PlantCell Reports
Plant Cell Reports (1993) 12:702-705
9 Springer-Verlag 1993
Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of C a t h a r a n t h u s r o s e u s P. R. H. Moreno, R. van der Heijden, and R. Verpoorte Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received May 3, 1993/Revised version received July 7, 1993 - Communicated by W. Barz
Abstract The effects of terpenoid precursor' feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.
Introduction Cell cultures of Catharanthus roseus produce a variety of monoterpenoid indole alkaloids. Ajmalicine, serpentine and catharanthine are usually the major constituents (Lounasmaa and Galambos 1989; Van der Heijden et aL 1989). The biosynthesis of these alkaloids depends on indole and terpenoid precursors supplied by two convergent metabolic pathways. The accumulation of these alkaloids is thus dependent on the regulation of the flow through the pathways leading to the indole and terpenoid precursors. An exogenous supply of a biosynthetic precursor to the culture medium may improve alkaloid accumulation where the productivity is limited by lack of that particular precursor. The effects of addition of precursors to cell suspension cultures of C. roseus were contradictory. Feeding of tryptamine, tryptophan or both to the medium resulted in different responses within the cells, in some cases increased alkaloid production (Zenk et al. 1977; Krueger and Carew 1978; Deus and Zenk 1982) and in others repressed (Drller et al. 1976) or C o r r e s p o n d e n c e to."
R. Verpoorte
unaffected (Mrrillon et al. 1986; Kargi and Ganapathi 1991). Addition of secologanin to C. roseus cell cultures increased alkaloid levels in some strains (Zenk et al. 1977; Mrrillon et al. 1986; Stafford and Smith 1986; Facchini and DiCosmo 1991). Alkaloid production by cell suspension cultures of C. roseus was increased by elicitation with fungal elicitors (DiCosmo and Misawa 1985; Eilert et al. 1986; Asada and Shuler 1989). After elicitor treatment the activity of enzymes involved in the biosynthesis of tryptamine were induced and tryptamine was accumulated (Eilert et al.~ 1986; Berlin et al. 1987; Moreno et al. 1991). A transient induction of tryptophan decarboxylase and strictosidine synthase genes was also observed after elicitation of cell suspension cultures C. roseus (Goddijn et al. 1992; Roewer et al. 1992). With the aim of increasing the yields, the combined effect of feeding terpenoid precursors and elicitation on alkaloid accumulation was investigated. In contrast to previous reports, the formation of strictosidine, the first alkaloidal product resulting from the feeding, was monitored. Material and Methods Cell Culture
Cell suspension cultures of Catharanthus roseus (L.) G. Don (cell line AlzA~)were grown in MS (Murashige and Skoog 1962) basal medium, supplemented with 30 g/1 sucrose (MS-H). Subculturing was done every 7 days by 5-fold dilution of the culture. Culture conditions: temperature 25+ I~ light 2800 lux 24 h/day, on gyratory shaker ca. 120 rpm. For the experiment 250 ml flasks containing 50 ml of MS-H medium were inoculated with 5-6 g cells from a seven-day-old culture. Each data point represented the average of three flasks. The feeding experiment with mevalonic acid, loganin and secologanin was performed simultaneously using' the same control group. Loganic acid feeding was performed separately having its own control group.
Feeding of Precursors to Cell Suspension
Secologanin (1 g), purchased from Aldrich (Milwaukee, Wise., USA),
703 was previously purified over a 100 g silica (Kieselgel 60, Merck, Darmstadt, Germany) column with 3 em diameter eluted with 100 ml fractions of 0, 5, 10 and 30% (vN) acetone in ethyl acetate, followed by 400 ml of 50% (vN) acetone in ethyl acetate. Mevalonie acid was prepared by adding appropriate amounts of mevalonic acid lactone (Sigma, St. Louis,/do, USA) to an equal molar aqueous solution of NaHCO 3. Aqueous solutions (100 pl) of loganin (Roth, Kaflsruhe, Germany), loganic acid (Extrasynthese, Lyon, France), purified secologanin and mevalonic acid were added, after filter sterilization, to suspension cultures of C. roseus (50 ml) 5 days after inoculation. Final concentration of precursors was 0.5 mM for loganin, logauic acid and secologanin and 3.3 mM for mevalonic acid. An equal volume of sterile distilled water was added to control flasks. Cultures were elicited 3 h after the addition of terpenoid precursors.
