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Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Stevia rebaudiana Bertoni
Plant Science 161 (2001) 155– 163 www.elsevier.com/locate/plantsci
Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Ste6ia rebaudiana Bertoni Nikolai Bondarev *, Oxana Reshetnyak, Alexander Nosov Timiryaze6 Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Received 7 August 2000; received in revised form 14 March 2001; accepted 14 March 2001
Abstract The composition and content of steviol-glycosides (SGs) 1 in in vitro cultures of stevia (Ste6ia rebaudiana Bertoni) were investigated. A comparative analysis of production of these compounds in intact plants, in vitro plants, dedifferentiated callus and suspension cultures, morphogenic callus and in vitro regenerated shoots were conducted. Qualitative composition of the SGs in in vitro plants was found to be identical to that of intact plants, but their content in the former plants appeared to be about five or six times lower. A significant decrease in this value was not observed upon long-term cultivation (for about 5 years) of the plants. Non-differentiated cell cultures, such as callus and cell suspension, were shown to synthesize only minor amounts of the SGs, and their content varied greatly during the growth cycle of the culture. Qualitative composition of the SGs in the cell cultures appeared to be highly scant as compared with that of the donor plants. No correlation between the SG content in organs of the donor plants and that in the cell cultures obtained was found. Factors determining plant cultivation conditions and influencing the accumulation of both, fresh and dry cell biomass were not able to completely induce the SG synthesis in non-differentiated cell cultures. This process was found to be restored only after the appearance of morphogenic structures and shoot formation. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ste6ia rebaudiana; In vitro; Callus and suspension cultures; Biosynthesis; Steviol-glycosides
1. Introduction Stevia (Ste6ia rebaudiana Bertoni) is a perennial plant belonging to the Compositae family. This species is characterized by a very limited range of natural habitats and is an endemic plant originating from Paraguay. Stevia leaves contain a number of diterpenoid steviol-glycosides (SGs) that are about 300 times sweeter than sucrose at their concentration of 4% (w/v) [1]. Major such types of compounds are stevioside and also rebaudiosides A and C (Fig. 1). Their individual content in
* Corresponding author. Tel.: + 7-95-9779445; fax: + 7-959778018. E-mail address: [email protected] (N. Bondarev). 1 So named diterpenoid glycoside, having steviol as an aglycone.
stevia leaves is known to amount to 1% and more per plant dry mass, whereas that of other SGs is considerably less [2,3]. These glycosides are nontoxic, non-mutagenic and low-caloric compounds, and, unlike traditional sugar substitutes such as xylitol or sorbitol, acquired tolerance to them does not occur [3,4]. Therefore, such compounds may be successfully used as sugar alternatives for patients suffering from diabetes and other diseases related to a disturbance in carbohydrate metabolism. In addition, the compounds in question may be successfully used in food industry. Stevia cell cultures in vitro represent a suitable model system to elucidate a number of the peculiarities of the metabolism of diterpenoid glycosides. Moreover, such investigations are of great importance for practice because cultured cells, tis-
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sues and organs of stevia may be used for production of non-caloric sugar substitutes. To date, however, diterpenoid steviol glycoside production in the in vitro stevia cultures is poorly understood, and the results obtained by different authors remain highly contradictory. Thus, Nabeta et al. [5], Suzuki et al. [6] and Miyagawa et al. [7] did not provide evidence for the presence of the SGs in the callus and suspension culture of S. rebaudiana. However, Lee et al. [8] found stevioside in the callus tissue of stevia. Hsing et al. [9] also found the stevioside in the callus tissue of stevia obtained from leaf explants after 70-day cultivation and reported that its content accounts for 16.24% of plant dry weight, and is two and four times higher than that in leaves and flowers of the same plant, respectively. On the other hand, Swanson et al. [10] did not find any presence of stevioside in both the in vitro callus and shoot cultures of stevia, and only rooted-shoot cultures
of the plant appeared to contain this glycoside. These authors concluded that only the plant shoots having both leaves and roots are able to synthesize the SGs. At the same time, Yamazaki and Flores [11] demonstrated that the shoot cultures of stevia are capable of producing stevioside in question. In the present study, we attempted to settle a number of questions that remain unclear so far: (1) are stevia cell, tissue, shoot cultures in vitro able to produce the SGs?; (2) how are qualitative composition and the content of the SGs in both the in vitro plants of stevia and its callus and suspension cultures?; (3) what are causes responsible for different biosynthetic activity in question of the cultures under consideration?; (4) does the biosynthetic activity of stevia cell cultures depend on the donor plant genotype or explant type?; and (5) is it possible to control the SG production via the change in the cultures cultivation conditions?
Fig. 1. Chemical structures of the steviol-glycosides and their aglycone-steviol.
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2. Materials and methods
2.1. Plant material Intact plants and in vitro plants of stevia (S. rebaudiana Bertoni), and also its callus and suspension cultures, were used in the present work. Stevia plants were represented by three clones (1, 2 and 3), and appropriated sterile plants were obtained from meristem of intact plants. Plants were cultivated in tubes on an agar-solidified Murashige-Skoog (MS) medium [12] in a growth chamber with 16-h light period and at an illuminance of 2000 lux. Stevia callus was generated from leaves (L) and stem (S) fragments of the in vitro plants. The stevia callus and suspension cultures were grown in the dark at 259 1°C and 70% air humidity on agar-solidified MS medium and liquid medium, respectively, containing 1 mg/l NAA and 0.5 mg/l BAP.
2.2. Extraction of the SGs Lyophilized and powered plant biomass (100– 500 mg) was placed in centrifuge tubes, treated twice with 100% methanol for 3 and 1 h on a magnetic stirrer, and centrifuged for 5 min at 240× g. The pooled supernatants obtained from each of the samples were dried under vacuum at 45–50°C on a rotor evaporator.
