment Thirty l i t r e a i r l i f t systems (LH Fermentation Co. L t d , Series 4000), m o d i f i e d w i t h 12 mm s t e r i l i s a b l e transfer and sample valves at the head and neck respectively were used in this study. Standard medium containing 4% (w/v) sucrose was used, with all other components set as published elsewhere (Fowler, 1983). The operating temperature of the system was 27°C.

over 18 minutes. These f i n a l conditions were maintained for a f u r t h e r 10 minutes. Alkaloid e l u t i o n was r o u t i n e l y monitored at 226 nm, and alkaloids t e n t a t i v e l y i d e n t i f i e d on the basis of r e t e n t i o n time and UV spectra. RESULTS AND DISCUSSION Suspensions of several cell l i n e s of C. roseus cell suspensions which n o r m a l l y - p r o ~ relatively low levels of catharanthine and ajmalicine after 13-15 days of i n c u b a t i o n responded to a pulse of VOSO4 with increased a l k a l o i d accumulation (Fig. la c; Table I ) . For example, the cell l i n e FD/ST - 84/07 normally produced undetectable or trace amounts of indole a l k a l o i d s a f t e r a 15 day c u l t u r e period (DiCosmo et a l . , in press), but f o l l o w i n g a pulse of -mg/l (final medium concentration) VOSO4, r a p i d l y accumulated ajmalicine and catharanthine. Similarly, cell l i n e LBE-I accumulated both catharanthine and ajmalicine (14.2 and 7.9 mg/l r e s p e c t i v e l y ) when not treated, but f o l l o w i n g addition of 50 mg/l VOSO4 these levels increased to 24.3 mg/l c a t h a r a n t h i n e and 14.3 mg/l a j m a l i c i n e . The s e n s i t i v i t y to VOSO4 of the cell l i n e s varied considerably (Table I ) . Seven l i n e s showed an i n c r e a s e in accumulation of catharanthine and/or ajmalicine, 2 c e l l lines did not respond, and in one c e l l l i n e , (LD-2), catharanthine accumulation was i n h i b i t e d . Table i

Analysis of Culture Growth Biomass was determined as fresh weight (Wilson et a l . , 1971). Residual glucose was estimated u ~ i n T a Yellow Springs glucose analyser - model 27. N i t r a t e and ammonium levels in the growth medium were r o u t i n e l y estimated in fermenter cultures using an Orion n i t r a t e ion electrode, model No. 83-07, and an Orion ammonium electrode, model No. 95-12, r e s p e c t i v e l y .

Cell Line

Quantitative determinations by the method of external standards were performed using a Waters 840 high performance l i q u i d chromatography (HPLC) system, equipped with a Hewlett-Packard 1040A UV detector. Separation was accomplished using a Brownlee RP-8 Spheri-5 column (22 x 0.46 cm). I n i t i a l conditions of 55:45 (v/v) methanol/water (containing 2.5 mM tetrabutylammonium phosphate) were maintained for 2 minutes, followed by a convex solvent gradient to 90:10 methanol/water

Cath (mg/l)

Aim (mg/l)

HJE

+VOSO4 Control

14.6 14.7

5.0 1.6

14.6 6.1

JWM

+VOSO4 Control

10.6 11.3

1.9 1.0

n.d. n.d.

JOH

+VOSO4 Control

19.1 15.7

35.8 26.7

5.0 7.0

JWO

+VOSO4 Control

13.7 13.0

3.9 n.d.

18.1 3.5

HWM

+VOSO4 Control

12.5 11.1

n.d. n .d.

n.d. n.d.

JWMC

+VOSO 4 Control

13.6 15.0

0.2 n.d.

n.d. n.d.

LBE-1

+VOSO4 Control

20.7 17.9

24.3 14.2

14.3 7.9

LD-2

+VOSO 4 Control

13.3 16.5

0.7 9.0

n.d. n.d.

JWM*

+VOSO4 Control

22.6 22.1

8.1 4.3

n.d. n.d.

4.4

4.5

Indole A l k a l o i d Analysis One gram (dry weight) of cell material was extracted in i0 ml of hot methanol by sonication ( i n a sonic bath) for 30 minutes. Following c e n t r i f u g a t i o n to sediment the cell debris, 5 ml of supernatant was reduced to dryness under vacuum, the r e s i d u e taken up in i ml of bicarbonate b u f f e r (pH i0) and p a r t i t i o n e d 3 times into ethyl acetate. The ethyl acetate phase was evaporated under vacuum, and the f i n a l e x t r a c t taken up in i ml of methanol. Following r e s o l u t i o n by t h i n - l a y e r chromatography (TLC) on a s i l i c a gel coated plate (Baker Si250) developed with ethyl acetate:methanol 9:1, alkaloids were t e n t a t i v e l y i d e n t i f i e d on the basis of Rf value and chromogenic reaction with ceric ammonium sulphate (CAS) spray reagent (Farnsworth et a l . , 1964).

