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Time-Course Studies on The Use of Secologanin by Catharanthus roseus Cells Cultured in vitro
J. PlantPhysiol. Vol. 134. pp. 608-612 (1989)
Time-Course Studies on The Use of Secologanin by Catharanthus roseus Cells Cultured in vitro F. NAUDASCHER 1, P. DOIREAUI, A. 1
2
GUILLOT
1 ,
C. VIEL2 , and M.
THIERSAULT
1
Faculte des Sciences-Laboratoire de Physiologie Vegetale, Parc de Grandmont, F-37200 Tours Faculte de Pharmacie-Laboratoire de Pharmacognosie, F-37042 Tours Cedex
Received October 14, 1988 . Accepted February 6,1989
Summary Secologanin was not detected in Catharanthus roseus cell suspensions which had not been supplied with this compound. Secologanin introduced into a suspension 10 days after the start of subculture rapidly disappeared from the medium. The intracellular content of secologanin peaked at 75 %, then decreased to 20 % of the amount provided and remained at this level. This observation might be explained by the existence of an inactive pool which would not have been fed in the control cells. During the first hours after secologanin had been added, about 40 % of the total quantity (in both cells and medium) seemed to temporarily disappear. Such a disappearance is governed by the presence of cells, for it does not occur in a culture medium deprived of cells. Re-conversion of secologanin into loganin, demethylation of secologanin or loganin, conjugation of loganin or loganic acid with p-coumaric acid do not account for this disappearance. No clear explanation of this disappearance phenomenon can be given at present. Similar results were obtained when secologanin was added 4 days after initiation of subculture. However, in this case, intracellular content peaked at 96 %; the final constant level was about 50 %. The results are discussed in light of previous reports describing alkaloid accumulation and time-courses of related enzyme activities in the same material.
Key words: Catharanthus roseus, cell suspensions, secologanin feeding.
Introduction The biosynthesis of indole alkaloids in Catharanthus roseus depends on indole and terpenoid precursors supplied by two convergent metabolic pathways. The accumulation of these alkaloids could thus apparently be affected by limiting the flow of indole and/or terpenoid precursors. Actually however, where some strains have shown a higher alkaloid accumulation following addition of tryptophan to the culture medium, other strains have not (Doller et al., 1976; Deus and Zenk, 1982). The C20 strain has been shown to exhibit no response to the introduction of tryptophan into the medium. Yet, the introduction of secologanin into the medium has been shown to greatly enhance the accumulation of alkaloids (Merillon et aI., 1986). This suggests that the flux of terpenoid precursors is limiting for alkaloid accumulation. Nevertheless, the amount of alkaloids accumulated by the cell suspensions is far less than the (C) 1989 by
Gustav Fischer Verlag. Stuttgart
amount of secologanin provided (about 0.1 J.tmole ajmalicine and serpentine for 25 J.tmoles secologanin). There are various hypotheses for this observation: alkaloid turnover, accumulation of some unmeasured alkaloids or diversion of secologanin into other metabolic pathways. The observation could also be explained by a relative inefficiency of the absorption system for secologanin and/or a rapid transformation of this compound in the culture medium. We investigated the latter two possibilities using cells in either growth or stationary phases.
Material and Methods Cells and culture conditions Cells of Catharanthus roseus G. Don (strain C20) were maintained as described by Merillon et al. (1983).
Use of secologanin by cell suspensions
Extraction and purification of secologanin Secologanin was extracted from young leafy shoots of Lonicera xylosteum L. as described by Kinast and Tietze (1976). The extract was chromatographed using a column containing Kieselgel Merck G60 (70-230 mesh) with the eluting mixture, CHCh/MeOH 9/1 VIV. The product obtained was a white foamish powder }..~~H = 235 nm (log E = 4.02); M.S. 70eV: 226 [M+9.6)(M+ corresponds to the aglycone according to Bentley and Johnstone (1967)] 209 (28.0), 195 (8.1),165 (46.7),157 (100),139 (81.3),125 (45.7), 97 (39.0). These values are similar to those obtained with a standard provided by Prof. Tietze.
Addition ofsecologanin to cell suspensions An aqueous solution of secologanin was sterilized by filtration and introduced under sterile conditions (100 ",I containing 25 ",moles per flask) in cell suspensions (50 ml per flask) after 4 days (= 4 d cells) or 10 days of subculture (10d cells).