2.3. Purification of the samples Samples were purified by polypropylene Sep-Pak cartridges filled with sorbent Separon™ SGX C-18 (60 mm; ‘Tessek LTD’, Czech). The dry remainder of the sample extracts was dissolved in distilled water and loaded into the cartridge pre-activated with 2 ml acetonitrile–water and 2 ml distilled water. The cartridge was washed with 10 ml distilled water, 5 ml of 20% (v/v) acetonitrile – water mixture and, finally, 7 ml of 80% (v/v) acetonitrile–water mixture. The latter fraction obtained was evaporated under vacuum at 50°C on a rotor evaporator. The dry remainder was dissolved in 85% (v/v) acetonitrile–water mixture and the solution obtained was passed through the microfilter with a pore diameter of 0.2 mm (KNPO ‘Diagnosticum’, Moscow). The preparations were used for determination of the composition and the content of the SGs by thin-layer
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chromatography (TLC) and high-performance liquid chromatography (HPLC).
2.4. Analysis of the SGs by the TLC Extracts were placed on glass plates with kieselgel (60 F524, 40×100×0.2 mm3; ‘Merck’, Germany) or silicagel (100×200×0.13 mm3; ‘Lyaene Kalur’, Haapsalu, Estonia). For qualitative analysis, solvent mixtures of chloroform– methanol (35:15 (v/v)), chloroform–isopropanol – acetic acid (20:20:5 (v/v)), chloroform–methanol– water (20:20:5 (v/v)), n-butanol –ethanol –water (20:5:10 (v/v)), n-butanol – acetic acid–diethyl ether – water (18:12:6:2 (v/v)), benzene–methanol– water (36:6:2 (v/v)), and chloroform– isopropanol –acetic acid–water (20:20:5:5 (v/v)) were used. For quantitative analysis, only the latter solvent mixture was used. The chromatograms were dried under air, sprayed by anise aldehyde or thymol solution [13] and dried in thermostat at 100–110°C for 10 min until the appearance of red –lilac spots. Densitometric quantification of five SGs (stevioside, rebaudiosides A, B and C, and steviolbioside) was made with chromatogram densitometer CD50 ‘Desaga’ (Heidelberg, Germany).
2.5. Assay of the SGs by the HPLC This assay was performed on a chromatographic apparatus (LKB, Bromma, Sweden) with 10-ml calibrated loop, detection at 210 nm. The samples were chromatographed on steel Ultra Pak column (TSK-OH-120, 4.6×250 mm2) with a particle size of 5 mm using an acetonitrile–water (85:15 (v/v)) mixture at a flow rate of 0.5 ml/min. The SGs included rebaudiosides A and C, and stevioside. Quantification of the fractions tested was carried out on the base of the calibrated data obtained with the use of standard SG samples. Standard samples of the SGs for both the TLC and HPLC were purchased from Arbuzov Institute of Organic and Physical Chemistry (branch of Russian Academy of Sciences, Kazan). The contents of stevioside and rebaudiosides A and C are represented by the data obtained by the HPLC, whereas those of rebaudioside B and steviolbioside are taken from the data obtained by the TLC. The data presented in tables and figure are means of the measurements. Taking into account the extraction and purification of the samples,
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Table 1 Steviol-glycoside content in leaves and stems of stevia intact plants represented by their different clones (mg/g dry weight) Clone
Plant organ
Stevioside
Rebaudioside A
Rebaudioside C
Rebaudioside B
Steviolbioside
Total SG content
1 1 2 3
Leaves Stems Leaves Leaves
24.9 4.5 21.6 30.2
12.0 2.6 9.5 0.4
4.6 0.4 3.2 0.2
2.2 1.0 1.1 5.4
0 0 0 0
43.7 8.5 35.4 36.2
standard errors of means did not exceed 5 and 10% for the HPLC and TLC assays, respectively. 3. Results
3.1. Composition and the content of SGs in plants, cell and tissue cultures of ste6ia
three times lower in stems than that in the same organs of intact plants. The composition of the SGs in these plant organs was about the same. Any significant decrease in the SG content in the stevia plants during their prolonged (about for 5 years) in vitro cultivation did not occur (Table 2).
3.1.1. Intact plants It was found that both leaves and stems of intact stevia plants contain stevioside and rebaudiosides A, B and C, but lack steviolbioside (Table 1). In stems, however, the SG content appeared to be several times lower than that in leaves. The qualitative composition of the SGs in all plant organs under study was about the same. On the other hand, the clones differed in both the total SG content and the quantitative relationship between individual SGs. The greatest amount of the SGs appeared to be present in leaves of the plant clone 1. Clones 2 and 3 did not differ in the total SG content. All the clones were found to be enriched by stevioside, which is present in about equal amounts, whereas the amounts of rebaudiosides A and C displayed the significant differences (Table 1). Thus, the content of the latter compounds in leaves of clones 1 and 2 accounted for more than 30% of the total SG content, while in the leaves of clone 3 this value was only about 2%. According to the data obtained, the plant clones differed also in some growth characteristics. In particular, dry mass of clone 1 appeared to be more than 30% greater as compared with that of clones 2 and 3, i.e. a positive correlation between dry leaf mass and the SG content was observed.
3.1.3. Callus cultures The TLC assay of the tissue extract samples obtained from various stevia callus strains cultivated for about 1 year provided evidence for the presence on the thin-layer plates of the spots, with Rf identical to Rf of the standard samples of steviolbioside, rebaudioside B and stevioside. Almost all the samples contained only minor amounts of steviolbioside, whereas rebaudioside B and stevioside were present in about equal amounts in the callus obtained from stems and petioles of plant clone 1 (data not shown). The remaining samples contained either only trace amounts of the SGs already mentioned, or they were not detected at all. The presence of rebaudiosides A and C in the callus cultures (nine strains) was not detected by the TLC and HPLC assays. SGs were detected using the solvent mixtures containing various type solvents. We assume that the stevia callus cultures are capable of synthesizing at least three SGs, such as steviolbioside, stevioside and rebaudioside B. At the same time, in most callus strains subcultivated for 2– 2.5 years, the SGs were not detected, and only one strain was found to contain trace amounts of rebaudioside B. Stevia callus cultures maintained for an appreciable length of time are not practically able to synthesize even a small amount of the SGs.
