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Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures
Plant Science 162 (2002) 165– 172 www.elsevier.com/locate/plantsci
Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures Graziella Collu, Aurelio Alonso Garcia, Robert van der Heijden, Robert Verpoorte * Gorlaeus Laboratories, Di6ision of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, PO Box 9502, 2300 RA Leiden, The Netherlands Received 9 February 2001; received in revised form 12 October 2001; accepted 12 October 2001
Abstract Cell cultures of three different plant families well known for their terpenoid biosynthesis (Apocynaceae, Diervillaceae and Rubiaceae) were compared with respect to their activity of the cytochrome P450 enzyme geraniol 10-hydroxylase (G10H). G10H activity was only detected in cell cultures of species belonging to the Apocynaceae family. The highest levels of G10H activity (240 pkatal/mg protein) were found in cell cultures of Catharanthus roseus. Further increase of G10H activity (335 pkatal/mg protein) could be achieved by transferring the C. roseus cell culture to an induction medium known to enhance alkaloid production. It was shown that G10H activity is correlated to the ability of the cells to accumulate terpenoid indole alkaloids, although it seemed that increased G10H is not the only requirement for increased alkaloid accumulation. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Catharanthus roseus; Terpenoid indole alkaloid biosynthesis; Geraniol 10-hydroxylase; Cytochrome P450
1. Introduction Iridoids are monoterpenes having an iridane skeleton. They occur wide-spread in nature, mainly in dicotyledonous plant families like Apocynaceae, Diervillaceae, Lamiaceae, Loganiaceae and Rubiaceae. For a long time, iridoids were not considered particularly important as a pharmacologically active class of compounds. Recently, however, more extensive studies have revealed that iridoids exhibit a wide range of bioactivity, for example, cardiovascular activity, antitumor activity and antiviral activity [1]. Chemical interest in the iridoids was stimulated because of the key role played by secologanin in the biosynthesis of the pharmaceutically interesting indole alkaloids and certain quinoline alkaloids found in Apocynaceae, Loganiaceae and Rubiaceae [2–4]. The biosynthetic pathways leading to the iridoids have been investigated intensively and it seems that * Corresponding author. Tel.: + 31-71-527-4510; fax: +31-71-5274511. E-mail address: [email protected] (R. Verpoorte).
there are two main routes. The biosynthesis begins with the conversion of geraniol into 10-hydroxygeraniol. Subsequently, the dialcohol is oxidised to the corresponding dialdehyde and finally the cyclopentane ring is formed by cyclisation, a process which divides the biosynthesis into two pathways depending on whether iridodial or the epimer 8-epi-iridodial is formed [5–7]. Iridodial is the precursor for secologanin, which is a key intermediate in the biosynthesis of alkaloids containing a monoterpenoid skeleton [7]. In studies on the biosynthesis of indole alkaloids in Catharanthus roseus (Fig. 1), it was shown that the biosynthesis of secologanin is a limiting factor in the accumulation of the terpenoid indole alkaloids [8,9]. A potential site for regulatory control in the secologanin biosynthesis was proposed for geraniol 10-hydroxylase (G10H), as this cytochrome P450 enzyme (P450) catalyses the first committed step in the pathway. An argument for this concept was provided by McFarlane et al. [10], who showed that G10H is reversibly, non-competitively inhibited by the terpenoid indole alkaloid catharanthine. Also the alkaloids, vinblastine and vindoline, had slight inhibitory effects on G10H. The Ki of catha-
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ranthine inhibition (1 mM) was in the range of reported concentrations of this alkaloid in C. roseus plants (0.3– 1 mM). It was proposed that a feedback inhibition mechanism is present that modulates the pathway. More evidence for a regulatory function for G10H was demonstrated by Schiel et al. [11], who showed that G10H activities were increased when C. roseus cell cultures were transferred to an induction medium, known to enhance the accumulation of alkaloids [11]. It
was also shown that there was a close relationship between G10H activity and alkaloid accumulation. Since G10H might represent an important regulatory enzyme in the biosynthesis of terpenoid indole alkaloids, we were interested to find the gene encoding this enzyme. This gene could then be used for more detailed analysis on G10H regulation. Until now the cloning of the G10H gene was hampered by the fact that a large number of related P450s are present in C. roseus. This
Fig. 1. Biosynthesis of terpenoid indole and related quinoline alkaloids in Catharanthus and Cinchona. Solid lines indicate single steps, dashed lines indicate multiple steps. MEP, 2-C-methyl-D-erythritol-4-phosphate (after DAC Hallard, Division of Pharmacognosy, Leiden, unpublished).
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Table 1 Culture conditions of the different plant cell suspension cultures Family
Genus, species and cultivar
Cell culture
Medium
Apocynaceae
Catharanthus roseus (L.)G.Don Tabernaemontana di6aricata (L.)R.Br. ex Roem. And Schult. Tabernaemontana elegans Stapf Tabernaemontana pandacaqui Poir. Rau6olfia sellowii Mu¨ ll.Arg. Weigela ‘Styriaca’ Cinchona calisaya Cinchona ‘Robusta’ Rubia tinctorum L.