Determination of secologanin and loganin contents in cells and culture media Cells and medium were separated by filtration through Whatman GF/C filters at various times after the addition of secologanin. The volume of the medium was measured, divided into 1 ml aliquots and stored at -30°C until used. The cells were quickly washed with cold water (+4 °C; 10 ml) and weighed. Next, the cells were extracted, first with boiling MeOH/CHCh 5/5 V/V, then MeOH and finally MeOH/H 20 5/5 VIV. The cell extracts were concentrated. Aliquots of media or cellular extracts were chromatographed through Lichroprep RP 8 40 - 63",m (MERCK) (35 g in a glass column i.d. 30 mm). The elution was carried out using successively: 150ml MeOH/H20 5/95 VIV, 150ml MeOH/H20 20/80 VIV, and 150 ml MeOH/H 20 60/40 VIV. The flow rate was 150 ml h -1. Carbohydrates were eluted in the 5/95 mixture, loganic and secologanic acids in the 20/80 and loganin and secologanin in the 60/40. Secologanin and loganin were measured as acetates by GC (glass column 3 m x 2 mm packed with 2 % OV 17 on Chromosorb WAW 100-120 mesh, DMCS treated; 245°C; N2 flow rate: 80 mI· min - 1). The recovery was about 97 % ± 3 %.
Testing of cellular extracts and media for the occurrence of loganic and secologanic acids The 20/80 fraction was tested for the occurrence of loganic and secologanic acids by TLC according to the methods of Coscia et al. (1969) and Guarnaccia and Coscia (1971) and by GC after methylation according to Battersby et al. (1969).
Testing of cellular extracts for the occurrence of logan in and loganic acid as p-coumaroyl·esters These compounds were analysed using methods described by Garcia and Chulia (1987).
Replication of results The results reported here are those from one experiment, but this experiment was replicated several times over a period of three years. Despite some inevitable variation, each replication produced similar results supporting the conclusions of the present study.
609
Results and Discussion 1. Conditions in cells grown without addition of secologanin Secologanin was never detected in Catharanthus roseus cell suspensions, regardless of the age of the subculture. The secologanin content of the control cells was thus in each case less than 0.5 nmol/10 6 cells (or around 0.1 J.tmol/flask at the beginning of the stationary phase) whereas tryptamine was in each case present in quantities between 10 and 25 nmol/ 106 cells (Doireau et aI., 1987).
2. Time-course of secologanin utilisation Secologanin was introduced into the suspensions as described in «Material and Methods». The addition was made during the growth phase, 4 days following initiation of the subculture (henceforth referred to as 4 d cells), or during the stationary phase, 10 days following initiation of the subculture (henceforth referred to as 10 d cells) (Merillon et aI., 1986; Doireau et aI., 1987). 2.1. Secologanin disappeared very rapidly from the culture medium (Fig. 1 Aa and Ba). Less than 10 % secologanin remained 4 hours after its addition, and its level was no longer measurable after 8 hours (4d cells) and 10 hours (10d cells). The rate of this disappearance may be related to rapid absorption of this substance by the cells and/or its conversion to other substances in the culture medium. 2.2. Secologanin content increased rapidly in the cells, although less rapidly than it disappeared from the medium (see Fig. 1 Aa and Ba). The secologanin content reached its maximum in 6 hours (4d cells) and in 8 hours (10d cells) after the introduction of secologanin. This maximum coincided with the point where the culture medium contained practically no more secologanin (2 - 3 % of the quantity introduced). In 4 d cells, the maximal quantity observed corresponded to 96 % of the secologanin added to the flask, in 10 d cells only 75 %. The secologanin content in 4 d cells then declined rapidly (in 4 hours) to a level 50 % of the added quantity. In contrast, secologanin content in 10 d cells declined slowly (in 30 hours) and the final level was only 20 % of the added quantity. In both cases, the final level remained constant for at least 60 hours. Thus, part of the secologanin seems to have eluded metabolism even though tryptamine and strictosidine synthase were present in the cells (Doireau et aI., 1987). The greatest utilisation in 10 d cells could be related to a higher strictosidine synthase activity in these cells (Doireau et al., 1987). Yet, it is surprising that the secologanin level remains constant in those cells up through the 14th day of the subculture, even though at this time, strictosidine synthase activity is growing, and the stationary phase is generally a period of alkaloid accumulation (Vinas and Pareilleux, 1982; Merillon et aI., 1986; Morris, P., 1986; Doireau et aI., 1987). The introduction of tryptophan together with secologanin produced little increase in alkaloid accumulation as compared with the introduction of secologanin alone (Merillon et al., 1986). This would seem to eliminate the hypothesis than the non-utilisation of part of the secologanin is due to a lack of indolic precursors. Furthermore, the tryptamine content remains high in the cells supplied with both secologanin and
610
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cases: more than 40 % for the 4 d cell suspensions in two hours, and 55 % for the 10 d in half an hour. However, 2 to 6 hours later in the 4 d cell suspensions (Fig. 1 Ab) and ~ to 8 hours later in the 10 d cell suspensions (see Fig. 1 Bb), the total quantity of secologanin per flask increased markedly. Two hypotheses are offered for this curious phenomenon. First, the addition of a large quantity of secologanin could have caused major metabolic perturbations leading to a high but momentary accumulation of endogenous secologanin. Second, part of the exogenous secologanin could have been converted (by a very rapid but slowly reversible process) into one or more forms undetected by our methods. It remains to be seen whether this reversible conversion is an intracellular or extracellular phenomenon. In an attempt to answer this question, a cells-media cross experiment was undertaken. 10 d cell suspensions were used because the disappearance of secologanin was the most rapid and intense in these suspensions.