3.1.2. In 6itro plants Table 2 shows that the SG content in the tube plants was five or six times lower in leaves and
3.1.4. Suspension cultures For the analysis of the SGs performed by the TLC and HPLC assays, four stevia suspension
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Table 2 Steviol-glycoside content in leaves and stems of stevia in vitro plantsa represented by their different clones (mg/g dry weight) Clone
Rebaudioside A
Rebaudioside C
Rebaudioside B Steviolbioside
Total SG content
Two-month plant culti6ation in 6itro 1 Leaves 3.4 1 Stems 1.7 2 Leaves 4.6 2 Stems 1.2
1.0 0.4 1.8 0.4
1.6 0.4 0.6 0.2
0.9 0.3 0.3 0.1
0 0 0 0
6.9 2.8 7.3 1.9
Fi6e-year plant culti6ation in 6itro 1 Leaves 3.3 1 Stems 0.8
1.9 0.6
0.7 0.1
0.2 0
0 0
6.1 1.5
a
Plant organ
Stevioside
Five-week-old plants.
cultures (1L, 2L, 2S and 3L) were used. The analysis was carried out at four different points of the stevia cultivation cycle. In addition, the cultural medium was examined for the possible presence of the SGs. The latter, however, were not detected in it. An accumulation dynamics of the SGs in S. rebaudiana suspension culture (strain 2S) during its cultivation cycle is shown in Fig. 2. It can be seen that a maximal content of the SGs (about 115 g per gram of plant dry mass) was found on the 14th day of the cultivation cycle, i.e. at the end of the exponential growth phase. However, because of overall considerable variation in the SG content during the cultivation cycle, this value significantly (about four times) decreased even on the 17th day of the culture growth. The results obtained in the course of the analysis of four stevia cultures on the 14th day of their cultivation are presented in Table 3. As follows from the latter, only minor amounts (no more than 15 g per gram dry plant mass) of the SGs under study (stevioside, rebaudiosides A and C) were found to be present in other cultures. The main glycoside that was detected in all of the cultures was stevioside; rebaudioside A was found to be present only in some strains, while rebaudioside C was absent completely (Table 3). According to the TLC assay, only trace amount of rebaudioside B was detected in the strain 2S. The same trace amount of steviolbioside was found in the given cultures but its content was higher during the first months of the cultivation cycle. It is important to note that no correlation between the SG contents in organs of the donor
plants and that in the cell cultures obtained was found.
3.2. Influence of the culti6ation factors on the SG accumulation in cell cultures Earlier, we found that a number of factors determining the plant cultivation conditions, such as temperature, light intensity and the presence of carbohydrates, mineral nutrients and plant growth regulators in the cultural medium, are able to considerably affect both fresh and dry
Fig. 2. The dynamics of S. rebaudiana suspension culture growth and the content of steviol-glycosides (stevioside, rebaudiosides A and C) in the cells (strain 2S) during their cultivation cycle.
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Table 3 Steviol-glycoside content in S. rebaudiana suspension cultures on the 14th day of their cultivation (mg/g dry weight) Strain
Stevioside
Rebaudiosid Rebaudiosid Total SG eA eC content
1L 2L 2S 3L
15 10 103 0
Trace 5 12 0
0 0 0 0
15 15 115 0
biomass of stevia cells [14,15]. These factors appeared to have practically no effect on the SG content in the stevia cultures under study (data not shown). In particular, despite the fact that we have observed both greening of the callus tissue subjected to white, red or blue light and production of chloroplasts in the callus cells, no marked accumulation of the SGs was detected.
3.3. Influence of cell differentiation on the SG content in in 6itro cultures Taking into account the results already mentioned, we attempted to elucidate whether the SG synthesis depends on cell differentiation. In order to check this assumption, a morphogenic stevia callus with well-developed shoots was obtained. As follows from Table 4, once the morphogenic structures and the shoot were generated, the SG content considerably increased. Thus, the SG content in the morphogenic callus and regenerated shoots appeared to be about four to five times and 37 times higher than that in the suspension culture, respectively, with the latter content accounting for more than one-third of the total SG amount found in stems of the donor in vitro plants.
4. Discussion The fact established in this study that the content of the SGs in stems of intact stevia plants appeared to be several times lower than that in their leaves is consistent with some reports in the literature [16]. In the present work, however, we were interested primarily in both the composition of the SGs and the content of such individual type compounds in leaves of different stevia clones. The intact plant clones under study were found to differ in both these characteristics. The latter are of considerable importance, since they allow one to select the plants containing the most desirable glycosides in terms of their gustatory, pharmacological and other properties. A positive correlation between the total SG content in stevia leaves and their dry mass is in accordance with the data obtained earlier by Metivier and Viana with respect to stevioside [17]. The cell stevia cultures appeared to contain the SGs about two or three orders of magnitude lower as compared with that in both intact plants and those grown in vitro. The relationship between individual glycosides remained unaltered. Considerable depletion of the spectrum of the synthesized SGs observed in the suspension cultures was expressed largely in the absence of the SGs occurring in the plants only in minimal amounts (Tables 1–3). Their synthesis was reduced to such an extent that their presence even in trace amounts could not be detected by HPLC. However, we found somewhat different pattern with regard to steviolbioside. This compound was not detected in both the intact and the tube plants, although its presence in stevia leaves was noted earlier by other investigators [1,18,19]. We have found that the callus and suspension stevia cultures during the
Table 4 The contents of three major steviol-glycosides in S. rebaudiana in vitro culturesa (mg/g dry weight) Culture (clone 1)
Stevioside
Rebaudioside A
Rebaudioside C
Total SG content
Plant organs Leaves Stems Callus Suspension culture Morphogenic callus Shoots morphogenic callus
3300 800 0 15 60 387
1900 600 0 Trace 23 112
700 100 0 0 0 53
5900 1500 0 15 83 552
a
Five-week-old cultures.