Elicitor Preparation and Elicitation Procedure Pythium aphanidermatum (Centraalbureau voor Schimmelcultures, Baam, The Netherlands) was grown on solid MS-H medium. From a seven-day-old culture, one emz mycelia was inoculated on 100 ml liquid MS-H medium. The cukure was kept in gyratory water-bath at 28~ for 7 days. The culture medium was filtered through a 0.2 ]am pore filter (Sartorius type N13107, Gtttingen, Germany) and autoclaved for 20 min (121~ The autoclaved cell-free filtrate was used as elicitor preparation. Elicitation was performed by adding 10 ml of elicitor preparation to 50 ml five-day-old cell suspension cultures. Control cultures received the same amount of sterile distilled water. Cultures were harvested 72 and 120 h after elicitation. Alkaloid Determination For ajmalicine and tryptamine determination freeze-dried cell material and culture meditma were extracted according to Schripsema and Verpoorte (1992) and analyzed by means of HPLC (Van der Heijden et al. 1987) equipped with photodiode-array detection. Strictosidine was extracted from 50 - 100 mg of lyophilized cell material with 15 ml ethanol using an Ystral homogenizer. After centrifugation at 4,000 g for 20 rain, 10 ml of the extract was taken and reduced to dryness under reduced pressure. To the dried extract 1 ml 1 M H3PO4was added and the suspension was thoroughly homogenized
using a Vortex mixer. It was then transferred to an Eppendorf tube and centrifuged for 5 rain at 14,000 g and 50 Ill of the supematant was injected in the HPLC system as above mentioned. Strictosidine synthesized as described by Pfit2ner and Zenk (1982) was used as reference compound. Results and Discussion
Feeding of the terpenoid precursors mevalonic acid, loganin, loganic acid or secologanin to suspension cultures of C. roseus did not affect the culture growth, as determined by dry weight concentrations measured at 72 and 120 h after feeding (Table 1 and 2). These results demonstrated no toxic effect for all precursors added to the cultures. Secologanin was added to the cultures during the growth-phase (day 5). Addition of secologanin to the cultures increased accumulation of strictosidine and ajmalicine (Figure 1). The accumulation of these two alkaloids was increased 11-fold 72 h after the feeding and about 5-fold after 120 h when this cell culture normally has reached about its maximum alkaloid contents (unpublished results). In this cell line strictosidine and ajmalicine were the main alkaloids, no serpentine nor catharanthine were found. Tryptamine was depleted 72 h after the addition of secologanin. An increase of the tryptamine level could be again observed 120 h after the feeding which might be due to a limitation in secologanin supply again (Naudascher et al. 1989a). As reported in Fig. 1, strictosidine was detected in higher concentration than ajmalicine. The strictosidine accumulation in the cells suggests the presence of a regulatory step in alkaloid biosynthesis after strictosidine formation.
Table 1. Effect of terpenoid precursors feeding and elicitation on biomass accumulation in C. roseus cell suspension cultures. The values show the averages and standard deviations of three measurements.