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tryptophan, although lower than in the control cells (MerilIon et al., 1986). These observations suggest that part of the secologanin was perhaps accumulated in an «inactive» pool. 2.3. The curves (see Fig. 1 Ab and Bb) illustrating changes in the total quantity of secologanin per flask (4d or lod cells + culture medium) clearly indicate a surprising phenomenon. At first, secologanin disappeared rapidly in both
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Use of secologanin by cell suspensions
611
Table 2: Time-course of secologanin contents in a cells-deprived medium. Cell suspension was cultured during 10 days, then cells were filtered off and secologanin (12.5 /-tmoles) was added into the medium.
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previous study of Lonicera cellular suspensions (T anahashi et aI., 1984). 3.2. Even though the C( + } cells had no secologanin available in the M( -} culture medium, they nonetheless exhibited a considerable increase in secologanin content during the two hours following their transfer to the M( - } medium. This would suggest that the C( +} cells, while in the M( + ) medium, had accumulated by an apparently reversible process, a significant quantity of secologanin in a form which our methods could not detect. 3.3. An important increase in secologanin content of C( - } cells was also observed after their transfer from the M( - } culture medium to the M( +}. 4 hours after change of medium, this increase culminated in a much higher value than the quantity present in the M( + } medium at time of transfer. This would again suggest that the M( + } culture medium, as the C( + } cells, contains secologanin in forms undetected by our methods. These undetected forms of secologanin would thus appear to exist in the cells as well as in the media supplied with secologanin. The following question is raised: is the occurrence of these undetected forms due to cellular activity, or to interaction with the culture medium?
4. Behavior of secologanin in a cell-deprived medium In response to the above question, a culture medium which contained cells for 10 days was filtered (40 - 90 Jlm) in order
to eliminate the cells. Secologanin was then introduced, and the medium was analysed at various times. Table 2 shows that no significant change occurred in the quantity of secologanin during 6 hours. A mean of 99 ± 4 % of secologanin was recovered, corresponding to the amount of secologanin which can be extracted from a fresh culture medium to which secologanin had previously been added. From this it follows that the occurrence of these undetected forms of secologanin is not due to a secologaninl medium interaction, but rather, in every case, to the presence of cells. 5. Attempts to identify the «undetected forms» of secologanin
Loganin and loganic acid can be converted in vivo into secologanin (Atta-ur-Rahman and Basha, 1983), but there is no data in the literature about the possibility of re-conversion of secologanin into loganin and loganic acid or the possibility of demethylation. On the other hand, loganin and loganic acid occur in Gentiana pedicellata as p-coumaroyl esters (Garcia and Chulia, 1986 and 1987). We investigated the possible occurrence of these various compounds in an attempt to identify the undetected forms of secologanin. Loganin was detected neither in the cells, nor in the media at any time during the experiments. The cells actually contained less than 0.05 Jlmole per flask (or less than 0.25 nmolel 106 cells during the stationary phase) and the culture media - less than 0.7 Jlmole per flask. Traces of loganic and secologanic acids too small to be measured were detected in the cells and media. Similarly, p-coumaroyl esters of loganin and loganic acid seemed to exist in the cells, but in quantities too small to be identified or measured with any certainty. These results appear to rule out that the conversion of loganic acid and loganin into secologanin is reversible even in the presence of an excess of secologanin (at least not under intracellular conditions). Thus, the temporary disappearance of secologanin in the cell suspensions could not be explained in this manner. 6. Conclusions
The weak alkaloid accumulation earlier observed to result from the introduction of secologanin (Merillon et aI., 1986) is not due to the relative inefficiency of the absorption system, since the secologanin furnished disappeared from the culture medium and accumulated in large quantities in the cell. Nor is it due to a degradation of secologanin in the culture medium. This is evidenced by the stability of secologanin introduced into a culture medium from which cells were removed by filtering a cell suspension.