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first months of their cultivation exhibited relatively active synthesis of steviolbiosides, but this process was not stable upon long-term cultivation of the cell cultures. Similar regularity concerning the synthesis of wider spectrum of secondary metabolism compounds as compared with that observed in the initial plants was established earlier for other plant cell cultures [20– 22]. In this case, some peculiarities appear to be inherent in plant secondary metabolism and represent distinct characteristics of both archaic plants and juvenile stages of the present plants [23,24]. The contradictory results concerning the biosynthesis and accumulation of the SGs in stevia cell cultures [5– 10] may be simply explained by the unstable level of these compounds during both prolonged plant maintenance in vitro (Section 3.1.3) or one cycle of plant subcultivation (Fig. 2). As revealed in this study, some increase in the SGs production and accumulation of the secondary compounds was observed after the transition from superficial to deep cultivation of stevia cells, and similar results were obtained upon cultivation of Dioscorea deltoidea and Rhaponticum carthamoides cells producing steroid glycosides [25] and ecdisteroides [26], respectively. All the responses are likely due to specific cell cultivation conditions underlying some changes in both the structure of the cultured cells and their supply with oxygen and nutrients. A sharp decline in the SG content observed in the cultured cells at the beginning of the stationary phase of their periodical cultivation was found to be not due to release of such compounds into the nutrient medium. The absence of stable SG accumulation in the cultured cells during their cultivation cycle is most probably due to the metabolic conversion of these compounds. Almost complete stopping of the SG production in the cultivated cells and the restoration of this process in the differentiated plant cultures, such as shoot, indicate that its intensity is dependent on the extent of culture differentiation (Table 4). This suggests that the SG production occurs in specialized cells or cellular structures. However, any evidence indicating the existence distinctive anatomic structures involved in both this process and the storing of the SGs, to our knowledge, are not available in the literature. On the other hand, alternative explanation of the finding in question is the following. A mesophyll of stevia leaves is
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known to be characterized by well-developed chloroplasts with grana and thylakoids [27]. These structures are probably responsible for the SG synthesis in stevia cells. Another situation most likely takes place in the callus stevia tissues obtained from leaf explants, since these tissues consist of dedifferentiated meristematic cells where all chloroplasts are reorganized into proplastids [28]. For instance, there is evidence that, in potato plants, chlorophyll concentration markedly decreases during the callus formation, whereas the level of this pigment is restored during the shoot formation [29]. Somatic dedifferentiated cells of the callus tissue most likely synthesize only small amounts of the SGs, since these cells are heterothrophic ones, even if they are cultivated in light, and therefore have poorly developed chloroplasts [27]. We suggest that the intensity of the SG synthesis depends on both the extent of development and functional activity of chloroplasts in cells of the cultures under study. This hypothesis is consistent with some reports in the literature indicating that the formation of such compounds, or at least some steps of this process, occurs namely in chloroplasts. Thus, in stevia leaf cells, these organelles were found to exhibit a high level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity [30] and, in addition, to contain the enzyme ent-kaurenoic acid 13-hydroxylase converting ent-kaurenoic acid to steviol [31]. The available data that phenol compounds are synthesized in chloroplasts of both the shoots and the callus tissue of Camellia sinensis [32,33] and terpenoid synthesis follows the same pathway [34] suggest that diterpenoid glycosides are produced in chloroplasts as well. The initial steps of SG synthesis possibly occur using an alternative glyceraldehyde-3-phosphate/pyruvate or 1-deoxy-Dxylulose-5-phosphate pathway also located in chloroplasts. The findings that, in higher plants, many isoprenoids including some diterpenes are synthesized via this pathway [35,36] are consistent with this proposal. Isopentenyl-diphosphate synthesis likely follows the same pathway rather than the mevalonate one. The fact that the chloroplastcontaining stevia callus cultivated in light did not synthesize a higher amount of the SGs as compared with that in the callus grown in the dark does not contradict our proposal. The apparent discrepancy is most likely explained by that prolonged synthesis of isoprenoids in chloroplasts, the
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process responsible for the accumulation in them of carotenoids, phytol and other compounds essential for photosynthesis, precedes the GS production [37].
Acknowledgements The authors thank Dr I.M. Andreev from the Institute for assistance in translating the manuscript from Russian into English.