Dry Weight
(g/L) time after elicitafion (hours) 72
120
no feeding treatment
secologanin
loganin
mevalonic acid
control
19.29 + 0.19
19,41 + 0.20
19.18 + 0.76
19.10 + 0.52
elicited
17.02 + 0.43
15,49 + 1.09
16.22 + 0.23
16.54 + 0.43
control
18.19 + 0.32
17.98 + 0.51
18.10 + 0.30
18.32 + 0.78
elicited
14.18 + 0.34
14.02 + 0.13
12.22 + 2.07
13.50 + 1.68
704 The cultures were elicited 3 h after addition of secologanin when most of the added compound is taken up by the cells (Naudascher et al. 1989a). Culture growth was inhibited after elicitation, but no excessive cell lysis was observed after 120 h with elicitor (Table 1). Elicitation of secologanin fed cells had not increased ajmalicine nor strictosidine production. The ajmaiicine contents in fed cells, both control and elicited, were the same 72 h after elicitation (Figure 1). In control cells ajmalicine accumulation increased as a function of time, while in elicited cells ajmalicine levels remained unchanged. Although, it was reported that elicitor treatment increased tryptamine accumulation (Seitz et al. 1989; Moreno et al., 1991), fed cells after elicitation presented much lower levels of tryptamine than non-fed elicited cells. A decrease of strictosidine contents was also observed after elicitation. No strictosidine could be detected in the cells 120 h after elicitation. Examination of the HPLC chromatogram showed that most of the other minor alkaloids disappeared after elicitor treatment (data not shown). Loganin was recognized as a precursor of secologanin (Inouye et al. 1986). Naudascher et al. (1989b) reported that loganin was rapidly taken up by C. roseus cells when added to cultures in growth phase. The accumulation of ajmalicine and strictosidine was circa 8fold increased in loganin treated cells 120 h after the feeding (Figure 1). Comparing loganin and secologanin feeding, the former treatment promoted a higher alkaloid accumulation. This could be due to a chemical instability of secologanin (Tietze 1983). Naudascher et al. (1989b) also observed that exogenous loganin was used in toto by C. roseus cells unlike exogenous secologanin (Naudascher et al. 1989a). Tryptamine was partially depleted when compared with non-fed ceils. The effect of elicitation on cells fed with loganin was similar to that observed with secologanin. Elicited cells presented similar amounts of ajmalicine as the respective control 72 h after elicitation. A non-significant decrease of ajmalicine was observed in elicited cells 120 h after elicitation, while control fed cells displayed a significant increase in ajmalicine production (Figure 1). The acidic iridoid, loganic acid, had been implicated in the biosynthesis of indole alkaloids in C. roseus by its natural occurrence and in vivo conversion to its methyl ester loganin (Guarnaccia et al. 1974). Although, no studies about the uptake of this compound by the cells were available, the addition of this iridoid was performed as described for secologanin and loganin. As for the other terpenoid precursors tested, no toxic effect was observed (Table 2). The accumulation of strictosidine and ajmalicine in the fed cells was much lower than observed for the feeding with secologanin or loganin (Figure 1). Ajmalicine contents 120 h after feeding loganic acid were similar to those found 72 h after addition of loganin or secologanin. Decrease of tryptamine levels could also be
observed, however, in a lesser extent than that observed for the other iridoids fed to the cells. Elicitation of loganic acid fed cells showed similar results as observed with loganin and secologanin. After elicitation cell growth was also inhibited, but no extreme cell lysis was observed (Table 2). Strictosidine was completely depleted in the elicited cells. The increase of tryptamine in elicited fed cells was lower than in non-fed
150-
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Figure 1. Effect of feeding terpenoid precursors and elicitation on indole alkaloid accumulation (cell + medium) of Catharanthus roseus cell suspension cultures.
705 Table 2. Effect of feeding loganic acid and elicitation on biomass accumulation in C. roseus cell suspension cultures. The values represent the averages and the standard deviations of three measurements.
elicitation was not interfering with the condensation of tryptamine with secologanin to form strictosidine. Thus, elicitor treatment of C. roseus cells seemed to stimulate the strictosidine catabolism.
Acknowledgements Dry Weight (e/L) time after elicitation
no feeding treatment
loganic acid
(hours) 72
120
The authors wish to thank Ir. L.H. Stevens and T.J.C. Luijendijk for the synthesis of strictosidine. P.R.H. Moreno is indebted to the CAPES (Brazil) for the Doctoral fellowship awarded. The research of Dr. R van der Heijden has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Science.