9Cr58 A12A2 Div BW101
MS9 MS6
Tel S4 Ori B101 Rsel Ws CiLed CiRo Rtinc
MS6 MS6 B5(15) B5(15) B5(25) B5(25) B5(12)
Diervillaceae Rubiaceae
Subculture scheme (days)
Dilution (factor)
7 7
4 2
7 7 7 7 10 7 7
2 2 2 2 2 2 2
The cell cultures were grown in 250 ml Erlenmeyer flasks under continuous light (2800 lux) at 25 °C on gyratory shakers at 120 rpm.
made a molecular approach using PCR primers designed on the highly conserved heme-binding domain of cytochrome P450 enzymes difficult [12]. In order to clone the gene it is, therefore, necessary to work out a purification method that will yield sufficient amounts of purified G10H to determine internal amino-acid sequences for the design of more specific PCR primers. A first step in the purification is the selection of a cell line that has a high G10H activity. The present study reports the screening of several different plant cell cultures for their G10H activity. Three plant families were selected, which are known to contain the iridoid pathway and from these families several species were selected for the screening experiment, C. roseus; Tabernaemontana di6aricata; T. elegans; T. pandacaqui and Rau6olfia sellowii (Apocynaceae), Weigela ‘Styriaca’ (Diervillaceae) and Cinchona calisaya, Cinchona ‘Robusta’ and Rubia tinctorum (Rubiaceae). After selecting the culture that has the highest G10H activity, we tried to further increase this activity by transferring the selected cell culture to different induction media. Finally, the relation between G10H activity and alkaloid accumulation in two C. roseus cell cultures with different alkaloid accumulating capacities was studied; one accumulates alkaloids, while in the other no alkaloids are detected. The results presented in this report give more insight in the proposed regulatory role of G10H in the biosynthesis of terpenoid indole alkaloids. 2. Materials and methods
2.1. Chemicals Geraniol (\99.5%) was obtained from Fluka (Buchs, Switzerland). 10-Hydroxygeraniol (97%) and tryptamine were purchased from Aldrich (WI, USA). Ajmalicine was from Roth (Karsruhe, Germany). All other chemicals were of the highest purity commercially available. Organic solvents were of analytical grade.
2.2. Cell culture media MS6 consisted of MS salts [13], 100 mg/l myo-inositol, 1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 2 mg/l glycine, 1 mg/l kinetin, 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 g/l sucrose. MS9 consisted of MS salts, 100 mg/l myo-inositol, 1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 2 mg/l glycine and 30 g/l sucrose. MS58 consisted of MS salts, 100 mg/l myo-inositol, 0.4 mg/l thiamine, 0.2 mg/l kinetin, 2 mg/l a-naphthaleneacetic acid (NAA), and 30 g/l sucrose. B5(12) consisted of B5 salts [14], 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 0.2 mg/l kinetin, 2 mg/l 2,4-D, 0.5 mg/l NAA, 0.5 mg/l indole-3-acetic acid (IAA) and 20 g/l sucrose. B5(15) consisted of B5 salts, 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 0.2 mg/l kinetin, 1 mg/l 2,4-D, and 40 g/l sucrose. B5(25) consisted of B5 salts, 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 50 mg/l L-cystein, 0.2 mg/l kinetin, 2 mg/l 2,4-D, and 20 g/l sucrose. IM78 consisted of IM2 salts [15], 100 mg/l myo-inositol, 0.1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, and 80 g/l sucrose. IM79 is as IM78, but depleted of all phosphate, nitrate and ammonium.
2.3. Cell cultures All cell suspension cultures were grown in 250 ml Erlenmeyer flasks under continuous light (2800 lux) at 25 °C on gyratory shakers at 120 rpm. Experiments were performed in duplicate.
2.4. Screening of different cell cultures for their G10H acti6ity Nine different plant cell suspension cultures were selected, all presumed to possess the iridoid pathway and consequently have G10H activity. They were
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grown and subcultured as described in Table 1. For the screening of G10H activity, Erlenmeyer flasks containing 50 ml of medium (Table 1) were inoculated with 5 g of cells (fresh weight, FCW) from a seven-days-old culture. For C. calisaya a 10-days-old culture was used.
2.5. Induction of G10H acti6ity For the induction experiment with the cell culture of C. roseus (9Cr58 A12A2), Erlenmeyer flasks containing 50 ml of IM78 or IM79 were inoculated with 5 g of FCW from the selected culture.
2.6. The relation between G10H acti6ity and alkaloid accumulation For this experiment two C. roseus cell cultures were selected. One cell culture (cell line 58CrPM) was grown and maintained in MS58 (a medium with growth hormones) since 1983. The other was initiated in 1988 by transferring the above mentioned C. roseus cell culture into MS9 medium (a medium without growth hormones). The thus obtained cell culture was further grown and maintained in MS9 (9Cr58 A12A2). 9Cr58 A12A2 developed in an alkaloid accumulating cell culture, while 58CrPM became a non-accumulating (below detection limit) cell culture. Both cell cultures were subcultured every 7 days by a 4-fold dilution and maintained under the same culture conditions. For the G10H-alkaloid relation experiment, Erlenmeyer flasks containing 50 ml of MS9 or MS58 were inoculated with 5 g of FCW from either the accumulating or non-accumulating C. roseus cell culture, respectively. To investigate whether transfer to induction medium could increase alkaloid accumulation and G10H activity, both cell cultures were also transferred to IM78 as described in the induction experiment.
2.7. Har6esting Cultures were harvested every second day (in duplicate). The cells were separated from the medium by filtration over a glass filter (G3) under suction, washed with water, weighed for FW determination, frozen in liquid nitrogen and stored at − 80 °C until use. The volume of the culture medium was measured and part of it was stored at −80 °C.