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[28] D. Yu, W. Hong, M. Chen, D. Wang, Ultrastructural study of mesophyll cells during their dedifferentiation in Ste6ia rebaudiana, Acta Bot. Sin. 35 (1993) 499 – 505. [29] E.A. Kazakova, The pigment composition of potato callus and regenerants in the ontogenesis, Bull. VIR 204 (1990) 70 – 75 (in Russian). [30] K.K. Kim, H. Yamashita, Y. Sawa, H. Shibata, A high activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in chloroplasts of Ste6ia rebaudiana Bertoni, Biosci. Biotech. Biochem. 60 (1996) 685 – 686. [31] K.K. Kim, Y. Sawa, H. Shibata, Hydroxylation of ent-Kaurenoic acid to steviol in Ste6ia rebaudiana Bertoni — purification and partial characterization of the enzyme, Arch. Bichem. Biophys. 332 (1996) 223 –230. [32] M.N. Zaprometov, Phenol compounds of plants: biosynthesis, transformation and functions, in: New Directions
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in Plant Physiology, Nauka, Moscow, 1985. [33] M.N. Zaprometov, N.V. Zagoskina, One more evidence for chloroplast involvement in the biosynthesis of phenol compounds, Russ. J. Plant Physiol. 34 (1987) 165 – 172. [34] V.A. Paseshnichenko, Regulation of terpenoid biosynthesis in plants and its relation to the biosynthesis of phenol compounds, Russ. J. Plant Physiol. 42 (1995) 699– 714. [35] V.A. Paseshnichenko, A new alternative non-mevalonate pathway of isoprenoid biosynthesis in eubacteria and plants, Biochemistry 63 (1998) 171 –182 (in Russian). [36] H.K. Lichtenthaler, The plants’ 1-deoxy-D-xylulose-5phosphate pathway for biosynthesis of isoprenoids, Fett/ Lipid 100 (1998) 128 –138. [37] T.W. Goodwin, E.I. Mercer, Introduction to Plant Biochemistry, vol. 2, Mir, Moscow, 1986.
Peculiarities of diterpenoid steviol glycoside production in in vitro cultures of Ste6ia rebaudiana Bertoni Nikolai Bondarev *, Oxana Reshetnyak, Alexander Nosov Timiryaze6 Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia Received 7 August 2000; received in revised form 14 March 2001; accepted 14 March 2001
Abstract The composition and content of steviol-glycosides (SGs) 1 in in vitro cultures of stevia (Ste6ia rebaudiana Bertoni) were investigated. A comparative analysis of production of these compounds in intact plants, in vitro plants, dedifferentiated callus and suspension cultures, morphogenic callus and in vitro regenerated shoots were conducted. Qualitative composition of the SGs in in vitro plants was found to be identical to that of intact plants, but their content in the former plants appeared to be about five or six times lower. A significant decrease in this value was not observed upon long-term cultivation (for about 5 years) of the plants. Non-differentiated cell cultures, such as callus and cell suspension, were shown to synthesize only minor amounts of the SGs, and their content varied greatly during the growth cycle of the culture. Qualitative composition of the SGs in the cell cultures appeared to be highly scant as compared with that of the donor plants. No correlation between the SG content in organs of the donor plants and that in the cell cultures obtained was found. Factors determining plant cultivation conditions and influencing the accumulation of both, fresh and dry cell biomass were not able to completely induce the SG synthesis in non-differentiated cell cultures. This process was found to be restored only after the appearance of morphogenic structures and shoot formation. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ste6ia rebaudiana; In vitro; Callus and suspension cultures; Biosynthesis; Steviol-glycosides
1. Introduction Stevia (Ste6ia rebaudiana Bertoni) is a perennial plant belonging to the Compositae family. This species is characterized by a very limited range of natural habitats and is an endemic plant originating from Paraguay. Stevia leaves contain a number of diterpenoid steviol-glycosides (SGs) that are about 300 times sweeter than sucrose at their concentration of 4% (w/v) [1]. Major such types of compounds are stevioside and also rebaudiosides A and C (Fig. 1). Their individual content in
* Corresponding author. Tel.: + 7-95-9779445; fax: + 7-959778018. E-mail address: [email protected] (N. Bondarev). 1 So named diterpenoid glycoside, having steviol as an aglycone.
stevia leaves is known to amount to 1% and more per plant dry mass, whereas that of other SGs is considerably less [2,3]. These glycosides are nontoxic, non-mutagenic and low-caloric compounds, and, unlike traditional sugar substitutes such as xylitol or sorbitol, acquired tolerance to them does not occur [3,4]. Therefore, such compounds may be successfully used as sugar alternatives for patients suffering from diabetes and other diseases related to a disturbance in carbohydrate metabolism. In addition, the compounds in question may be successfully used in food industry. Stevia cell cultures in vitro represent a suitable model system to elucidate a number of the peculiarities of the metabolism of diterpenoid glycosides. Moreover, such investigations are of great importance for practice because cultured cells, tis-
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sues and organs of stevia may be used for production of non-caloric sugar substitutes. To date, however, diterpenoid steviol glycoside production in the in vitro stevia cultures is poorly understood, and the results obtained by different authors remain highly contradictory. Thus, Nabeta et al. [5], Suzuki et al. [6] and Miyagawa et al. [7] did not provide evidence for the presence of the SGs in the callus and suspension culture of S. rebaudiana. However, Lee et al. [8] found stevioside in the callus tissue of stevia. Hsing et al. [9] also found the stevioside in the callus tissue of stevia obtained from leaf explants after 70-day cultivation and reported that its content accounts for 16.24% of plant dry weight, and is two and four times higher than that in leaves and flowers of the same plant, respectively. On the other hand, Swanson et al. [10] did not find any presence of stevioside in both the in vitro callus and shoot cultures of stevia, and only rooted-shoot cultures
of the plant appeared to contain this glycoside. These authors concluded that only the plant shoots having both leaves and roots are able to synthesize the SGs. At the same time, Yamazaki and Flores [11] demonstrated that the shoot cultures of stevia are capable of producing stevioside in question. In the present study, we attempted to settle a number of questions that remain unclear so far: (1) are stevia cell, tissue, shoot cultures in vitro able to produce the SGs?; (2) how are qualitative composition and the content of the SGs in both the in vitro plants of stevia and its callus and suspension cultures?; (3) what are causes responsible for different biosynthetic activity in question of the cultures under consideration?; (4) does the biosynthetic activity of stevia cell cultures depend on the donor plant genotype or explant type?; and (5) is it possible to control the SG production via the change in the cultures cultivation conditions?
Fig. 1. Chemical structures of the steviol-glycosides and their aglycone-steviol.