References control
17.64 + 0.59
17.79 +_ 0.35
elicited
16.72 + 0.46
15.79 + 0.34
control
19.31 + 2.86
16.96 +
0.18
elicited
14.47 + 0.30
13.62 +
0.34
m
cells. Contrary to the results with the other iridoids added, ajmalicine levels increased 120 h after elicitation. Mevalonic acid is the basic precursor for all terpenoids, including iridoids like secologanin. The incorporation of mevalonic acid in terpenoid indole alkaloids was already reported by Guamaccia et al. (1974). Mevalonic acid was added to the cultures at a concentration (3.3 mM) which was reported to saturate the C~0 (steroid) biosynthetic pathway in Nicotiana tabacum suspension cultures (Threlfall and Whitehead 1988). As shown in Figure 1, C. roseus cultures showed no significant increase in alkaloid levels after mevalonate feeding. Similarly, Krueger and Carew (1978) reported no increase in alkaloid production when mevalonic acid was fed at a concentration of 0.76 mM. Elicitor treatment resulted in an increase of the tryptamine levels. Tryptamine levels were similar for both fed and non-fed cells. By adding secologanin and its precursors loganin and loganic acid to C. roseus cell suspension cultures, alkaloid accumulation was increased. The major product observed was strictosidine. Feeding of mevalonic acid, the early precursor of the terpenoid pathway, did not increase alkaloid production. These results suggests one or more limiting steps in the terpenoid pathway which should be located between mevalonic acid and loganic acid. Elicitation increased tryptamine accumulation but ajmalicine levels remained constant. The combination of elicitation with feeding terpenoid precursors did not improve alkaloid accumulation. After the treatment with elicitor,strictosidine was depleted from the cells. As elicited ceils fed with iridoid precursors showed lower levels of tryptamine than non-fed elicited cells, probably
-Asada M, Shuler ML (1989) Appl Microbiol Biotechnol 30:475-481. -Berlin J, Mollenschott C, DiCosmo F (1987) Z Naturforsch 42c:11011108. -Deus B, Zenk MH (1982) Biotechnol Bioeng 24:1965-1974. -Dicosmo F, Misawa M (1985) Trends Biotechnol 3:318-322. -Drller G, Alfermann AW, Reinhard E (1976) Planta Med 30:14-20. -Eilert U, Constabel F, Kurz WGW (1986) J Plant Physiol 126:11-22. -Facehini PJ, DiCosmo F (1991) Appl Microbiol Biotechnol 35:382392. -Goddijn OJM, de Kamp RF, Zanetti A, Schilperoort R.A, Hoge JHC (1992) Plant Mol Biol 18:1113-1120. -Guamaccia R, Botta L, Coscia CJ (1974) J Amer Chem Soc 96:70797084. -Inouye H, Uesato S (1986) In: Herz W, Grisebach H, Kirby GW, Tamm Ch, (eds) Progress in the Chemistry of Natural Products, vol 50. Springer-Vedag, Wien New York, pp 169-236. -Kargi F, Ganapathi B (1991) Enzyme Microb Technol 13:643-647. -Krueger RJ, Carew DP (1978) J Nat Prod 41:327-331. -Lounasmaa M, Galambos J (1989) In: Herz W, Grisebach H, Kirby GW, Tamm Ch, (eds) Progress in the Chemistry of Natural Products, vol 55. Springer-Verlag, Wien New York, lap 89-I 15. -Mrrillon JM, Doireau P, Guillot A, Chrnieux JC, Rideau M (1986) Plant Cell Rep 5:23-26. -Moreno PRH, Poulsen C, Van der Heijden R, Verpoorte R (1991) Planta Med 57 (suppl.2): A103. -Murashige T, Skoog F (1962) Physiol Plant 18:100-127. -Naudascher F, Doireau P, Guillot A, Viel C, Thiersault M (1989a) J Plant Physiol 134:608-612. -Naudascher F, Doireau P, Guillot A, Thiersault M (1989b) J Plant Physiol 135:366-368. -Pfitzner U, Zenk MH (1982) Planta Med 46:10-14. -Roewer IA, Cloutier N, Nessler CL, De Luca V (1992) Plant Cell Rep 11:86-89.