FW (g160 ml)

FD/ST - +VOSO4 84/07 Control

The e f f e c t of cell l i n e on response to vanadyl sulphate. Cells were treated on day 10 with 50 mg/l vanadyl sulphate and harvested 3 days later.

144 The VOSO4 concentration required for maximum a l k a l o i d accumulation d i f f e r e d f o r each cell l i n e (Fig. la c). Maximum s t i m u l a t i o n of catharanthine in the 3 l i n e s tested occurred between i0 and 50 mg/l. For cell l i n e JWM*, the maximum s t i m u l a t i o n was achieved when the f i n a l concentration of VOSO4 was 10 mg/l; 200 mg/l VOSO4 i n h i b i t e d catharanthine production. However in cell l i n e FD/ST-84/07 up to 500 mg/l VOSO4 was f o u n d to s t i m u l a t e alkaloid accumulation.

(a) ~11~

Table z

Line JWM*

(b)

Vanadyl sulphate mg/I

~ 2 o ~

II. 10 20 30 40

I 50 con

Line FD/ST-84/07 I

47

Day 2 + VOSO4 Control

18.2 20.0

8.9 8.8

1.2 1.4

Day 5 + VOSO4 Control

21.1 21.4

16.9 14.8

3.1 1.7

Day 10 + VOSO4 Control

24.8 23,1

21.6 13.5

4.2 3.6

A 30 l i t r e culture of cell l i n e JWM* was treated w i t h VOSO4. When added on day 5 ( e a r l y exponential phase), the increase in catharanthine y i e l d was s l i g h t , and the a l k a l o i d production p r o f i l e was s i m i l a r to that expected in untreated c e l l cultures of t h i s l i n e (Fig. 2). However, when VOS04 was added on day 7 (mid-exponential phase), catharanthine yields increased s u b s t a n t i a l l y from less than 1 mg/l to 6 mg/l w i t h i n 24 hours of treatment and rose f u r t h e r to reach a concentration of i i mg/l by day i i (Fig. 3).

Vanadyl sulphate mg/I

(C) ~ 5

Aim

(mg/l)

The e f f e c t of c e l l age on response to vanadyl sulphate (50 mg/l) by c e l l l i n e JOH. Cells were harvested 3 days a f t e r treatment.

~ 0 10 20 go 100200con •

Cath (mg/l)

FW (g/60 ml )

Time of induction

Vanadyl sulphate

0 0

Fig. -

-

I

50 mg/I pulse

I

50

con Vanadyl sulphate mg/I

-12 -11

100 300 500

S e n s i t i v i t y of three c e l l vanadyl sulphate treatment.

~ lines

to

-10

500

•~ 400

"8 ~ 0

-7~, .6~

"~ = 300 I,I. 20O

Cells responded to VOSO4 treatment at specific times during t h e i r culture cycle. For example (Table 2), the cell l i n e JOH accumulated maximum l e v e l s of catharanthine when treated on day 10 ( s t a t i o n a r y phase). When VOSO4 was added on day 2 (lag phase) or day 5 (exponential phase), there was no i n c r e a s e in c a t h a r a n t h i n e y i e l d . Ajmalicine accumulation was not stimulated in these c e l l s . A s i m i l a r e f f e c t of age on response to VOSO4 was demonstrated in other cell l i n e s (data not shown). The pronounced e f f e c t of cell age on s e n s i t i v i t y to VOSO4 is in marked contrast to the action of some fungal e l i c i t o r s which become less e f f e c t i v e as the c e l l s age (DiCosmo et a l . , in press), whereas o t h e r compounds--wTTl stimulate accumulation at any stage of growth (Smith et a l . , 1986). This suggests that VOSO4 may have ¥-~stinct mode of a c t i o n and t h a t the accumulation of i n d o l e a l k a l o i d s f o l l o w i n g treatment with VOSO4 might not be a simple stress response. Vanadium has been shown to e i t h e r inhibit or a c t i v a t e a range of enzymes, particularly phosphohydrolases of the plasma membrane (Macara, 1980; Sze, 1984).

~°Je~°--°--°--°~°--

o °//" o~O ~0~/

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A__A~A~A~

8

14

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2

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16

18

Intervals in Days

Fi~. 2 Treatment of a 30 l i t r e culture of cell l i n e JWM* on day 5 with 50 mg/l vanadyl sulphate.

Since total a l k a l o i d y i e l d is related to biomass concentration, we attempted to increase alkaloid y i e l d by t r e a t i n g the cultures with VOSO4 on day ii, j u s t as stationary phase, and therefore maximum cell biomass was reached. Biosynthesis cannot occur without a carbon-source, and the glucose content of the medium (from hydrolysis of the sucrose supplied) is normally depleted by day I0 (Fig. 2). To maintain the a v a i l a b i l i t y of glucose, therefore, pulses of a p r e s t e r i l i s e d

145 In Figs. i 4 standard deviation bars were omitted for c l a r i t y , but variations for a l l measurements were t y p i c a l l y 1%.