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2. Materials and methods
2.1. Plant material Intact plants and in vitro plants of stevia (S. rebaudiana Bertoni), and also its callus and suspension cultures, were used in the present work. Stevia plants were represented by three clones (1, 2 and 3), and appropriated sterile plants were obtained from meristem of intact plants. Plants were cultivated in tubes on an agar-solidified Murashige-Skoog (MS) medium [12] in a growth chamber with 16-h light period and at an illuminance of 2000 lux. Stevia callus was generated from leaves (L) and stem (S) fragments of the in vitro plants. The stevia callus and suspension cultures were grown in the dark at 259 1°C and 70% air humidity on agar-solidified MS medium and liquid medium, respectively, containing 1 mg/l NAA and 0.5 mg/l BAP.
2.2. Extraction of the SGs Lyophilized and powered plant biomass (100– 500 mg) was placed in centrifuge tubes, treated twice with 100% methanol for 3 and 1 h on a magnetic stirrer, and centrifuged for 5 min at 240× g. The pooled supernatants obtained from each of the samples were dried under vacuum at 45–50°C on a rotor evaporator.
2.3. Purification of the samples Samples were purified by polypropylene Sep-Pak cartridges filled with sorbent Separon™ SGX C-18 (60 mm; ‘Tessek LTD’, Czech). The dry remainder of the sample extracts was dissolved in distilled water and loaded into the cartridge pre-activated with 2 ml acetonitrile–water and 2 ml distilled water. The cartridge was washed with 10 ml distilled water, 5 ml of 20% (v/v) acetonitrile – water mixture and, finally, 7 ml of 80% (v/v) acetonitrile–water mixture. The latter fraction obtained was evaporated under vacuum at 50°C on a rotor evaporator. The dry remainder was dissolved in 85% (v/v) acetonitrile–water mixture and the solution obtained was passed through the microfilter with a pore diameter of 0.2 mm (KNPO ‘Diagnosticum’, Moscow). The preparations were used for determination of the composition and the content of the SGs by thin-layer
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chromatography (TLC) and high-performance liquid chromatography (HPLC).
2.4. Analysis of the SGs by the TLC Extracts were placed on glass plates with kieselgel (60 F524, 40×100×0.2 mm3; ‘Merck’, Germany) or silicagel (100×200×0.13 mm3; ‘Lyaene Kalur’, Haapsalu, Estonia). For qualitative analysis, solvent mixtures of chloroform– methanol (35:15 (v/v)), chloroform–isopropanol – acetic acid (20:20:5 (v/v)), chloroform–methanol– water (20:20:5 (v/v)), n-butanol –ethanol –water (20:5:10 (v/v)), n-butanol – acetic acid–diethyl ether – water (18:12:6:2 (v/v)), benzene–methanol– water (36:6:2 (v/v)), and chloroform– isopropanol –acetic acid–water (20:20:5:5 (v/v)) were used. For quantitative analysis, only the latter solvent mixture was used. The chromatograms were dried under air, sprayed by anise aldehyde or thymol solution [13] and dried in thermostat at 100–110°C for 10 min until the appearance of red –lilac spots. Densitometric quantification of five SGs (stevioside, rebaudiosides A, B and C, and steviolbioside) was made with chromatogram densitometer CD50 ‘Desaga’ (Heidelberg, Germany).
2.5. Assay of the SGs by the HPLC This assay was performed on a chromatographic apparatus (LKB, Bromma, Sweden) with 10-ml calibrated loop, detection at 210 nm. The samples were chromatographed on steel Ultra Pak column (TSK-OH-120, 4.6×250 mm2) with a particle size of 5 mm using an acetonitrile–water (85:15 (v/v)) mixture at a flow rate of 0.5 ml/min. The SGs included rebaudiosides A and C, and stevioside. Quantification of the fractions tested was carried out on the base of the calibrated data obtained with the use of standard SG samples. Standard samples of the SGs for both the TLC and HPLC were purchased from Arbuzov Institute of Organic and Physical Chemistry (branch of Russian Academy of Sciences, Kazan). The contents of stevioside and rebaudiosides A and C are represented by the data obtained by the HPLC, whereas those of rebaudioside B and steviolbioside are taken from the data obtained by the TLC. The data presented in tables and figure are means of the measurements. Taking into account the extraction and purification of the samples,
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Table 1 Steviol-glycoside content in leaves and stems of stevia intact plants represented by their different clones (mg/g dry weight) Clone
Plant organ
Stevioside
Rebaudioside A
Rebaudioside C
Rebaudioside B
Steviolbioside
Total SG content
1 1 2 3
Leaves Stems Leaves Leaves
24.9 4.5 21.6 30.2
12.0 2.6 9.5 0.4
4.6 0.4 3.2 0.2
2.2 1.0 1.1 5.4
0 0 0 0
43.7 8.5 35.4 36.2
standard errors of means did not exceed 5 and 10% for the HPLC and TLC assays, respectively. 3. Results
3.1. Composition and the content of SGs in plants, cell and tissue cultures of ste6ia
three times lower in stems than that in the same organs of intact plants. The composition of the SGs in these plant organs was about the same. Any significant decrease in the SG content in the stevia plants during their prolonged (about for 5 years) in vitro cultivation did not occur (Table 2).
3.1.1. Intact plants It was found that both leaves and stems of intact stevia plants contain stevioside and rebaudiosides A, B and C, but lack steviolbioside (Table 1). In stems, however, the SG content appeared to be several times lower than that in leaves. The qualitative composition of the SGs in all plant organs under study was about the same. On the other hand, the clones differed in both the total SG content and the quantitative relationship between individual SGs. The greatest amount of the SGs appeared to be present in leaves of the plant clone 1. Clones 2 and 3 did not differ in the total SG content. All the clones were found to be enriched by stevioside, which is present in about equal amounts, whereas the amounts of rebaudiosides A and C displayed the significant differences (Table 1). Thus, the content of the latter compounds in leaves of clones 1 and 2 accounted for more than 30% of the total SG content, while in the leaves of clone 3 this value was only about 2%. According to the data obtained, the plant clones differed also in some growth characteristics. In particular, dry mass of clone 1 appeared to be more than 30% greater as compared with that of clones 2 and 3, i.e. a positive correlation between dry leaf mass and the SG content was observed.