2.8. Determination of G10H acti6ities Membrane fractions were prepared as described previously [16] and stored at − 80 °C until use. Protein was determined according to Bradford [17] using bovine serum albumin (BSA) as a standard. G10H activities were determined as described [16]
2.9. Alkaloid analysis The alkaloid extraction was performed as described before [18]. Freeze-dried cell material (50 mg) was suspended in 0.5 ml water and mixed thoroughly with 5 ml dichloromethane for 1 min. After centrifugation the organic layer was removed and the residue was again extracted with 5 ml dichloromethane and centrifuged. Then both organic phases were combined. For the extraction of tryptamine, 0.5 ml 1 M NaOH was added to the residue and again an extraction with 5 ml dichloromethane was performed. The culture medium (2 ml) was extracted with 4 ml dichloromethane by vortexing for 1 min. The organic phases were evaporated separately under vacuum and the residues were dissolved in 0.5 ml high performance liquid chromatography (HPLC) eluent (50 mM sodium phosphate (pH 3.9)–acetonitrile– 2methoxyethanol (80:15:5, v/v). The alkaloids were identified by HPLC analysis equipped with a photodiode-array detector [19] and by TLC analysis using different spray-reagents for visualisation [20].
3. Results and discussion
3.1. Screening of different cell cultures for their G10H acti6ity From the nine different cell cultures studied, only in three G10H activity was detected, C. roseus; T. di6aricata and R. sellowii. These species all belong to the Apocynaceae family. The species belonging to the families Diervillaceae and Rubiaceae did not show any detectable G10H activity. Yet, the cultures showed normal growth profiles (data not shown). Probably, these cell cultures do either not express the gene encoding the G10H enzyme or the enzyme is not active under the chosen conditions. The highest G10H activity of 240 pkatal/mg protein was found in the C. roseus cell cultures during the exponential phase of growth (Fig. 2A; 4 days after inoculation); subsequently the G10H activity decreased gradually. R. sellowii cell cultures reached a maximum G10H activity of 45 pkatal/mg protein in the lag-phase (Fig. 2B; 2 days after inoculation). When these cultures reached the stationary phase of growth, the G10H activity had almost disappeared. In T. di6aricata the G10H activity was highest in the exponential phase of growth (Fig. 2C; 14 pkatal/mg protein after 10 days of inoculation). G10H activities in cell cultures of C. roseus and T. di6aricata have been reported before. For C. roseus cell cultures G10H activities have been measured ranging from 5 to 80 pkatal/mg protein depending on the media in which the cells are grown [11,21,22]. The C. roseus
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cell culture used in the screening experiment exhibited a three times higher G10H activity than the maximum activity reported in literature. Also for T. di6aricata cell cultures differences in G10H activity according to the medium composition have been reported. Here the activity ranged from 4 to 42 pkatal/mg protein [21,23]. The T. di6aricata cell culture used in the screening experiment showed a G10H activity similar to those reported in literature. In the other two Tabernaemontana species, T. pandacaqui and T. elegans, we could not detect any G10H activity, although they were grown in the same medium as T. di6aricata. Also earlier work in our laboratory on the T. pandacaqui cell culture failed to detect any G10H activity [21]. The involvement of the iridoid pathway in the biosynthesis of the indole alkaloids in Rau6olfia species has been demonstrated by labelling experiments with 10-hydroxygeraniol and crude enzyme extracts from R. serpentina [24]. However, to our knowledge, this is the first report showing G10H activity in a Rau6olfia cell culture. Also for the Cinchona alkaloids it has been established that the iridoid pathway plays a role in supplying secologanin which then condenses with tryptamine in the same way as for the indole alkaloids [3]. However, the pathway is more complicated and includes rearrangements both in the iridoid moiety and in the indole part of the structure. Furthermore, the alkaloid biosynthesis in Cinchona has to compete for some precursors with the anthraquinone biosynthesis [25]. Efforts to produce the Cinchona alkaloids by means of in vitro cultured cells have so far not been very successful, mainly because the cell cultures suffered from a rapid decrease of alkaloid productivity upon prolonged subculturing [3]. In the screening experiment no G10H activities could be detected in the Cinchona cell cultures. Earlier research in our group on the Cinchona cell cultures showed that other enzymes involved in the biosynthesis of the alkaloids suffered from a decrease in activity upon prolonged subculturing [26,27]. In summary, it seems that the Cinchona cell cultures not only show instability with respect to alkaloid production but
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also with respect to enzyme activities. R. tinctorum and Weigela ‘Styriaca’ plants do not produce alkaloids because they lack the indole pathway. Although these plants do produce iridoids, their cell cultures do not show any G10H activity. In conclusion, the highest G10H activities are found in C. roseus cell cultures. Hence, we selected this cell culture for further investigation on induction of G10H activity.
3.2. Induction of G10H acti6ity As stated in the introduction G10H activities can be increased when cell cultures are transferred to an induction medium, known to enhance alkaloid accumulation. Therefore, we investigated if transferring our selected C. roseus cells to an induction medium could further increase G10H activity. When the selected cell culture of C. roseus (9Cr58 A12A2) was grown in MS9 growth medium, an increase of G10H activity was observed during the first days of growth as was shown in Fig. 2A, after which the activity declined steadily. After transferring this cell culture to induction medium IM78, the same rapid increase of G10H activity was observed during the first days of growth. However, unlike in MS9 growth medium, the G10H activity continued to increase and reached a maximum after 10 days of inoculation (Fig. 3B; 335 pkatal/mg protein). When the selected C. roseus cell culture was transferred to an induction medium depleted of nitrate, phosphate and ammonium (IM79), which all are components known to inhibit alkaloid accumulation [28], the G10H activity increased 1.3-fold (300 pkatal/mg protein after 6 days of culture). Subsequently the activity declined rapidly after 6 days. The results above show that the induced G10H activity levels (335 pkatal/mg protein) obtained after transferring a C. roseus cell line grown in MS9 growth medium to induction medium IM78, make this cell material the best source for G10H purification.
Fig. 2. Biomass accumulation (fresh weight, g/l culture, ) and geraniol 10-hydroxylase activity (pkatal/mg protein, ) in cell cultures of Catharanthus roseus (A); R. selowii (B); and Tabernaemontana di6aricata (C). Indicated are the mean values; bars represent individual values.