-Schripsema J, Verpcorte R (1992) Planta Med 58:245-249. -Seitz HU, Constabel F, De Luca V, Kurz WGW (1989) Plant Cell Tiss Org Cuk 18:71-78. -Stafford A, Smith L (1986) In: Morris P, Scragg AM, Stafford A, Fowler MW, (eds) Secondary Metabolism in Plant Cell Cultures, Cambridge University Press, Cambridge, pp. 250-256. -ThrelfaU DR, Whitehead IM (1988) Phytochemisuy 27:2567-2580. -Tietze L-F (1983) Angew Chem Int Ed Engl 22:828-841. -Van der Heijden R, Lamping PJ, Out PP, Wijnsma R, VeRaoorte (1987) J Chromatogr 396: 286-295. -Van der Heijden R, Verpoorte R, Ten Hoopen HJG (1989) Plant Cell Tiss Org Cult 18:231-280. -Zenk MH, E1-Shagi H, Arens H, Sfrckigt J, Weiler EW, Deus B (1977) In: Barz W, Reinhard E, Zenk MH, (eds) Plant Tissue Culture and its Biotechnological Application. Springer Verlag, Berlin, pp. 27-44.
Plant Cell Reports (1993) 12:702-705
9 Springer-Verlag 1993
Effect of terpenoid precursor feeding and elicitation on formation of indole alkaloids in cell suspension cultures of C a t h a r a n t h u s r o s e u s P. R. H. Moreno, R. van der Heijden, and R. Verpoorte Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received May 3, 1993/Revised version received July 7, 1993 - Communicated by W. Barz
Abstract The effects of terpenoid precursor' feeding and elicitation by a biotic elicitor on alkaloid production of Catharanthus roseus suspension cultures were studied. After addition of secologanin, loganin or loganic acid an increase in the accumulation of ajmalicine and strictosidine and a decrease of tryptamine level was observed in non-elicited cells. Elicitation increased tryptamine accumulation in non-fed cells but it did not further increase alkaloid accumulation in precursor-fed cells. A decrease of tryptamine level was also observed, despite the induction of the tryptamine pathway after elicitation. Feeding mevalonic acid did not increase alkaloid accumulation in any studied case.
Introduction Cell cultures of Catharanthus roseus produce a variety of monoterpenoid indole alkaloids. Ajmalicine, serpentine and catharanthine are usually the major constituents (Lounasmaa and Galambos 1989; Van der Heijden et aL 1989). The biosynthesis of these alkaloids depends on indole and terpenoid precursors supplied by two convergent metabolic pathways. The accumulation of these alkaloids is thus dependent on the regulation of the flow through the pathways leading to the indole and terpenoid precursors. An exogenous supply of a biosynthetic precursor to the culture medium may improve alkaloid accumulation where the productivity is limited by lack of that particular precursor. The effects of addition of precursors to cell suspension cultures of C. roseus were contradictory. Feeding of tryptamine, tryptophan or both to the medium resulted in different responses within the cells, in some cases increased alkaloid production (Zenk et al. 1977; Krueger and Carew 1978; Deus and Zenk 1982) and in others repressed (Drller et al. 1976) or C o r r e s p o n d e n c e to."