3.1.3. Callus cultures The TLC assay of the tissue extract samples obtained from various stevia callus strains cultivated for about 1 year provided evidence for the presence on the thin-layer plates of the spots, with Rf identical to Rf of the standard samples of steviolbioside, rebaudioside B and stevioside. Almost all the samples contained only minor amounts of steviolbioside, whereas rebaudioside B and stevioside were present in about equal amounts in the callus obtained from stems and petioles of plant clone 1 (data not shown). The remaining samples contained either only trace amounts of the SGs already mentioned, or they were not detected at all. The presence of rebaudiosides A and C in the callus cultures (nine strains) was not detected by the TLC and HPLC assays. SGs were detected using the solvent mixtures containing various type solvents. We assume that the stevia callus cultures are capable of synthesizing at least three SGs, such as steviolbioside, stevioside and rebaudioside B. At the same time, in most callus strains subcultivated for 2– 2.5 years, the SGs were not detected, and only one strain was found to contain trace amounts of rebaudioside B. Stevia callus cultures maintained for an appreciable length of time are not practically able to synthesize even a small amount of the SGs.
3.1.2. In 6itro plants Table 2 shows that the SG content in the tube plants was five or six times lower in leaves and
3.1.4. Suspension cultures For the analysis of the SGs performed by the TLC and HPLC assays, four stevia suspension
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Table 2 Steviol-glycoside content in leaves and stems of stevia in vitro plantsa represented by their different clones (mg/g dry weight) Clone
Rebaudioside A
Rebaudioside C
Rebaudioside B Steviolbioside
Total SG content
Two-month plant culti6ation in 6itro 1 Leaves 3.4 1 Stems 1.7 2 Leaves 4.6 2 Stems 1.2
1.0 0.4 1.8 0.4
1.6 0.4 0.6 0.2
0.9 0.3 0.3 0.1
0 0 0 0
6.9 2.8 7.3 1.9
Fi6e-year plant culti6ation in 6itro 1 Leaves 3.3 1 Stems 0.8
1.9 0.6
0.7 0.1
0.2 0
0 0
6.1 1.5
a
Plant organ
Stevioside
Five-week-old plants.
cultures (1L, 2L, 2S and 3L) were used. The analysis was carried out at four different points of the stevia cultivation cycle. In addition, the cultural medium was examined for the possible presence of the SGs. The latter, however, were not detected in it. An accumulation dynamics of the SGs in S. rebaudiana suspension culture (strain 2S) during its cultivation cycle is shown in Fig. 2. It can be seen that a maximal content of the SGs (about 115 g per gram of plant dry mass) was found on the 14th day of the cultivation cycle, i.e. at the end of the exponential growth phase. However, because of overall considerable variation in the SG content during the cultivation cycle, this value significantly (about four times) decreased even on the 17th day of the culture growth. The results obtained in the course of the analysis of four stevia cultures on the 14th day of their cultivation are presented in Table 3. As follows from the latter, only minor amounts (no more than 15 g per gram dry plant mass) of the SGs under study (stevioside, rebaudiosides A and C) were found to be present in other cultures. The main glycoside that was detected in all of the cultures was stevioside; rebaudioside A was found to be present only in some strains, while rebaudioside C was absent completely (Table 3). According to the TLC assay, only trace amount of rebaudioside B was detected in the strain 2S. The same trace amount of steviolbioside was found in the given cultures but its content was higher during the first months of the cultivation cycle. It is important to note that no correlation between the SG contents in organs of the donor
plants and that in the cell cultures obtained was found.
3.2. Influence of the culti6ation factors on the SG accumulation in cell cultures Earlier, we found that a number of factors determining the plant cultivation conditions, such as temperature, light intensity and the presence of carbohydrates, mineral nutrients and plant growth regulators in the cultural medium, are able to considerably affect both fresh and dry
Fig. 2. The dynamics of S. rebaudiana suspension culture growth and the content of steviol-glycosides (stevioside, rebaudiosides A and C) in the cells (strain 2S) during their cultivation cycle.
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Table 3 Steviol-glycoside content in S. rebaudiana suspension cultures on the 14th day of their cultivation (mg/g dry weight) Strain
Stevioside
Rebaudiosid Rebaudiosid Total SG eA eC content
1L 2L 2S 3L
15 10 103 0
Trace 5 12 0
0 0 0 0
15 15 115 0
biomass of stevia cells [14,15]. These factors appeared to have practically no effect on the SG content in the stevia cultures under study (data not shown). In particular, despite the fact that we have observed both greening of the callus tissue subjected to white, red or blue light and production of chloroplasts in the callus cells, no marked accumulation of the SGs was detected.
3.3. Influence of cell differentiation on the SG content in in 6itro cultures Taking into account the results already mentioned, we attempted to elucidate whether the SG synthesis depends on cell differentiation. In order to check this assumption, a morphogenic stevia callus with well-developed shoots was obtained. As follows from Table 4, once the morphogenic structures and the shoot were generated, the SG content considerably increased. Thus, the SG content in the morphogenic callus and regenerated shoots appeared to be about four to five times and 37 times higher than that in the suspension culture, respectively, with the latter content accounting for more than one-third of the total SG amount found in stems of the donor in vitro plants.
