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Development of a novel system for producing ajmalicine and serpentine using direct culture of leaves in Catharanthus roseus intact plant
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 99, No. 3, 208–215. 2005 DOI: 10.1263/jbb.99.208
© 2005, The Society for Biotechnology, Japan
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Development of a Novel System for Producing Ajmalicine and Serpentine Using Direct Culture of Leaves in Catharanthus roseus Intact Plant AKIRA IWASE,1 HIDEKI AOYAGI,1 MASARU OHME-TAKAGI,2 AND HIDEO TANAKA1* Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan1 and Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba, Ibaraki 305-8562, Japan2 Received 10 December 2004/Accepted 11 January 2005
Due to problems of production instability, the production of plant secondary metabolites using dedifferentiated cells (callus) is not always feasible on an industrial scale. To propose a new methodology, which does not use dedifferentiated cells, a novel system for producing useful secondary metabolites using the direct culture of intact plant leaves was developed. Catharanthus roseus was used as a model medicinal plant to produce terpenoid indole alkaloids (TIAs) by suspension culture of the leaves in the phytohormone-free MS liquid medium. Adjustment of the osmotic pressure (993 kPa at 25°C) in the medium, light irradiation (60 mmol m–2 s–1) and addition of glucose (10 g/l) were effective to promote the production of TIAs such as ajmalicine (Aj) and serpentine (Sp). On the basis of semi-quantitative RT-PCR analyses, it was revealed that the culture conditions promoted gene expression of enzymes in the TIA pathway in the cultured leaves. By feeding glucose (10 g/l) on day 10 of the culture period, Aj was produced at a concentration of about 18 mg/l and Sp was produced at a concentration about 11-fold that of the control. These results represent the first step in the development of a novel production system for plant secondary metabolites. [Key words: Catharanthus roseus, cell culture, terpenoid indole alkaloid, secondary metabolite, biosynthesis]
sion of transgenes in calli (9). Moreover, it has been reported that the production instability occurred in a transgenic callus even though the transgenes were stably expressed (10). Thus, useful metabolite production using a callus is a risky technology. Much attention therefore has been focused on the development of a useful metabolite production system using genetically stable cells instead of a callus. The biosynthesis and accumulation of secondary metabolites in higher plants take place in highly differentiated cells during their developmental processes (11, 12). Therefore, many approaches using differentiated cells, such as those of multiple shoots, adventitious roots and hairy roots, have been proposed. However, establishment of cultured organs is not always feasible for all plants. Moreover, the procedure to select cultured organs with high productivity is timeconsuming and involves the induction of a callus using adequate phytohormones or Agrobacterium infection. Cells that constitute an organ of an intact plant have the ability to produce secondary metabolites. In other words, the organ is considered to represent a state of immobilization with differentiated cells at high density with the ability to stably produce secondary metabolites due to the cell to cell communication.
From the end of the 1960s, the production of useful metabolites (mainly secondary metabolites) using plant cell culture (callus) technology has been widely investigated (1). As a callus can grow indefinitely and culture conditions for its growth can be easily controlled, the technology has great potential for producing useful metabolites on an industrial scale. However, there are only a few cases of practical applications, such as the production of shikonin (2), tuberose polysaccharides (3), and paclitaxel (reviewed in Ref. 4). One of the main problems is instability associated with calli for useful metabolite production (5). In general, the ability of a callus to produce secondary metabolites is lost during the induction of callus (being in a dedifferentiated state) formation from an intact plant (being in a differentiated state) using phytohormones. Even if a cell line with high productivity is screened using various selection techniques, a loss in productivity frequently occurs over time during subculturing (6). Such callus instability might be due to genetic and epigenetic changes such as chromosome breakage (7) and DNA methylation (8). The phenomenon of DNA methylation has also been reported to be involved in the expres* Corresponding author. e-mail: [email protected] phone: +81-(0)29-853-7297; 7212 fax: +81-(0)29-853-4605 208
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We considered that if the optimal culture conditions for maximizing the secondary metabolite production ability of intact plant organs are established, a simple and stable system for producing secondary metabolites might be developed. On the basis of this idea, the direct culture of intact plant organs was investigated using phytohormone-free liquid medium. Catharanthus roseus (L.) G. Don, which has been widely employed for the production of useful metabolites (13), was used in this study as a model medicinal plant. C. roseus biosynthesizes terpenoid indole alkaloids (TIAs) such as dimeric alkaloid vinblastine (anticancer activity), monomeric ajmalicine (anti-hypertension activity) and its oxidized form, serpentine (tranquillizer activity). In this paper, we propose a novel methodology, which does not use a callus (dedifferentiated cells), for producing useful secondary metabolites using intact plant organs. As a model, a system for the production of ajmalicine (Aj) and serpentine (Sp) by the direct culture of leaves of C. roseus was developed.