612
F. NAUDASCHER, P. DOIREAU, A. GUILLOT, C. VIEL, and M. THIERSAULT
The phenomenon could possibly stem from part of the secologanin being trapped into an inactive pool. The intracellular localization of such a pool is currently under study. Furthermore, our observations suggested that the cells converted secologanin through a reversible process into other forms that these experiments have been unable to reveal. Moreover, one cannot completely rule out the hypothesis that endogenous secologanin accumulated following metabolic perturbations related to massive absorption of exogenous secologanin. Further study of these points will be undertaken as soon as radioactive secologanin becomes available. Acknowledgements The authors wish to thank Dr Tietze for supplying pure secologanin, T. Becue for registering the mass spectra and L. Troup for the preparation of the english manuscript.
References ATTA-UR-RAHMAN and A. BASHA: Biosynthesis of indole alkaloids. Clarendon Press, Oxford (1983). BATTERSBY, A. R., A. R. BURNETT, and P. G. PARSONS: Alkaloid biosynthesis. Part XIV. Secologanin: its conversion into ipecoside and its role as biological precursor of the indole alkaloids. J. Chern. Soc., C, 1187 -1192 (1969). BENTLEY, T. W. and R. A. W. JOHNSTONE: Aspects of mass spectra of organic compounds. Part IV. Cyclopentane monoterpenes of the iridoid group. J. Chern. Soc., C, 2234-2240 (1967). COSCIA, C. J., R. GUARNACCIA, and L. BOTTA: Monoterpene biosynthesis. I. Occurrence and mevalonoid origin of gentiopicroside and loganic acid in Swertia caroliniensis. Biochemistry, 8, 5036-5043 (1969).
DEUS, B. and M. H. ZENK: Exploitation of plant cells for the production of natural compounds. Biotechn. Bioengin., 24, 1965 -1974 (1982). DOIREAU, P., J. M. MERILLON, A. GUILLOT, M. RIDEAU, J. C. CHENIEUX, and M. BRILLARD: Time-course studies on indole alkaloid accumulation and changes in tryptophan decarboxylase and strictosidine synthase activities: a comparison in three strains of Catharanthus roseus cells. Planta Med., 53, 364-367 (1987). DOELLER, G., A. W. ALFERMANN, and E. REINHARD: Produktion von Indolalkaloiden in Calluskulturen von Catharanthus roseus. Planta Med., 30, 14-20 (1976). GARCIA, J. and A. J. CHULIA: Loganin and new iridoid glucosides in Gentiana pedicellata. Planta Med., 327 -329 (1986). - - 4'-p-coumaroyl iridoid glucosides from Gentiana pedicellata. Planta Med., 101-103 (1987). GUARNACCIA, R. and C. J. COSCIA: Occurrence and biosynthesis of secologanic acid in Vinca rosea. J. Am. Chern. Soc., 93, 6320-6321 (1971). KINAST, G. and L. F. TIETZE: Enzymatische Spaltung von Secologanin. Ein Modell zur Biogenese der Indolalkaloide. Chern. Ber., 109,3640-3645 (1976). MERILLON, J. M., J. C. CHENIEUX, and M. RIDEAU: Time-course of growth, evolution of sugar-nitrogen metabolism and accumulation of alkaloids in a cell suspension of Catharanthus roseus. Planta Med., 47, 169-176 (1983). MERILLON, J. M., P. DOIREAU, A. GUILLOT, J. C. CHENIEUX, and M. RIDEAU: Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media. Plant Cell Rep., 5, 23-26 (1986). MORRIS, P.: Regulation of product synthesis in cell cultures of Catharanthus roseus. II. Comparison of production media. Planta Med., 121-126 (1986). TANAHASHI, T., N. NAGAKURA, H. INOUYE, and M. H. ZENK: Radioimmunoassay for the determination of loganin and the biotransformation of loganin to secologanin by plant cell cultures. Phytochemistry, 23, 1917 -1922 (1984). VINAS, R. and A. PAREILLEUX: Production d'alcaloides par des suspensions cellulaires de Catharanthus roseus cultivees in vitro. Physiol. veg., 20, 219-225 (1982).