4. Discussion The fact established in this study that the content of the SGs in stems of intact stevia plants appeared to be several times lower than that in their leaves is consistent with some reports in the literature [16]. In the present work, however, we were interested primarily in both the composition of the SGs and the content of such individual type compounds in leaves of different stevia clones. The intact plant clones under study were found to differ in both these characteristics. The latter are of considerable importance, since they allow one to select the plants containing the most desirable glycosides in terms of their gustatory, pharmacological and other properties. A positive correlation between the total SG content in stevia leaves and their dry mass is in accordance with the data obtained earlier by Metivier and Viana with respect to stevioside [17]. The cell stevia cultures appeared to contain the SGs about two or three orders of magnitude lower as compared with that in both intact plants and those grown in vitro. The relationship between individual glycosides remained unaltered. Considerable depletion of the spectrum of the synthesized SGs observed in the suspension cultures was expressed largely in the absence of the SGs occurring in the plants only in minimal amounts (Tables 1–3). Their synthesis was reduced to such an extent that their presence even in trace amounts could not be detected by HPLC. However, we found somewhat different pattern with regard to steviolbioside. This compound was not detected in both the intact and the tube plants, although its presence in stevia leaves was noted earlier by other investigators [1,18,19]. We have found that the callus and suspension stevia cultures during the
Table 4 The contents of three major steviol-glycosides in S. rebaudiana in vitro culturesa (mg/g dry weight) Culture (clone 1)
Stevioside
Rebaudioside A
Rebaudioside C
Total SG content
Plant organs Leaves Stems Callus Suspension culture Morphogenic callus Shoots morphogenic callus
3300 800 0 15 60 387
1900 600 0 Trace 23 112
700 100 0 0 0 53
5900 1500 0 15 83 552
a
Five-week-old cultures.
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first months of their cultivation exhibited relatively active synthesis of steviolbiosides, but this process was not stable upon long-term cultivation of the cell cultures. Similar regularity concerning the synthesis of wider spectrum of secondary metabolism compounds as compared with that observed in the initial plants was established earlier for other plant cell cultures [20– 22]. In this case, some peculiarities appear to be inherent in plant secondary metabolism and represent distinct characteristics of both archaic plants and juvenile stages of the present plants [23,24]. The contradictory results concerning the biosynthesis and accumulation of the SGs in stevia cell cultures [5– 10] may be simply explained by the unstable level of these compounds during both prolonged plant maintenance in vitro (Section 3.1.3) or one cycle of plant subcultivation (Fig. 2). As revealed in this study, some increase in the SGs production and accumulation of the secondary compounds was observed after the transition from superficial to deep cultivation of stevia cells, and similar results were obtained upon cultivation of Dioscorea deltoidea and Rhaponticum carthamoides cells producing steroid glycosides [25] and ecdisteroides [26], respectively. All the responses are likely due to specific cell cultivation conditions underlying some changes in both the structure of the cultured cells and their supply with oxygen and nutrients. A sharp decline in the SG content observed in the cultured cells at the beginning of the stationary phase of their periodical cultivation was found to be not due to release of such compounds into the nutrient medium. The absence of stable SG accumulation in the cultured cells during their cultivation cycle is most probably due to the metabolic conversion of these compounds. Almost complete stopping of the SG production in the cultivated cells and the restoration of this process in the differentiated plant cultures, such as shoot, indicate that its intensity is dependent on the extent of culture differentiation (Table 4). This suggests that the SG production occurs in specialized cells or cellular structures. However, any evidence indicating the existence distinctive anatomic structures involved in both this process and the storing of the SGs, to our knowledge, are not available in the literature. On the other hand, alternative explanation of the finding in question is the following. A mesophyll of stevia leaves is
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known to be characterized by well-developed chloroplasts with grana and thylakoids [27]. These structures are probably responsible for the SG synthesis in stevia cells. Another situation most likely takes place in the callus stevia tissues obtained from leaf explants, since these tissues consist of dedifferentiated meristematic cells where all chloroplasts are reorganized into proplastids [28]. For instance, there is evidence that, in potato plants, chlorophyll concentration markedly decreases during the callus formation, whereas the level of this pigment is restored during the shoot formation [29]. Somatic dedifferentiated cells of the callus tissue most likely synthesize only small amounts of the SGs, since these cells are heterothrophic ones, even if they are cultivated in light, and therefore have poorly developed chloroplasts [27]. We suggest that the intensity of the SG synthesis depends on both the extent of development and functional activity of chloroplasts in cells of the cultures under study. This hypothesis is consistent with some reports in the literature indicating that the formation of such compounds, or at least some steps of this process, occurs namely in chloroplasts. Thus, in stevia leaf cells, these organelles were found to exhibit a high level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity [30] and, in addition, to contain the enzyme ent-kaurenoic acid 13-hydroxylase converting ent-kaurenoic acid to steviol [31]. The available data that phenol compounds are synthesized in chloroplasts of both the shoots and the callus tissue of Camellia sinensis [32,33] and terpenoid synthesis follows the same pathway [34] suggest that diterpenoid glycosides are produced in chloroplasts as well. The initial steps of SG synthesis possibly occur using an alternative glyceraldehyde-3-phosphate/pyruvate or 1-deoxy-Dxylulose-5-phosphate pathway also located in chloroplasts. The findings that, in higher plants, many isoprenoids including some diterpenes are synthesized via this pathway [35,36] are consistent with this proposal. Isopentenyl-diphosphate synthesis likely follows the same pathway rather than the mevalonate one. The fact that the chloroplastcontaining stevia callus cultivated in light did not synthesize a higher amount of the SGs as compared with that in the callus grown in the dark does not contradict our proposal. The apparent discrepancy is most likely explained by that prolonged synthesis of isoprenoids in chloroplasts, the
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process responsible for the accumulation in them of carotenoids, phytol and other compounds essential for photosynthesis, precedes the GS production [37].
Acknowledgements The authors thank Dr I.M. Andreev from the Institute for assistance in translating the manuscript from Russian into English.
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