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Fig. 3. G10H activity (pkatal/mg protein, ) and ajmalicine (mg/g DW, ) and tryptamine (mg/g DW, ) accumulation in cell cultures of Catharanthus roseus (9Cr58 A12A2) grown in growth medium without growth hormones MS9 (A) and in induction medium IM78 (B). Indicated are the mean values; bars represent individual values.
3.3. The relation between G10H acti6ity and alkaloid accumulation Finally, we studied the relation between G10H activity and alkaloid accumulation in two C. roseus cell cultures with different alkaloid accumulating capacities. One cell culture accumulates alkaloids (9Cr58 A12A2), while in the other (58CrPM) no alkaloids are detected (below detection limit). We followed the G10H activity pattern in these cell cultures when grown in normal growth media and when transferred to induction medium IM78. This would give more information about the proposed regulatory role of G10H in the biosynthesis of terpenoid indole alkaloids. When the non-accumulating cell culture of C. roseus (58CrPM) was grown in normal growth medium (MS58), only low levels of G10H activity were detected (Fig. 4A; 5 pkatal/mg protein after 4 days of culture). This cell line produced high amounts of tryptamine (maximum 760 mg/g DW after 12 days of culture). However, no terpenoid indole alkaloids were found. When this culture was transferred to induction medium IM78 medium, the G10H activity increased approximately 10-fold (Fig. 4B; 70 pkatal/mg protein after 4 days of culture). Concomitantly, the amount of tryptamine decreased 10-fold and small amounts of strictosidine could be detected (maximum 39 mg/g DW after 14 days of culture). However, no other alkaloids like ajmalicine were formed, neither in the cells nor in the medium. Therefore, it seems that the formed strictosidine is not further metabolised into other alkaloids. These results imply that the induction of G10H seems to be required before terpenoid indole alkaloid production can occur. However, probably a step beyond strictosidine is limiting for the production of other alkaloids. Hence induction of G10H is not the only requirement necessary to produce other terpenoid indole alkaloids then strictosidine.
When the alkaloid accumulating cell culture of C. roseus (9Cr58 A12A2) was grown in normal growth medium (MS9), a specific G10H activity of 230 pkatal/ mg protein was measured after 4 days of culture (Fig. 3A). This cell culture also accumulates tryptamine (maximum 400 mg/g DW after 14 days of culture) and the alkaloid ajmalicine reached a maximum just after the peak of G10H activity (maximunm 142 mg/g DW after 10 days of culture). Also another alkaloid, vindolinine, was detected in this cell line. One may speculate that in this cell culture tryptamine is accumulated because insufficient amounts of secologanin are available from the iridoid pathway to condense with the available tryptamine. After transferring the 9Cr58 A12A2 culture to induction medium IM78, the G10H activity increased 1.5fold (Fig. 2B; 335 pkatal/mg protein after 10 days of culture) and the amount of tryptamine decreased concomitantly. Surprisingly, no increase in alkaloid concentrations could be detected in the cells. However, analysis of the medium showed that different indole alkaloids such as ajmalicine, vindolinine and tabersonine were excreted into the medium (e.g. after 18 days, 2.7 mg ajmalicine/l medium). This excretion of alkaloids into the medium could be explained by the acidification of the medium and the hypothesis that alkaloids accumulate by an ion-trap mechanism in the place with the lowest pH [29]. Transferring the cells to induction medium caused a gradual decrease of the medium pH, ending approximately 1 pH U lower after 20 days compared with cells growing in normal growth medium. Thus, also in the alkaloid accumulating C. roseus cell culture, it is clear that the G10H activity is correlated to the ability of the cells to accumulate alkaloids. Overall, our results support the proposed regulatory role of the G10H enzyme in the iridoid and alkaloid biosynthesis [10,11,21]. However, apparently increased
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Fig. 4. G10H activity (pkatal/mg protein, ) and ajmalicine (mg/g DW, ) and tryptamine (mg/g DW, ) accumulation in cell cultures of Catharanthus roseus (58CrPM) grown in growth medium with growth hormones MS58 (A) and in induction medium IM78 (B). Indicated are the mean values; bars represent individual values.
G10H activity is not the only requirement for increased alkaloid accumulation. The possibility that other enzymes as well will limit terpenoid indole alkaloid accumulation is not ruled out. Moreover, this screening experiment has resulted in the selection of a cell line (C. roseus (9Cr58 A12A2) in induction medium) that produced a four times higher specific G10H activity compared with the values reported in literature, which makes this cell material a good source for G10H purification.
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Activities of enzymes involved in indole alkaloid biosynthesis in suspension cultures of Catharanthus, Cinchona and Tabernaemontana species, Plant Physiol. Biochem. 30 (1992) 675 –681. [27] C. Poulsen, R. Verpoorte, Activities of chorismate utilizing enzymes and of enzymes involved in indole alkaloid biosynthesis in cell suspension cultures, Plant Physiol. Biochem. 30 (1992) 105 – 113. [28] K.H. Knobloch, J. Berlin, Influence of medium composition on the formation of secondary compounds in cell suspension cultures of Catharanthus roseus (L.)G.Don, Z. Naturforsch. 35c (1980) 551 – 556. [29] T.J.M. Blom, M.I. Sierra, T.B. van Vliet, M.E.I. Franke-van Dijk, P. de Koning, F. van Iren, R. Verpoorte, K.R. Libbenga, Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.)G.Don. and its conversion into serpentine, Planta 183 (1991) 170 – 177.