MATERIALS AND METHODS Plant material C. roseus (L.) G. Don was propagated from cuttings, which were obtained from naturally grown plants in Okinawa Prefecture, Japan. It was grown in a growth cabinet (temperature, 25°C; photoperiod, 16 h; dark period, 8 h; light intensity, 60 mmol m–2 s–1). The fully expanded leaves, third to the sixth from the shoot apical meristem, were used for the experiments. Cultivation and analysis Opposite leaves were separated from the stem on the left and right as shown in Fig. 1, a and b. The leaves were washed with distilled water (D.W.) containing 0.005% (v/v) Polyoxyethylene sorbitan monooleate (Wako Pure Chemical Industries, Osaka) and then, their surfaces were sterilized using
FIG. 1. Schematic diagram of the method used for symmetric cutting and cultivation of C. roseus leaves. a, Left side leaf; b, right side leaf; c or e, left side of a leaf; d or f, right side of a leaf.
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both 70% EtOH for 1 min and 2% chlorine sodium hypochlorite solution (Wako) for 15 min. Finally, the leaves were washed three times with sterile D.W. After the surfaces of the leaves were wiped with sterile paper, these leaves were cut and separated at the midrib into left and right sections (Fig. 1, c–f). Suspension culture was carried out in 200-ml Erlenmeyer flasks on a rotary shaker (120 rpm) at 25°C. One gram of the fresh leaves was used to inoculate 50 ml of phytohormonefree MS medium (14) containing 10 g/l glucose. The pH of the medium was adjusted to 5.7 before autoclaving at 121°C for 20 min. After the cultivation, the total medium volume was measured and the leaves were separated from the medium using a 32-mm stainless mesh. The leaves were washed with a large amount of D.W. and then frozen in liquid nitrogen and freeze-dried for 72 h. The weight of leaves was measured and the dry cell concentration was calculated. The residual glucose concentration was measured with the Glucose test kit Wako (Wako). Peroxidase activity was measured according to the procedure described previously (15). Osmotic pressure measurement Osmotic pressure of the medium was measured by a Digital Micro Osmometer OM801 (Vogel, Giessen, Germany) based on the freezing point depression system. In order to measure the osmotic pressure of C. roseus leaves, sufficient water was supplied to the intact plant 24 h before the measurement. The leaves, third to the sixth from the shoot apical meristem, were frozen in the liquid nitrogen and mashed, centrifuged at 9000´g for 5 min, and then the supernatant was used for the osmotic pressure measurement. TIAs extraction and analysis Both intracellular and extracellular alkaloid levels were determined. The extraction of TIAs was performed using the method reported previously (16). To quantify the TIAs, HPLC analysis (17) was performed with the LC-VP HPLC system (Shimadzu, Kyoto). Amount of serpentine was expressed as its HPLC chromatogram area and the total TIAs was expressed as the total area on the HPLC chromatogram. To identify the alkaloids, TLC (Silica Gel 60 F254 Plates; Merck & Co., Frankfurt, Germany) analysis was performed by the method reported previously (18, 19). The peak fractions containing TIA were isolated by the HPLC system, and were identified on the basis of RF values, fluorescence under UV irradiation, and chromogenic reaction with 1% ceric ammonium sulfate and Dragendorff’s reagents. Scanning electron microscopy Leaves were fixed in FAA solution (2.5% formaldehyde, 2.5% acetic acid, 45% ethanol and 50% D.W.) under vacuum for 30 min. They were then dehydrated in an ethanol series (50% to 100%), and put into 100% isoamyl acetate. They were dried at the critical point (WCP-2 Critical Point Dryer; Hitachi, Tokyo). After the samples were sputtered with PtPd for 2 min (Ion Sputter; Hitachi), they were examined by scanning electron microscopy (JSM-6330F Field Emission Scanning Electron Microscope; JEOL, Tokyo) at 5 kV. RNA analyses RT-PCR was performed by the method reported previously (20). RNA was isolated from cultured or uncultured leaves using the TRIzol RNA isolation method. First-strand cDNA was prepared from 5 mg of total RNA with the Ready-ToGo T-primed First-Strand Kit (Amersham Bioscience, Piscataway, NJ, USA) according to the manufacturer’s instructions. Gene expression of five key enzymes in the TIA pathway, namely geraniol 10-hydroxylase (G10H), cytochrome P450-reductase (CPR), tryptophan decarboxylase (TDC), strictosidine synthase (STD) and strictosidine-D-glucosidase (SGD), was checked (Fig. 2). The gene of C. roseus putative 60-kDa chaperonin beta subunit (CHAP) was used as an internal control. This gene was expressed constitutively in cultured leaves under our culture conditions. PCR was performed using Taq DNA polymerase (Takara Bio, Shiga) with genespecific primers under the following conditions: 95°C for 1 min, followed by 35 cycles of the next three steps, 95°C for 1 min, 55°C