Time-Course Studies on The Use of Secologanin by Catharanthus roseus Cells Cultured in vitro F. NAUDASCHER 1, P. DOIREAUI, A. 1
2
GUILLOT
1 ,
C. VIEL2 , and M.
THIERSAULT
1
Faculte des Sciences-Laboratoire de Physiologie Vegetale, Parc de Grandmont, F-37200 Tours Faculte de Pharmacie-Laboratoire de Pharmacognosie, F-37042 Tours Cedex
Received October 14, 1988 . Accepted February 6,1989
Summary Secologanin was not detected in Catharanthus roseus cell suspensions which had not been supplied with this compound. Secologanin introduced into a suspension 10 days after the start of subculture rapidly disappeared from the medium. The intracellular content of secologanin peaked at 75 %, then decreased to 20 % of the amount provided and remained at this level. This observation might be explained by the existence of an inactive pool which would not have been fed in the control cells. During the first hours after secologanin had been added, about 40 % of the total quantity (in both cells and medium) seemed to temporarily disappear. Such a disappearance is governed by the presence of cells, for it does not occur in a culture medium deprived of cells. Re-conversion of secologanin into loganin, demethylation of secologanin or loganin, conjugation of loganin or loganic acid with p-coumaric acid do not account for this disappearance. No clear explanation of this disappearance phenomenon can be given at present. Similar results were obtained when secologanin was added 4 days after initiation of subculture. However, in this case, intracellular content peaked at 96 %; the final constant level was about 50 %. The results are discussed in light of previous reports describing alkaloid accumulation and time-courses of related enzyme activities in the same material.
Key words: Catharanthus roseus, cell suspensions, secologanin feeding.
Introduction The biosynthesis of indole alkaloids in Catharanthus roseus depends on indole and terpenoid precursors supplied by two convergent metabolic pathways. The accumulation of these alkaloids could thus apparently be affected by limiting the flow of indole and/or terpenoid precursors. Actually however, where some strains have shown a higher alkaloid accumulation following addition of tryptophan to the culture medium, other strains have not (Doller et al., 1976; Deus and Zenk, 1982). The C20 strain has been shown to exhibit no response to the introduction of tryptophan into the medium. Yet, the introduction of secologanin into the medium has been shown to greatly enhance the accumulation of alkaloids (Merillon et aI., 1986). This suggests that the flux of terpenoid precursors is limiting for alkaloid accumulation. Nevertheless, the amount of alkaloids accumulated by the cell suspensions is far less than the (C) 1989 by
Gustav Fischer Verlag. Stuttgart
amount of secologanin provided (about 0.1 J.tmole ajmalicine and serpentine for 25 J.tmoles secologanin). There are various hypotheses for this observation: alkaloid turnover, accumulation of some unmeasured alkaloids or diversion of secologanin into other metabolic pathways. The observation could also be explained by a relative inefficiency of the absorption system for secologanin and/or a rapid transformation of this compound in the culture medium. We investigated the latter two possibilities using cells in either growth or stationary phases.
Material and Methods Cells and culture conditions Cells of Catharanthus roseus G. Don (strain C20) were maintained as described by Merillon et al. (1983).
Use of secologanin by cell suspensions
Extraction and purification of secologanin Secologanin was extracted from young leafy shoots of Lonicera xylosteum L. as described by Kinast and Tietze (1976). The extract was chromatographed using a column containing Kieselgel Merck G60 (70-230 mesh) with the eluting mixture, CHCh/MeOH 9/1 VIV. The product obtained was a white foamish powder }..~~H = 235 nm (log E = 4.02); M.S. 70eV: 226 [M+9.6)(M+ corresponds to the aglycone according to Bentley and Johnstone (1967)] 209 (28.0), 195 (8.1),165 (46.7),157 (100),139 (81.3),125 (45.7), 97 (39.0). These values are similar to those obtained with a standard provided by Prof. Tietze.
Addition ofsecologanin to cell suspensions An aqueous solution of secologanin was sterilized by filtration and introduced under sterile conditions (100 ",I containing 25 ",moles per flask) in cell suspensions (50 ml per flask) after 4 days (= 4 d cells) or 10 days of subculture (10d cells).
Determination of secologanin and loganin contents in cells and culture media Cells and medium were separated by filtration through Whatman GF/C filters at various times after the addition of secologanin. The volume of the medium was measured, divided into 1 ml aliquots and stored at -30°C until used. The cells were quickly washed with cold water (+4 °C; 10 ml) and weighed. Next, the cells were extracted, first with boiling MeOH/CHCh 5/5 V/V, then MeOH and finally MeOH/H 20 5/5 VIV. The cell extracts were concentrated. Aliquots of media or cellular extracts were chromatographed through Lichroprep RP 8 40 - 63",m (MERCK) (35 g in a glass column i.d. 30 mm). The elution was carried out using successively: 150ml MeOH/H20 5/95 VIV, 150ml MeOH/H20 20/80 VIV, and 150 ml MeOH/H 20 60/40 VIV. The flow rate was 150 ml h -1. Carbohydrates were eluted in the 5/95 mixture, loganic and secologanic acids in the 20/80 and loganin and secologanin in the 60/40. Secologanin and loganin were measured as acetates by GC (glass column 3 m x 2 mm packed with 2 % OV 17 on Chromosorb WAW 100-120 mesh, DMCS treated; 245°C; N2 flow rate: 80 mI· min - 1). The recovery was about 97 % ± 3 %.