Activity of the cytochrome P450 enzyme geraniol 10-hydroxylase and alkaloid production in plant cell cultures Graziella Collu, Aurelio Alonso Garcia, Robert van der Heijden, Robert Verpoorte * Gorlaeus Laboratories, Di6ision of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, PO Box 9502, 2300 RA Leiden, The Netherlands Received 9 February 2001; received in revised form 12 October 2001; accepted 12 October 2001
Abstract Cell cultures of three different plant families well known for their terpenoid biosynthesis (Apocynaceae, Diervillaceae and Rubiaceae) were compared with respect to their activity of the cytochrome P450 enzyme geraniol 10-hydroxylase (G10H). G10H activity was only detected in cell cultures of species belonging to the Apocynaceae family. The highest levels of G10H activity (240 pkatal/mg protein) were found in cell cultures of Catharanthus roseus. Further increase of G10H activity (335 pkatal/mg protein) could be achieved by transferring the C. roseus cell culture to an induction medium known to enhance alkaloid production. It was shown that G10H activity is correlated to the ability of the cells to accumulate terpenoid indole alkaloids, although it seemed that increased G10H is not the only requirement for increased alkaloid accumulation. © 2002 Published by Elsevier Science Ireland Ltd. Keywords: Catharanthus roseus; Terpenoid indole alkaloid biosynthesis; Geraniol 10-hydroxylase; Cytochrome P450
1. Introduction Iridoids are monoterpenes having an iridane skeleton. They occur wide-spread in nature, mainly in dicotyledonous plant families like Apocynaceae, Diervillaceae, Lamiaceae, Loganiaceae and Rubiaceae. For a long time, iridoids were not considered particularly important as a pharmacologically active class of compounds. Recently, however, more extensive studies have revealed that iridoids exhibit a wide range of bioactivity, for example, cardiovascular activity, antitumor activity and antiviral activity [1]. Chemical interest in the iridoids was stimulated because of the key role played by secologanin in the biosynthesis of the pharmaceutically interesting indole alkaloids and certain quinoline alkaloids found in Apocynaceae, Loganiaceae and Rubiaceae [2–4]. The biosynthetic pathways leading to the iridoids have been investigated intensively and it seems that * Corresponding author. Tel.: + 31-71-527-4510; fax: +31-71-5274511. E-mail address: [email protected] (R. Verpoorte).
there are two main routes. The biosynthesis begins with the conversion of geraniol into 10-hydroxygeraniol. Subsequently, the dialcohol is oxidised to the corresponding dialdehyde and finally the cyclopentane ring is formed by cyclisation, a process which divides the biosynthesis into two pathways depending on whether iridodial or the epimer 8-epi-iridodial is formed [5–7]. Iridodial is the precursor for secologanin, which is a key intermediate in the biosynthesis of alkaloids containing a monoterpenoid skeleton [7]. In studies on the biosynthesis of indole alkaloids in Catharanthus roseus (Fig. 1), it was shown that the biosynthesis of secologanin is a limiting factor in the accumulation of the terpenoid indole alkaloids [8,9]. A potential site for regulatory control in the secologanin biosynthesis was proposed for geraniol 10-hydroxylase (G10H), as this cytochrome P450 enzyme (P450) catalyses the first committed step in the pathway. An argument for this concept was provided by McFarlane et al. [10], who showed that G10H is reversibly, non-competitively inhibited by the terpenoid indole alkaloid catharanthine. Also the alkaloids, vinblastine and vindoline, had slight inhibitory effects on G10H. The Ki of catha-
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ranthine inhibition (1 mM) was in the range of reported concentrations of this alkaloid in C. roseus plants (0.3– 1 mM). It was proposed that a feedback inhibition mechanism is present that modulates the pathway. More evidence for a regulatory function for G10H was demonstrated by Schiel et al. [11], who showed that G10H activities were increased when C. roseus cell cultures were transferred to an induction medium, known to enhance the accumulation of alkaloids [11]. It
was also shown that there was a close relationship between G10H activity and alkaloid accumulation. Since G10H might represent an important regulatory enzyme in the biosynthesis of terpenoid indole alkaloids, we were interested to find the gene encoding this enzyme. This gene could then be used for more detailed analysis on G10H regulation. Until now the cloning of the G10H gene was hampered by the fact that a large number of related P450s are present in C. roseus. This
Fig. 1. Biosynthesis of terpenoid indole and related quinoline alkaloids in Catharanthus and Cinchona. Solid lines indicate single steps, dashed lines indicate multiple steps. MEP, 2-C-methyl-D-erythritol-4-phosphate (after DAC Hallard, Division of Pharmacognosy, Leiden, unpublished).
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Table 1 Culture conditions of the different plant cell suspension cultures Family
Genus, species and cultivar
Cell culture
Medium
Apocynaceae
Catharanthus roseus (L.)G.Don Tabernaemontana di6aricata (L.)R.Br. ex Roem. And Schult. Tabernaemontana elegans Stapf Tabernaemontana pandacaqui Poir. Rau6olfia sellowii Mu¨ ll.Arg. Weigela ‘Styriaca’ Cinchona calisaya Cinchona ‘Robusta’ Rubia tinctorum L.