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TABLE. 1. Relative amounts (left : right) of F.C.W., D.C.W. and total HPLC peak area in the opposite leaves of C. roseus Relative amount (left side : right side) F.C.W. D.C.W. HPLC area a :b 1.008 ± 0.050 0.999 ± 0.042 1.001 ± 0.090 c :d 1.011 ± 0.046 0.990 ± 0.047 1.016 ± 0.137 Opposite leaves means left (a) and right (b) side leaves and in the left (c) and right (d) side of each leaf as shown in Fig. 1. Data were expressed as mean ± S.D. (a:b, n = 6; c:d, n = 9). Opposite leaf sections
FIG. 2. Schematic diagram of the main TIA biosynthetic pathway and enzymes. Solid arrows, Single enzymatic conversions. Dotted arrows, Multiple enzymatic conversions. Abbreviations of enzymes in the figure are as follows: CPR, cytochrome P450-reductase; G10H, geraniol 10-hydroxylase; TDC, tryptophan decarboxylase; STR, strictosidine synthase; SGD, strictosidine-D-glucosidase.
for 1 min and 72°C for 1 min, then 72°C for 4 min. The DNA products of the RT-PCR were cloned using the TA cloning vector (pGEM-T Easy Vector System; Promega, Fitchburg, WI, USA), and then sequenced (3100-Avant Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). Primers used were as follows, 5¢-ATGGATTCTAGCTCGGA GAAGTTG-3¢ and 5¢-TCACCAGACATCTCGGAGATACC-3¢ for Cpr (GenBank accession no. X69791), 5¢-ATGGATTACCTTAC CATAATATT-3¢ and 5¢-TTAAAGGGTGCTTGGTACAGCAC-3¢ for G10h (AJ251269), 5¢-ATGGGCAGCATTGATTCAACAAAT3¢ and 5¢-TCATCAAGCTTCTTTGAGCAAATC-3¢ for Tdc (X67662), 5¢-ATGGCAAACTTTTCTGAATCTAAA-3¢ and 5¢TAGCTAGAAACATAAGAATTTCCC-3¢ for Str (X61932), 5¢ATGGGATCTAAAGATGATCAGTCC-3¢ and 5¢-TTAGTATTTTT GCTTCTTGACTAAC-3¢ for Sgd (AF112888), and 5¢-ACTCTTG TGGTGAATAAACTTC-3¢ and 5¢-TTTGAGCTCCGTTTCAGTT TGC-3¢ for Chap (AF329435).
RESULTS Development of a method for culturing the leaves of C. roseus In this study, the leaf was selected as the optimal organ because it is a renewable resource, and is considered to be appropriate for practical use, i.e., separation of roots or stems from intact plants causes fatal damage. Moreover, using the leaf had advantages for setting up the experimental control as described below in detail. In general, the productivity of secondary metabolites in the leaves of higher plants is different based on position of leaves, age, each individual plant and environmental conditions. In order to evaluate the cultivation of leaves of an in-
tact plant, it is necessary to set appropriate experimental controls. We focused on the symmetrical feature of C. roseus leaves (Fig. 1). The relative ratios (left leaf : right leaf) of fresh cell weight (F.C.W.), dry cell weight (D.C.W.) and TIAs in opposite leaves were approximately one, suggesting that there were no marked differences between the leaves (Table 1). HPLC chromatograms showed that the compositions of the TIAs in the left and right side leaves of C. roseus were almost the same (data not shown). These tendencies were also observed between the left and right side of each leaf. On the basis of these results, we constructed a new culture system using the leaves (Fig. 1). After cutting and separating opposite leaves (Fig. 1, a and b), the surfaces of the leaves were sterilized, and then these leaves were cut symmetrically as shown in Fig. 1, c, d, e and f. One piece of leaf (Fig. 1, c) was used for the control without cultivation. The F.C.W., D.C.W., residual glucose concentration, peroxidase activity and TIA amount were measured during cultivation and these were compared to those of the control. It was thus possible to evaluate the cultivation using the leaves of an intact plant. One gram of fresh C. roseus leaves was cultivated in 50 ml of phytohormone-free MS10G medium (MS liquid medium containing 10 g/l glucose) using 200-ml Erlenmeyer flasks on a rotary shaker (120 rpm) under dark conditions. The leaves consumed glucose completely over 12 d of cultivation (Fig. 3A). The D.C.W. of the leaves was increased about twofold over 12 d of cultivation (Fig. 3B). The mesophyll cells in a cross section of a leaf were elongated and the thickness of the section increased from approximately 100 mm to 160 mm over 12 d of cultivation (Fig. 4A, C). The epidermal cells of the leaf were also expanded (Fig. 4B, D). It was found that the size of stomata was not changed but the epidermal cells around stomata were expanded. The leaf cells could consume glucose and grow, suggesting that leaves separated from the intact plant maintain primary metabolism even in liquid culture conditions. However, the total amount of TIAs (including both intracellular and extracellular TIA) was decreased to less than half of that in control leaves (0 d). Culture conditions for producing TIAs To produce TIAs in cultured leaves, various culture conditions were examined. First, the effect of osmotic pressure in the medium on TIA production was investigated. The leaves of intact C. roseus are aerial, so the leaves suspended in liquid medium are exposed to considerable osmotic stress. The leaves cultivated in liquid culture under isotonic conditions (namely, the osmotic pressure in the medium is equal to that found intracellularly) were assumed to be damaged little by osmotic stress. The osmotic pressure in the vacuoles of vari-
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FIG. 5. Effects of osmotic pressure (mannitol addition) on total TIA amount (relative HPLC area) of C. roseus cultured leaves after cultivation for 14 d. The relative amount was calculated from the value of peak area after cultivation divided by the value of peak area before cultivation. Dotted line, Relative amount = 1. Data are expressed as mean± SD (n = 3).
FIG. 3. (A) Time course of glucose concentration during the cultivation of C. roseus leaves (1 g fresh weight) in MS phytohormone-free medium with 10 g/l glucose. (B) Initial (0 d) and final (14 d) D.C.W. of C. roseus leaves after cultivation in the medium. Data are expressed as mean±SD (n = 10).
FIG. 6. Effects of light irradiation and glucose addition on total TIA amount of C. roseus leaves after cultivation for 14 d. a, Continuous dark; b, 7 d light and 7 d dark; c, 7 d dark and 7 d light; d, continuous light; e, continuous light without glucose addition. For experiments a–d, 10 g/l glucose and 40 g/l mannitol were added to the MS medium without phytohormones. However, for experiment e, glucose was not added and the mannitol concentration was increased to 50 g/l. Dotted line, Relative amount = 1. Data are expressed as mean ± SD (n = 3).
FIG. 4. Scanning electron micrographs of a cross-section of C. roseus leaves (A, C) and surface (B, D). (A, B) Control leaf (0 d). (C, D) Cultured leaf (12 d). Scale bar: 50 mm.
ous plants is ordinarily from 500 to 1500 kPa (21). As a result of our experiments, the average value of the osmotic pressure in C. roseus leaf cells was found to be approximately 993 kPa at 25°C. Therefore, MS10G + 40M (addition of 40 g/l mannitol to the medium; the osmotic pressure was 993 kPa at 25°C) was prepared as the isotonic medium. The leaves cultivated in the isotonic medium did not exhibit a
decrease in the total amount of TIAs (Fig. 5). However, in the case of both the low osmotic pressure medium (D.W.; the osmotic pressure is almost 0 kPa at 25°C and MS10G + 0M, no addition of mannitol; the osmotic pressure was about 375 kPa at 25°C) and high osmotic pressure medium (MS10G + 70M, addition of 70 g/l mannitol to the medium; the osmotic pressure was about 1439 kPa at 25°C), the total amount of TIAs in the leaves was decreased. Leaves may retain their secondary metabolism ability in liquid culture under optimal osmotic conditions. Adequate control of the osmotic pressure in the medium was necessary for producing TIAs in this culture system. Secondly, the effect of light irradiation on the production of TIAs was investigated. As light irradiation is known to promote secondary metabolism in many plants, it was also
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FIG. 7. Expression profiles of genes encoding enzymes in the TIA biosynthetic pathway (Fig. 2) were analyzed by semi-quantitative RT-PCR. Dark (Fig. 6, a) and light (Fig. 6, d) culture conditions were compared. Chap was used as an internal control of gene expression. Chap, Catharanthus roseus putative 60 kDa chaperonin beta subunit.