Vanadyl sulphate 50 m g / I pulse

I

-12 -11 -10

"~ 500

82;

.$ 400

~

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300

u.

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°~°~

£/\//

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j.o/e/s/ 1

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Fig. 3

2

4

6

8 10 12 Intervals in Days

14

16

18

Treatment of a 30 l i t r e culture of cell l i n e JWM* on day 7 with 50 mg/l vanadyl sulphate.

glucose solution were added to the culture daily from day 7. The concentration of these pulses was set to give a f i n a l medium composition of 6.66 g/l on days 7-11, and 13.3 g/l on days 12-14. There was, however, no increase in catharanthine productCon when VOSO4 was added (Fig. 4), so c l e a r l y factors other than glucose deprivation must be involved in the c r i t i c a l timing of VOSO4 s t i m u l a t i o n of a l k a l o i d accumulation. This is presently being investigated. Glucose pulses A A A A A B B B

I

ll 2 11

Vanadyl sulphate 50 mg/I pulse

~o

"~ 500 •

I

/o

•$ 4 0 0 .

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/07

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lb

1'2

1:~

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Intervals in Days

Fig. 4

Treatment of a 30 l i t r e culture of cell l i n e JWM* on day i i with 50 mg/l vanadyl sulphate. Pulses of glucose solution to give medium concentrations of: A) B)

6.66 mg/l 13.33 mg/l

Although quantitative analysis of only catharanthine and ajmalicine were performed, e l e v a t e d l e v e l s of other i n d o l e a l k a l o i d s (detected by t h e i r positive chromogenic reaction with CAS, and by HPLC) were frequently detected whenever the c e l l s u s p e n s i o n s responded p o s i t i v e l y to VOSO4 treatment. Tabersonine and tryptamine h a v e been t e n t a t i v e l y i d e n t i f i e d . Alkaloids were only detected in trace amounts in the ~pent culture medium.

Increased i n t r a c e l l u l a r accumulation of indole alkaloids in response to VOSO4 treatment in C. roseus cell suspension cultures may be useful Tor~ commercial production of secondary plant metabolites. We have demonstrated t h a t catharanthine and ajmalicine can be produced in a bioreactor of at least 30 1 capacity in only 10-15 days, in c o n c e n t r a t i o n s which would normally be attained only after 20-30 days of culture. Furthermore, the process involves stimulation by a d i s c r e t e , inexpensive, chemically defined compound which appears to be non-toxic to the c e l l s at the concentrations used. Such a procedure must compare favourably as a potential commercial process with the use of microbially-derived e l i c i t o r s which, although well documented as stimulators of plant secondary metabolism, remain uncharacterised chemically. ACKNOWLEDGEMENTS J.l. Smith, N.J. Smart, M. Misawa and W. G. W. Kurz acknowledge p a r t i a l funding by a Program for Industry/Laboratory Projects (PILP) grant. Research by S. G. T a l l e v i and F. DiCosmo was funded by an NSERC operating grant, an NSERC Biotechnology Strategic Grant, the University of Toronto and the Bickell Foundation. In addition, we would l i k e to thank O. Anson, A. C a s t i l l o , D. Champagne and C. Watson for t h e i r excellent technical assistance. REFERENCES Arfmann HA, Kohl W, Wray V (1985) Z Naturforsch 40 c:21-25. Ayabe S, lida K, Furuya T (1986) Plant Cell Reports 3:186-189. DiCosmo F, Towers GHN (1984) Recent Adv in Phytochem 18:97-175. DiCosmo F, Misawa M (1985) TIBTECH 3:318-322. DiCosmo F, Quesnel A, Misawa M, Tallevi SG (In press). Appl Biochem and Biotech E i l e r t U, Ehmke A, Wolters B (1984) Planta Medica:508-512. E i l e r t U, Kurz WGW, Constabel, F (1985) J Plant Physiol 119:65-76. Farnsworth NR, Blomster RN, Damratoski D, Meer WA, Cammarato LV (1964) Lloydia 27:302-314. Fowler MW, (1983) In: Mantell SH, Smith H (eds), Plant Biotechnology Cambridge University Press pp 3-8. Hattori T, Ohta Y (1985) Plant Cell Physiol 26:1101-1110. Lee S-L, Cheng K-D, Scott AI (1981) Phytochem 20:1841-1843. Macara IG (1980) TIBS 5:92-94. Morris P (1986) Planta Medica:121-126. Murashige TO, Skoog F (1962) Physiol Plant 15:473-497. Rokem JS, Tal B, Goldberg I (1985) J Nat Prods 48:210-222. Schenk RU, Hildebrandt AC (1972) Can J Bot 50:199-204 Smith, J l , Smart NJ, Quesnel AA, Misawa M, Kurz W (1986). In: Somers DA, Gengenbach BG, Biesboer DD, Hackett WP, Green CE, (eds) Abstracts VI International Congress of Plant Tissue and Cell Culture p 248. Sze H (1984) Physiol Plant 61:683-691. Wilson SB, King PJ, Street HE (1971) J Exp Bot 22:177-207