9Cr58 A12A2 Div BW101
MS9 MS6
Tel S4 Ori B101 Rsel Ws CiLed CiRo Rtinc
MS6 MS6 B5(15) B5(15) B5(25) B5(25) B5(12)
Diervillaceae Rubiaceae
Subculture scheme (days)
Dilution (factor)
7 7
4 2
7 7 7 7 10 7 7
2 2 2 2 2 2 2
The cell cultures were grown in 250 ml Erlenmeyer flasks under continuous light (2800 lux) at 25 °C on gyratory shakers at 120 rpm.
made a molecular approach using PCR primers designed on the highly conserved heme-binding domain of cytochrome P450 enzymes difficult [12]. In order to clone the gene it is, therefore, necessary to work out a purification method that will yield sufficient amounts of purified G10H to determine internal amino-acid sequences for the design of more specific PCR primers. A first step in the purification is the selection of a cell line that has a high G10H activity. The present study reports the screening of several different plant cell cultures for their G10H activity. Three plant families were selected, which are known to contain the iridoid pathway and from these families several species were selected for the screening experiment, C. roseus; Tabernaemontana di6aricata; T. elegans; T. pandacaqui and Rau6olfia sellowii (Apocynaceae), Weigela ‘Styriaca’ (Diervillaceae) and Cinchona calisaya, Cinchona ‘Robusta’ and Rubia tinctorum (Rubiaceae). After selecting the culture that has the highest G10H activity, we tried to further increase this activity by transferring the selected cell culture to different induction media. Finally, the relation between G10H activity and alkaloid accumulation in two C. roseus cell cultures with different alkaloid accumulating capacities was studied; one accumulates alkaloids, while in the other no alkaloids are detected. The results presented in this report give more insight in the proposed regulatory role of G10H in the biosynthesis of terpenoid indole alkaloids. 2. Materials and methods
2.1. Chemicals Geraniol (\99.5%) was obtained from Fluka (Buchs, Switzerland). 10-Hydroxygeraniol (97%) and tryptamine were purchased from Aldrich (WI, USA). Ajmalicine was from Roth (Karsruhe, Germany). All other chemicals were of the highest purity commercially available. Organic solvents were of analytical grade.
2.2. Cell culture media MS6 consisted of MS salts [13], 100 mg/l myo-inositol, 1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 2 mg/l glycine, 1 mg/l kinetin, 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 g/l sucrose. MS9 consisted of MS salts, 100 mg/l myo-inositol, 1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, 2 mg/l glycine and 30 g/l sucrose. MS58 consisted of MS salts, 100 mg/l myo-inositol, 0.4 mg/l thiamine, 0.2 mg/l kinetin, 2 mg/l a-naphthaleneacetic acid (NAA), and 30 g/l sucrose. B5(12) consisted of B5 salts [14], 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 0.2 mg/l kinetin, 2 mg/l 2,4-D, 0.5 mg/l NAA, 0.5 mg/l indole-3-acetic acid (IAA) and 20 g/l sucrose. B5(15) consisted of B5 salts, 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 0.2 mg/l kinetin, 1 mg/l 2,4-D, and 40 g/l sucrose. B5(25) consisted of B5 salts, 100 mg/l myo-inositol, 10 mg/l thiamine, 1 mg/l pyridoxine, 1 mg/l nicotinic acid, 50 mg/l L-cystein, 0.2 mg/l kinetin, 2 mg/l 2,4-D, and 20 g/l sucrose. IM78 consisted of IM2 salts [15], 100 mg/l myo-inositol, 0.1 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid, and 80 g/l sucrose. IM79 is as IM78, but depleted of all phosphate, nitrate and ammonium.
2.3. Cell cultures All cell suspension cultures were grown in 250 ml Erlenmeyer flasks under continuous light (2800 lux) at 25 °C on gyratory shakers at 120 rpm. Experiments were performed in duplicate.
2.4. Screening of different cell cultures for their G10H acti6ity Nine different plant cell suspension cultures were selected, all presumed to possess the iridoid pathway and consequently have G10H activity. They were
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grown and subcultured as described in Table 1. For the screening of G10H activity, Erlenmeyer flasks containing 50 ml of medium (Table 1) were inoculated with 5 g of cells (fresh weight, FCW) from a seven-days-old culture. For C. calisaya a 10-days-old culture was used.
2.5. Induction of G10H acti6ity For the induction experiment with the cell culture of C. roseus (9Cr58 A12A2), Erlenmeyer flasks containing 50 ml of IM78 or IM79 were inoculated with 5 g of FCW from the selected culture.
2.6. The relation between G10H acti6ity and alkaloid accumulation For this experiment two C. roseus cell cultures were selected. One cell culture (cell line 58CrPM) was grown and maintained in MS58 (a medium with growth hormones) since 1983. The other was initiated in 1988 by transferring the above mentioned C. roseus cell culture into MS9 medium (a medium without growth hormones). The thus obtained cell culture was further grown and maintained in MS9 (9Cr58 A12A2). 9Cr58 A12A2 developed in an alkaloid accumulating cell culture, while 58CrPM became a non-accumulating (below detection limit) cell culture. Both cell cultures were subcultured every 7 days by a 4-fold dilution and maintained under the same culture conditions. For the G10H-alkaloid relation experiment, Erlenmeyer flasks containing 50 ml of MS9 or MS58 were inoculated with 5 g of FCW from either the accumulating or non-accumulating C. roseus cell culture, respectively. To investigate whether transfer to induction medium could increase alkaloid accumulation and G10H activity, both cell cultures were also transferred to IM78 as described in the induction experiment.
2.7. Har6esting Cultures were harvested every second day (in duplicate). The cells were separated from the medium by filtration over a glass filter (G3) under suction, washed with water, weighed for FW determination, frozen in liquid nitrogen and stored at − 80 °C until use. The volume of the culture medium was measured and part of it was stored at −80 °C.