R. Verpoorte
unaffected (Mrrillon et al. 1986; Kargi and Ganapathi 1991). Addition of secologanin to C. roseus cell cultures increased alkaloid levels in some strains (Zenk et al. 1977; Mrrillon et al. 1986; Stafford and Smith 1986; Facchini and DiCosmo 1991). Alkaloid production by cell suspension cultures of C. roseus was increased by elicitation with fungal elicitors (DiCosmo and Misawa 1985; Eilert et al. 1986; Asada and Shuler 1989). After elicitor treatment the activity of enzymes involved in the biosynthesis of tryptamine were induced and tryptamine was accumulated (Eilert et al.~ 1986; Berlin et al. 1987; Moreno et al. 1991). A transient induction of tryptophan decarboxylase and strictosidine synthase genes was also observed after elicitation of cell suspension cultures C. roseus (Goddijn et al. 1992; Roewer et al. 1992). With the aim of increasing the yields, the combined effect of feeding terpenoid precursors and elicitation on alkaloid accumulation was investigated. In contrast to previous reports, the formation of strictosidine, the first alkaloidal product resulting from the feeding, was monitored. Material and Methods Cell Culture
Cell suspension cultures of Catharanthus roseus (L.) G. Don (cell line AlzA~)were grown in MS (Murashige and Skoog 1962) basal medium, supplemented with 30 g/1 sucrose (MS-H). Subculturing was done every 7 days by 5-fold dilution of the culture. Culture conditions: temperature 25+ I~ light 2800 lux 24 h/day, on gyratory shaker ca. 120 rpm. For the experiment 250 ml flasks containing 50 ml of MS-H medium were inoculated with 5-6 g cells from a seven-day-old culture. Each data point represented the average of three flasks. The feeding experiment with mevalonic acid, loganin and secologanin was performed simultaneously using' the same control group. Loganic acid feeding was performed separately having its own control group.
Feeding of Precursors to Cell Suspension
Secologanin (1 g), purchased from Aldrich (Milwaukee, Wise., USA),
703 was previously purified over a 100 g silica (Kieselgel 60, Merck, Darmstadt, Germany) column with 3 em diameter eluted with 100 ml fractions of 0, 5, 10 and 30% (vN) acetone in ethyl acetate, followed by 400 ml of 50% (vN) acetone in ethyl acetate. Mevalonie acid was prepared by adding appropriate amounts of mevalonic acid lactone (Sigma, St. Louis,/do, USA) to an equal molar aqueous solution of NaHCO 3. Aqueous solutions (100 pl) of loganin (Roth, Kaflsruhe, Germany), loganic acid (Extrasynthese, Lyon, France), purified secologanin and mevalonic acid were added, after filter sterilization, to suspension cultures of C. roseus (50 ml) 5 days after inoculation. Final concentration of precursors was 0.5 mM for loganin, logauic acid and secologanin and 3.3 mM for mevalonic acid. An equal volume of sterile distilled water was added to control flasks. Cultures were elicited 3 h after the addition of terpenoid precursors.
Elicitor Preparation and Elicitation Procedure Pythium aphanidermatum (Centraalbureau voor Schimmelcultures, Baam, The Netherlands) was grown on solid MS-H medium. From a seven-day-old culture, one emz mycelia was inoculated on 100 ml liquid MS-H medium. The cukure was kept in gyratory water-bath at 28~ for 7 days. The culture medium was filtered through a 0.2 ]am pore filter (Sartorius type N13107, Gtttingen, Germany) and autoclaved for 20 min (121~ The autoclaved cell-free filtrate was used as elicitor preparation. Elicitation was performed by adding 10 ml of elicitor preparation to 50 ml five-day-old cell suspension cultures. Control cultures received the same amount of sterile distilled water. Cultures were harvested 72 and 120 h after elicitation. Alkaloid Determination For ajmalicine and tryptamine determination freeze-dried cell material and culture meditma were extracted according to Schripsema and Verpoorte (1992) and analyzed by means of HPLC (Van der Heijden et al. 1987) equipped with photodiode-array detection. Strictosidine was extracted from 50 - 100 mg of lyophilized cell material with 15 ml ethanol using an Ystral homogenizer. After centrifugation at 4,000 g for 20 rain, 10 ml of the extract was taken and reduced to dryness under reduced pressure. To the dried extract 1 ml 1 M H3PO4was added and the suspension was thoroughly homogenized