Testing of cellular extracts and media for the occurrence of loganic and secologanic acids The 20/80 fraction was tested for the occurrence of loganic and secologanic acids by TLC according to the methods of Coscia et al. (1969) and Guarnaccia and Coscia (1971) and by GC after methylation according to Battersby et al. (1969).
Testing of cellular extracts for the occurrence of logan in and loganic acid as p-coumaroyl·esters These compounds were analysed using methods described by Garcia and Chulia (1987).
Replication of results The results reported here are those from one experiment, but this experiment was replicated several times over a period of three years. Despite some inevitable variation, each replication produced similar results supporting the conclusions of the present study.
609
Results and Discussion 1. Conditions in cells grown without addition of secologanin Secologanin was never detected in Catharanthus roseus cell suspensions, regardless of the age of the subculture. The secologanin content of the control cells was thus in each case less than 0.5 nmol/10 6 cells (or around 0.1 J.tmol/flask at the beginning of the stationary phase) whereas tryptamine was in each case present in quantities between 10 and 25 nmol/ 106 cells (Doireau et aI., 1987).
2. Time-course of secologanin utilisation Secologanin was introduced into the suspensions as described in «Material and Methods». The addition was made during the growth phase, 4 days following initiation of the subculture (henceforth referred to as 4 d cells), or during the stationary phase, 10 days following initiation of the subculture (henceforth referred to as 10 d cells) (Merillon et aI., 1986; Doireau et aI., 1987). 2.1. Secologanin disappeared very rapidly from the culture medium (Fig. 1 Aa and Ba). Less than 10 % secologanin remained 4 hours after its addition, and its level was no longer measurable after 8 hours (4d cells) and 10 hours (10d cells). The rate of this disappearance may be related to rapid absorption of this substance by the cells and/or its conversion to other substances in the culture medium. 2.2. Secologanin content increased rapidly in the cells, although less rapidly than it disappeared from the medium (see Fig. 1 Aa and Ba). The secologanin content reached its maximum in 6 hours (4d cells) and in 8 hours (10d cells) after the introduction of secologanin. This maximum coincided with the point where the culture medium contained practically no more secologanin (2 - 3 % of the quantity introduced). In 4 d cells, the maximal quantity observed corresponded to 96 % of the secologanin added to the flask, in 10 d cells only 75 %. The secologanin content in 4 d cells then declined rapidly (in 4 hours) to a level 50 % of the added quantity. In contrast, secologanin content in 10 d cells declined slowly (in 30 hours) and the final level was only 20 % of the added quantity. In both cases, the final level remained constant for at least 60 hours. Thus, part of the secologanin seems to have eluded metabolism even though tryptamine and strictosidine synthase were present in the cells (Doireau et aI., 1987). The greatest utilisation in 10 d cells could be related to a higher strictosidine synthase activity in these cells (Doireau et al., 1987). Yet, it is surprising that the secologanin level remains constant in those cells up through the 14th day of the subculture, even though at this time, strictosidine synthase activity is growing, and the stationary phase is generally a period of alkaloid accumulation (Vinas and Pareilleux, 1982; Merillon et aI., 1986; Morris, P., 1986; Doireau et aI., 1987). The introduction of tryptophan together with secologanin produced little increase in alkaloid accumulation as compared with the introduction of secologanin alone (Merillon et al., 1986). This would seem to eliminate the hypothesis than the non-utilisation of part of the secologanin is due to a lack of indolic precursors. Furthermore, the tryptamine content remains high in the cells supplied with both secologanin and
610
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cases: more than 40 % for the 4 d cell suspensions in two hours, and 55 % for the 10 d in half an hour. However, 2 to 6 hours later in the 4 d cell suspensions (Fig. 1 Ab) and ~ to 8 hours later in the 10 d cell suspensions (see Fig. 1 Bb), the total quantity of secologanin per flask increased markedly. Two hypotheses are offered for this curious phenomenon. First, the addition of a large quantity of secologanin could have caused major metabolic perturbations leading to a high but momentary accumulation of endogenous secologanin. Second, part of the exogenous secologanin could have been converted (by a very rapid but slowly reversible process) into one or more forms undetected by our methods. It remains to be seen whether this reversible conversion is an intracellular or extracellular phenomenon. In an attempt to answer this question, a cells-media cross experiment was undertaken. 10 d cell suspensions were used because the disappearance of secologanin was the most rapid and intense in these suspensions.