2.8. Determination of G10H acti6ities Membrane fractions were prepared as described previously [16] and stored at − 80 °C until use. Protein was determined according to Bradford [17] using bovine serum albumin (BSA) as a standard. G10H activities were determined as described [16]
2.9. Alkaloid analysis The alkaloid extraction was performed as described before [18]. Freeze-dried cell material (50 mg) was suspended in 0.5 ml water and mixed thoroughly with 5 ml dichloromethane for 1 min. After centrifugation the organic layer was removed and the residue was again extracted with 5 ml dichloromethane and centrifuged. Then both organic phases were combined. For the extraction of tryptamine, 0.5 ml 1 M NaOH was added to the residue and again an extraction with 5 ml dichloromethane was performed. The culture medium (2 ml) was extracted with 4 ml dichloromethane by vortexing for 1 min. The organic phases were evaporated separately under vacuum and the residues were dissolved in 0.5 ml high performance liquid chromatography (HPLC) eluent (50 mM sodium phosphate (pH 3.9)–acetonitrile– 2methoxyethanol (80:15:5, v/v). The alkaloids were identified by HPLC analysis equipped with a photodiode-array detector [19] and by TLC analysis using different spray-reagents for visualisation [20].
3. Results and discussion
3.1. Screening of different cell cultures for their G10H acti6ity From the nine different cell cultures studied, only in three G10H activity was detected, C. roseus; T. di6aricata and R. sellowii. These species all belong to the Apocynaceae family. The species belonging to the families Diervillaceae and Rubiaceae did not show any detectable G10H activity. Yet, the cultures showed normal growth profiles (data not shown). Probably, these cell cultures do either not express the gene encoding the G10H enzyme or the enzyme is not active under the chosen conditions. The highest G10H activity of 240 pkatal/mg protein was found in the C. roseus cell cultures during the exponential phase of growth (Fig. 2A; 4 days after inoculation); subsequently the G10H activity decreased gradually. R. sellowii cell cultures reached a maximum G10H activity of 45 pkatal/mg protein in the lag-phase (Fig. 2B; 2 days after inoculation). When these cultures reached the stationary phase of growth, the G10H activity had almost disappeared. In T. di6aricata the G10H activity was highest in the exponential phase of growth (Fig. 2C; 14 pkatal/mg protein after 10 days of inoculation). G10H activities in cell cultures of C. roseus and T. di6aricata have been reported before. For C. roseus cell cultures G10H activities have been measured ranging from 5 to 80 pkatal/mg protein depending on the media in which the cells are grown [11,21,22]. The C. roseus
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cell culture used in the screening experiment exhibited a three times higher G10H activity than the maximum activity reported in literature. Also for T. di6aricata cell cultures differences in G10H activity according to the medium composition have been reported. Here the activity ranged from 4 to 42 pkatal/mg protein [21,23]. The T. di6aricata cell culture used in the screening experiment showed a G10H activity similar to those reported in literature. In the other two Tabernaemontana species, T. pandacaqui and T. elegans, we could not detect any G10H activity, although they were grown in the same medium as T. di6aricata. Also earlier work in our laboratory on the T. pandacaqui cell culture failed to detect any G10H activity [21]. The involvement of the iridoid pathway in the biosynthesis of the indole alkaloids in Rau6olfia species has been demonstrated by labelling experiments with 10-hydroxygeraniol and crude enzyme extracts from R. serpentina [24]. However, to our knowledge, this is the first report showing G10H activity in a Rau6olfia cell culture. Also for the Cinchona alkaloids it has been established that the iridoid pathway plays a role in supplying secologanin which then condenses with tryptamine in the same way as for the indole alkaloids [3]. However, the pathway is more complicated and includes rearrangements both in the iridoid moiety and in the indole part of the structure. Furthermore, the alkaloid biosynthesis in Cinchona has to compete for some precursors with the anthraquinone biosynthesis [25]. Efforts to produce the Cinchona alkaloids by means of in vitro cultured cells have so far not been very successful, mainly because the cell cultures suffered from a rapid decrease of alkaloid productivity upon prolonged subculturing [3]. In the screening experiment no G10H activities could be detected in the Cinchona cell cultures. Earlier research in our group on the Cinchona cell cultures showed that other enzymes involved in the biosynthesis of the alkaloids suffered from a decrease in activity upon prolonged subculturing [26,27]. In summary, it seems that the Cinchona cell cultures not only show instability with respect to alkaloid production but
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also with respect to enzyme activities. R. tinctorum and Weigela ‘Styriaca’ plants do not produce alkaloids because they lack the indole pathway. Although these plants do produce iridoids, their cell cultures do not show any G10H activity. In conclusion, the highest G10H activities are found in C. roseus cell cultures. Hence, we selected this cell culture for further investigation on induction of G10H activity.
3.2. Induction of G10H acti6ity As stated in the introduction G10H activities can be increased when cell cultures are transferred to an induction medium, known to enhance alkaloid accumulation. Therefore, we investigated if transferring our selected C. roseus cells to an induction medium could further increase G10H activity. When the selected cell culture of C. roseus (9Cr58 A12A2) was grown in MS9 growth medium, an increase of G10H activity was observed during the first days of growth as was shown in Fig. 2A, after which the activity declined steadily. After transferring this cell culture to induction medium IM78, the same rapid increase of G10H activity was observed during the first days of growth. However, unlike in MS9 growth medium, the G10H activity continued to increase and reached a maximum after 10 days of inoculation (Fig. 3B; 335 pkatal/mg protein). When the selected C. roseus cell culture was transferred to an induction medium depleted of nitrate, phosphate and ammonium (IM79), which all are components known to inhibit alkaloid accumulation [28], the G10H activity increased 1.3-fold (300 pkatal/mg protein after 6 days of culture). Subsequently the activity declined rapidly after 6 days. The results above show that the induced G10H activity levels (335 pkatal/mg protein) obtained after transferring a C. roseus cell line grown in MS9 growth medium to induction medium IM78, make this cell material the best source for G10H purification.