using a Vortex mixer. It was then transferred to an Eppendorf tube and centrifuged for 5 rain at 14,000 g and 50 Ill of the supematant was injected in the HPLC system as above mentioned. Strictosidine synthesized as described by Pfit2ner and Zenk (1982) was used as reference compound. Results and Discussion
Feeding of the terpenoid precursors mevalonic acid, loganin, loganic acid or secologanin to suspension cultures of C. roseus did not affect the culture growth, as determined by dry weight concentrations measured at 72 and 120 h after feeding (Table 1 and 2). These results demonstrated no toxic effect for all precursors added to the cultures. Secologanin was added to the cultures during the growth-phase (day 5). Addition of secologanin to the cultures increased accumulation of strictosidine and ajmalicine (Figure 1). The accumulation of these two alkaloids was increased 11-fold 72 h after the feeding and about 5-fold after 120 h when this cell culture normally has reached about its maximum alkaloid contents (unpublished results). In this cell line strictosidine and ajmalicine were the main alkaloids, no serpentine nor catharanthine were found. Tryptamine was depleted 72 h after the addition of secologanin. An increase of the tryptamine level could be again observed 120 h after the feeding which might be due to a limitation in secologanin supply again (Naudascher et al. 1989a). As reported in Fig. 1, strictosidine was detected in higher concentration than ajmalicine. The strictosidine accumulation in the cells suggests the presence of a regulatory step in alkaloid biosynthesis after strictosidine formation.
Table 1. Effect of terpenoid precursors feeding and elicitation on biomass accumulation in C. roseus cell suspension cultures. The values show the averages and standard deviations of three measurements.
Dry Weight
(g/L) time after elicitafion (hours) 72
120
no feeding treatment
secologanin
loganin
mevalonic acid
control
19.29 + 0.19
19,41 + 0.20
19.18 + 0.76
19.10 + 0.52
elicited
17.02 + 0.43
15,49 + 1.09
16.22 + 0.23
16.54 + 0.43
control
18.19 + 0.32
17.98 + 0.51
18.10 + 0.30
18.32 + 0.78
elicited
14.18 + 0.34
14.02 + 0.13
12.22 + 2.07
13.50 + 1.68
704 The cultures were elicited 3 h after addition of secologanin when most of the added compound is taken up by the cells (Naudascher et al. 1989a). Culture growth was inhibited after elicitation, but no excessive cell lysis was observed after 120 h with elicitor (Table 1). Elicitation of secologanin fed cells had not increased ajmalicine nor strictosidine production. The ajmaiicine contents in fed cells, both control and elicited, were the same 72 h after elicitation (Figure 1). In control cells ajmalicine accumulation increased as a function of time, while in elicited cells ajmalicine levels remained unchanged. Although, it was reported that elicitor treatment increased tryptamine accumulation (Seitz et al. 1989; Moreno et al., 1991), fed cells after elicitation presented much lower levels of tryptamine than non-fed elicited cells. A decrease of strictosidine contents was also observed after elicitation. No strictosidine could be detected in the cells 120 h after elicitation. Examination of the HPLC chromatogram showed that most of the other minor alkaloids disappeared after elicitor treatment (data not shown). Loganin was recognized as a precursor of secologanin (Inouye et al. 1986). Naudascher et al. (1989b) reported that loganin was rapidly taken up by C. roseus cells when added to cultures in growth phase. The accumulation of ajmalicine and strictosidine was circa 8fold increased in loganin treated cells 120 h after the feeding (Figure 1). Comparing loganin and secologanin feeding, the former treatment promoted a higher alkaloid accumulation. This could be due to a chemical instability of secologanin (Tietze 1983). Naudascher et al. (1989b) also observed that exogenous loganin was used in toto by C. roseus cells unlike exogenous secologanin (Naudascher et al. 1989a). Tryptamine was partially depleted when compared with non-fed ceils. The effect of elicitation on cells fed with loganin was similar to that observed with secologanin. Elicited cells presented similar amounts of ajmalicine as the respective control 72 h after elicitation. A non-significant decrease of ajmalicine was observed in elicited cells 120 h after elicitation, while