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See experimental procedure represented in Fig. 2. Both cells and media were analysed at different times. The results of the analyses are shown in Fig. 3 and Table 1; respectively. The following was noted: 3.1. Cells which had absorbed secologanin for one hour [C( +)] and which were subsequently transferred to a medium without secologanin [M( -)] did not release secologanin into the culture medium, since no secologanin could be detected there. This observation is in agreement with a
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Fig. 1: Time-course of secologanin contents in cell suspensions after secologanin (25/-1moles) was introduced. This compound was added 4 days (A) or 10 days (B) after the start of the subculture. Results in % of the quantity introduced. a: in cells and in the medium .- - - - -., b: total quantity (cells + medium) e--e.
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tryptophan, although lower than in the control cells (MerilIon et al., 1986). These observations suggest that part of the secologanin was perhaps accumulated in an «inactive» pool. 2.3. The curves (see Fig. 1 Ab and Bb) illustrating changes in the total quantity of secologanin per flask (4d or lod cells + culture medium) clearly indicate a surprising phenomenon. At first, secologanin disappeared rapidly in both
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Use of secologanin by cell suspensions
611
Table 2: Time-course of secologanin contents in a cells-deprived medium. Cell suspension was cultured during 10 days, then cells were filtered off and secologanin (12.5 /-tmoles) was added into the medium.
IJmoles
Time (hours) Secologanin (% of the quantity introduced)
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Fig. 3: Time-course of secologanin contents in cells after cells-media crossing. The cells-media crossing defined the time zero (see Fig. 2), •. ... .• C( + ) in M( -) . - . C( - ) in M( + ). Table 1: Time-course of secologanin contents in the media after cells-media crossing. Time (hours) M(+) /-tmol
M(-) /-tmol
o 2.88
2 1.93
3
4 to 32
1.49
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The cells-media crossing defined the time zero (see Fig. 2).
previous study of Lonicera cellular suspensions (T anahashi et aI., 1984). 3.2. Even though the C( + } cells had no secologanin available in the M( -} culture medium, they nonetheless exhibited a considerable increase in secologanin content during the two hours following their transfer to the M( - } medium. This would suggest that the C( +} cells, while in the M( + ) medium, had accumulated by an apparently reversible process, a significant quantity of secologanin in a form which our methods could not detect. 3.3. An important increase in secologanin content of C( - } cells was also observed after their transfer from the M( - } culture medium to the M( +}. 4 hours after change of medium, this increase culminated in a much higher value than the quantity present in the M( + } medium at time of transfer. This would again suggest that the M( + } culture medium, as the C( + } cells, contains secologanin in forms undetected by our methods. These undetected forms of secologanin would thus appear to exist in the cells as well as in the media supplied with secologanin. The following question is raised: is the occurrence of these undetected forms due to cellular activity, or to interaction with the culture medium?
4. Behavior of secologanin in a cell-deprived medium In response to the above question, a culture medium which contained cells for 10 days was filtered (40 - 90 Jlm) in order
to eliminate the cells. Secologanin was then introduced, and the medium was analysed at various times. Table 2 shows that no significant change occurred in the quantity of secologanin during 6 hours. A mean of 99 ± 4 % of secologanin was recovered, corresponding to the amount of secologanin which can be extracted from a fresh culture medium to which secologanin had previously been added. From this it follows that the occurrence of these undetected forms of secologanin is not due to a secologaninl medium interaction, but rather, in every case, to the presence of cells. 5. Attempts to identify the «undetected forms» of secologanin
Loganin and loganic acid can be converted in vivo into secologanin (Atta-ur-Rahman and Basha, 1983), but there is no data in the literature about the possibility of re-conversion of secologanin into loganin and loganic acid or the possibility of demethylation. On the other hand, loganin and loganic acid occur in Gentiana pedicellata as p-coumaroyl esters (Garcia and Chulia, 1986 and 1987). We investigated the possible occurrence of these various compounds in an attempt to identify the undetected forms of secologanin. Loganin was detected neither in the cells, nor in the media at any time during the experiments. The cells actually contained less than 0.05 Jlmole per flask (or less than 0.25 nmolel 106 cells during the stationary phase) and the culture media - less than 0.7 Jlmole per flask. Traces of loganic and secologanic acids too small to be measured were detected in the cells and media. Similarly, p-coumaroyl esters of loganin and loganic acid seemed to exist in the cells, but in quantities too small to be identified or measured with any certainty. These results appear to rule out that the conversion of loganic acid and loganin into secologanin is reversible even in the presence of an excess of secologanin (at least not under intracellular conditions). Thus, the temporary disappearance of secologanin in the cell suspensions could not be explained in this manner. 6. Conclusions
The weak alkaloid accumulation earlier observed to result from the introduction of secologanin (Merillon et aI., 1986) is not due to the relative inefficiency of the absorption system, since the secologanin furnished disappeared from the culture medium and accumulated in large quantities in the cell. Nor is it due to a degradation of secologanin in the culture medium. This is evidenced by the stability of secologanin introduced into a culture medium from which cells were removed by filtering a cell suspension.