Fig. 2. Biomass accumulation (fresh weight, g/l culture, ) and geraniol 10-hydroxylase activity (pkatal/mg protein, ) in cell cultures of Catharanthus roseus (A); R. selowii (B); and Tabernaemontana di6aricata (C). Indicated are the mean values; bars represent individual values.
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Fig. 3. G10H activity (pkatal/mg protein, ) and ajmalicine (mg/g DW, ) and tryptamine (mg/g DW, ) accumulation in cell cultures of Catharanthus roseus (9Cr58 A12A2) grown in growth medium without growth hormones MS9 (A) and in induction medium IM78 (B). Indicated are the mean values; bars represent individual values.
3.3. The relation between G10H acti6ity and alkaloid accumulation Finally, we studied the relation between G10H activity and alkaloid accumulation in two C. roseus cell cultures with different alkaloid accumulating capacities. One cell culture accumulates alkaloids (9Cr58 A12A2), while in the other (58CrPM) no alkaloids are detected (below detection limit). We followed the G10H activity pattern in these cell cultures when grown in normal growth media and when transferred to induction medium IM78. This would give more information about the proposed regulatory role of G10H in the biosynthesis of terpenoid indole alkaloids. When the non-accumulating cell culture of C. roseus (58CrPM) was grown in normal growth medium (MS58), only low levels of G10H activity were detected (Fig. 4A; 5 pkatal/mg protein after 4 days of culture). This cell line produced high amounts of tryptamine (maximum 760 mg/g DW after 12 days of culture). However, no terpenoid indole alkaloids were found. When this culture was transferred to induction medium IM78 medium, the G10H activity increased approximately 10-fold (Fig. 4B; 70 pkatal/mg protein after 4 days of culture). Concomitantly, the amount of tryptamine decreased 10-fold and small amounts of strictosidine could be detected (maximum 39 mg/g DW after 14 days of culture). However, no other alkaloids like ajmalicine were formed, neither in the cells nor in the medium. Therefore, it seems that the formed strictosidine is not further metabolised into other alkaloids. These results imply that the induction of G10H seems to be required before terpenoid indole alkaloid production can occur. However, probably a step beyond strictosidine is limiting for the production of other alkaloids. Hence induction of G10H is not the only requirement necessary to produce other terpenoid indole alkaloids then strictosidine.
When the alkaloid accumulating cell culture of C. roseus (9Cr58 A12A2) was grown in normal growth medium (MS9), a specific G10H activity of 230 pkatal/ mg protein was measured after 4 days of culture (Fig. 3A). This cell culture also accumulates tryptamine (maximum 400 mg/g DW after 14 days of culture) and the alkaloid ajmalicine reached a maximum just after the peak of G10H activity (maximunm 142 mg/g DW after 10 days of culture). Also another alkaloid, vindolinine, was detected in this cell line. One may speculate that in this cell culture tryptamine is accumulated because insufficient amounts of secologanin are available from the iridoid pathway to condense with the available tryptamine. After transferring the 9Cr58 A12A2 culture to induction medium IM78, the G10H activity increased 1.5fold (Fig. 2B; 335 pkatal/mg protein after 10 days of culture) and the amount of tryptamine decreased concomitantly. Surprisingly, no increase in alkaloid concentrations could be detected in the cells. However, analysis of the medium showed that different indole alkaloids such as ajmalicine, vindolinine and tabersonine were excreted into the medium (e.g. after 18 days, 2.7 mg ajmalicine/l medium). This excretion of alkaloids into the medium could be explained by the acidification of the medium and the hypothesis that alkaloids accumulate by an ion-trap mechanism in the place with the lowest pH [29]. Transferring the cells to induction medium caused a gradual decrease of the medium pH, ending approximately 1 pH U lower after 20 days compared with cells growing in normal growth medium. Thus, also in the alkaloid accumulating C. roseus cell culture, it is clear that the G10H activity is correlated to the ability of the cells to accumulate alkaloids. Overall, our results support the proposed regulatory role of the G10H enzyme in the iridoid and alkaloid biosynthesis [10,11,21]. However, apparently increased
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Fig. 4. G10H activity (pkatal/mg protein, ) and ajmalicine (mg/g DW, ) and tryptamine (mg/g DW, ) accumulation in cell cultures of Catharanthus roseus (58CrPM) grown in growth medium with growth hormones MS58 (A) and in induction medium IM78 (B). Indicated are the mean values; bars represent individual values.
G10H activity is not the only requirement for increased alkaloid accumulation. The possibility that other enzymes as well will limit terpenoid indole alkaloid accumulation is not ruled out. Moreover, this screening experiment has resulted in the selection of a cell line (C. roseus (9Cr58 A12A2) in induction medium) that produced a four times higher specific G10H activity compared with the values reported in literature, which makes this cell material a good source for G10H purification.
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Activities of enzymes involved in indole alkaloid biosynthesis in suspension cultures of Catharanthus, Cinchona and Tabernaemontana species, Plant Physiol. Biochem. 30 (1992) 675 –681. [27] C. Poulsen, R. Verpoorte, Activities of chorismate utilizing enzymes and of enzymes involved in indole alkaloid biosynthesis in cell suspension cultures, Plant Physiol. Biochem. 30 (1992) 105 – 113. [28] K.H. Knobloch, J. Berlin, Influence of medium composition on the formation of secondary compounds in cell suspension cultures of Catharanthus roseus (L.)G.Don, Z. Naturforsch. 35c (1980) 551 – 556. [29] T.J.M. Blom, M.I. Sierra, T.B. van Vliet, M.E.I. Franke-van Dijk, P. de Koning, F. van Iren, R. Verpoorte, K.R. Libbenga, Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cells of Catharanthus roseus (L.)G.Don. and its conversion into serpentine, Planta 183 (1991) 170 – 177.