control fed cells displayed a significant increase in ajmalicine production (Figure 1). The acidic iridoid, loganic acid, had been implicated in the biosynthesis of indole alkaloids in C. roseus by its natural occurrence and in vivo conversion to its methyl ester loganin (Guarnaccia et al. 1974). Although, no studies about the uptake of this compound by the cells were available, the addition of this iridoid was performed as described for secologanin and loganin. As for the other terpenoid precursors tested, no toxic effect was observed (Table 2). The accumulation of strictosidine and ajmalicine in the fed cells was much lower than observed for the feeding with secologanin or loganin (Figure 1). Ajmalicine contents 120 h after feeding loganic acid were similar to those found 72 h after addition of loganin or secologanin. Decrease of tryptamine levels could also be
observed, however, in a lesser extent than that observed for the other iridoids fed to the cells. Elicitation of loganic acid fed cells showed similar results as observed with loganin and secologanin. After elicitation cell growth was also inhibited, but no extreme cell lysis was observed (Table 2). Strictosidine was completely depleted in the elicited cells. The increase of tryptamine in elicited fed cells was lower than in non-fed
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(hours}
[ ~ tryptamine l ~ strictosidine [ ~ ajrnal}clne
Figure 1. Effect of feeding terpenoid precursors and elicitation on indole alkaloid accumulation (cell + medium) of Catharanthus roseus cell suspension cultures.
705 Table 2. Effect of feeding loganic acid and elicitation on biomass accumulation in C. roseus cell suspension cultures. The values represent the averages and the standard deviations of three measurements.
elicitation was not interfering with the condensation of tryptamine with secologanin to form strictosidine. Thus, elicitor treatment of C. roseus cells seemed to stimulate the strictosidine catabolism.
Acknowledgements Dry Weight (e/L) time after elicitation
no feeding treatment
loganic acid
(hours) 72
120
The authors wish to thank Ir. L.H. Stevens and T.J.C. Luijendijk for the synthesis of strictosidine. P.R.H. Moreno is indebted to the CAPES (Brazil) for the Doctoral fellowship awarded. The research of Dr. R van der Heijden has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Science.
References control
17.64 + 0.59
17.79 +_ 0.35
elicited
16.72 + 0.46
15.79 + 0.34
control
19.31 + 2.86
16.96 +
0.18
elicited
14.47 + 0.30
13.62 +
0.34
m
cells. Contrary to the results with the other iridoids added, ajmalicine levels increased 120 h after elicitation. Mevalonic acid is the basic precursor for all terpenoids, including iridoids like secologanin. The incorporation of mevalonic acid in terpenoid indole alkaloids was already reported by Guamaccia et al. (1974). Mevalonic acid was added to the cultures at a concentration (3.3 mM) which was reported to saturate the C~0 (steroid) biosynthetic pathway in Nicotiana tabacum suspension cultures (Threlfall and Whitehead 1988). As shown in Figure 1, C. roseus cultures showed no significant increase in alkaloid levels after mevalonate feeding. Similarly, Krueger and Carew (1978) reported no increase in alkaloid production when mevalonic acid was fed at a concentration of 0.76 mM. Elicitor treatment resulted in an increase of the tryptamine levels. Tryptamine levels were similar for both fed and non-fed cells. By adding secologanin and its precursors loganin and loganic acid to C. roseus cell suspension cultures, alkaloid accumulation was increased. The major product observed was strictosidine. Feeding of mevalonic acid, the early precursor of the terpenoid pathway, did not increase alkaloid production. These results suggests one or more limiting steps in the terpenoid pathway which should be located between mevalonic acid and loganic acid. Elicitation increased tryptamine accumulation but ajmalicine levels remained constant. The combination of elicitation with feeding terpenoid precursors did not improve alkaloid accumulation. After the treatment with elicitor,strictosidine was depleted from the cells. As elicited ceils fed with iridoid precursors showed lower levels of tryptamine than non-fed elicited cells, probably
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