612
F. NAUDASCHER, P. DOIREAU, A. GUILLOT, C. VIEL, and M. THIERSAULT
The phenomenon could possibly stem from part of the secologanin being trapped into an inactive pool. The intracellular localization of such a pool is currently under study. Furthermore, our observations suggested that the cells converted secologanin through a reversible process into other forms that these experiments have been unable to reveal. Moreover, one cannot completely rule out the hypothesis that endogenous secologanin accumulated following metabolic perturbations related to massive absorption of exogenous secologanin. Further study of these points will be undertaken as soon as radioactive secologanin becomes available. Acknowledgements The authors wish to thank Dr Tietze for supplying pure secologanin, T. Becue for registering the mass spectra and L. Troup for the preparation of the english manuscript.
References ATTA-UR-RAHMAN and A. BASHA: Biosynthesis of indole alkaloids. Clarendon Press, Oxford (1983). BATTERSBY, A. R., A. R. BURNETT, and P. G. PARSONS: Alkaloid biosynthesis. Part XIV. Secologanin: its conversion into ipecoside and its role as biological precursor of the indole alkaloids. J. Chern. Soc., C, 1187 -1192 (1969). BENTLEY, T. W. and R. A. W. JOHNSTONE: Aspects of mass spectra of organic compounds. Part IV. Cyclopentane monoterpenes of the iridoid group. J. Chern. Soc., C, 2234-2240 (1967). COSCIA, C. J., R. GUARNACCIA, and L. BOTTA: Monoterpene biosynthesis. I. Occurrence and mevalonoid origin of gentiopicroside and loganic acid in Swertia caroliniensis. Biochemistry, 8, 5036-5043 (1969).
DEUS, B. and M. H. ZENK: Exploitation of plant cells for the production of natural compounds. Biotechn. Bioengin., 24, 1965 -1974 (1982). DOIREAU, P., J. M. MERILLON, A. GUILLOT, M. RIDEAU, J. C. CHENIEUX, and M. BRILLARD: Time-course studies on indole alkaloid accumulation and changes in tryptophan decarboxylase and strictosidine synthase activities: a comparison in three strains of Catharanthus roseus cells. Planta Med., 53, 364-367 (1987). DOELLER, G., A. W. ALFERMANN, and E. REINHARD: Produktion von Indolalkaloiden in Calluskulturen von Catharanthus roseus. Planta Med., 30, 14-20 (1976). GARCIA, J. and A. J. CHULIA: Loganin and new iridoid glucosides in Gentiana pedicellata. Planta Med., 327 -329 (1986). - - 4'-p-coumaroyl iridoid glucosides from Gentiana pedicellata. Planta Med., 101-103 (1987). GUARNACCIA, R. and C. J. COSCIA: Occurrence and biosynthesis of secologanic acid in Vinca rosea. J. Am. Chern. Soc., 93, 6320-6321 (1971). KINAST, G. and L. F. TIETZE: Enzymatische Spaltung von Secologanin. Ein Modell zur Biogenese der Indolalkaloide. Chern. Ber., 109,3640-3645 (1976). MERILLON, J. M., J. C. CHENIEUX, and M. RIDEAU: Time-course of growth, evolution of sugar-nitrogen metabolism and accumulation of alkaloids in a cell suspension of Catharanthus roseus. Planta Med., 47, 169-176 (1983). MERILLON, J. M., P. DOIREAU, A. GUILLOT, J. C. CHENIEUX, and M. RIDEAU: Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media. Plant Cell Rep., 5, 23-26 (1986). MORRIS, P.: Regulation of product synthesis in cell cultures of Catharanthus roseus. II. Comparison of production media. Planta Med., 121-126 (1986). TANAHASHI, T., N. NAGAKURA, H. INOUYE, and M. H. ZENK: Radioimmunoassay for the determination of loganin and the biotransformation of loganin to secologanin by plant cell cultures. Phytochemistry, 23, 1917 -1922 (1984). VINAS, R. and A. PAREILLEUX: Production d'alcaloides par des suspensions cellulaires de Catharanthus roseus cultivees in vitro. Physiol. veg., 20, 219-225 (1982).