FIG. 8. Time courses of glucose and dry cell concentration (A), ajmalicine concentration (B), relative amount of serpentine (C) and peroxidase activity (D) during the cultivation of C. roseus leaves in MS + glucose (10 g/l) + mannitol (40 g/l) medium without phytohormones under continuous light irradiation. Open bars show extracellular, while closed bars show intracellular concentration, relative amount and activity. n.d. Not detected. Data are expressed as mean±SD (n=3).
expected to be effective to promote TIA production in this culture system. TIA production in the leaves was promoted by light irradiation (60 mmol m–2 s–1) (Fig. 6, b–d). Under continuous light conditions (Fig. 6, d), the total amount of TIAs was increased about 2.8-fold compared to that in the control. Moreover, it was clear from the HPLC chromatogram that the production of Aj and Sp was enhanced significantly by light irradiation (data not shown). Thirdly, the effect of the addition of glucose under light irradiation on the production of TIAs was investigated. TIAs were produced under the conditions listed for Fig. 6, d. However, in the absence of glucose (containing only 50 g/l mannitol), the total amount of TIA did not increase (Fig. 6, e) and their composition did not change (data not shown). This result suggested that the addition of glucose was needed for TIA production. Expression of enzyme genes in the TIA pathway The changes in the expression of enzyme genes in the TIA biosynthetic pathway were analyzed and compared under light and dark conditions by semi-quantitative RT-PCR analysis (Fig. 7). Under light conditions, Aj and Sp production was promoted (Fig. 6, d), whereas it was not in the dark (Fig. 6, a). G10h was expressed only on 0 d (control) under dark conditions during 14 d of cultivation. In the case of light conditions, G10h was continuously expressed at high level until 12 d of cultivation. The transcript levels of Cpr during cultivation was higher under the light conditions than in the dark. The transcript level of Tdc (not detected at 0 d) was also higher under the light conditions than in the dark. Expression of the Str gene was detected on 0 d (control), but it was not detected during the cultivation as in the case of G10h. The Sgd gene was only induced under light conditions. From these results, it was clear that the culture conditions (MS10G + 40M under light irradiation) promoted the expression of genes encoding enzymes in the TIA pathway. Effect of the addition of glucose to the medium on TIA production Figure 8 shows the time courses of glucose concentration, dry cell concentration, Aj concentration, and relative amount of Sp and peroxidase activity during the cultivation of C. roseus leaves in MS10G + 40M medium under continuous light irradiation.
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FIG. 10. HPLC chromatogram of alkaloids in cultured leaves. The culture conditions were as described in Fig. 9, c. The arrows show peaks of TIAs. Vd, Vindoline; Aj, ajmalicine; Ca, catharanthine; Sp, serpentine.
ample, the concentration of vindoline was decreased slightly and catharanthine was produced in small amount. FIG. 9. Effects of glucose feeding method on (A) ajmalicine and (B) serpentine production. a, Control leaves; b, MS + glucose (10 g/l) + mannitol (40 g/l) (12-d batch culture); c, MS + glucose (10 g/l) + mannitol (40 g/l) (18-d fed-batch culture). Glucose (10 g/l) was added on day 10; d, MS+glucose (20 g/l)+mannitol (30 g/l) (22-d batch culture); e, MS + glucose (10 g/l) + mannitol (40 g/l) (28-d fed-batch culture). Glucose (10 g/l) was added on days 10 and 18; f, MS+glucose (30 g/l)+ mannitol (10 g/l) (28-d batch culture). n.d., Not detected. Data are expressed as mean ± SD (n = 3).
The dry cell concentration was gradually increased during 7 d of cultivation and then leveled off. Glucose was consumed completely by 12 d (Fig. 8A). Aj was produced and reached the maximum concentration (7 mg/l) at 7 d, when the cell growth stopped, and then decreased (Fig. 8B). The Sp amount started to increase after 7 d, and reached the maximum value (about 7-fold that of the control) at 12 d, when the glucose was consumed completely (Fig. 8C). Both alkaloids were mainly accumulated within the cells. As peroxidase is considered to convert Aj into Sp in C. roseus (22), the peroxidase activity was measured during cultivation (Fig. 8D). The change in peroxidase activity was correlated with Sp production. As the lack of glucose in the medium was likely to decrease the amounts of Aj and Sp (Fig. 8), the effect of the addition of glucose to the medium on the production of Aj and Sp in cultured leaves was investigated. Two types of fed-batch cultures (glucose was fed at 10 d [Fig. 9, c]; fed at 10 d and 18 d [Fig. 9, e]) and batch cultures (using medium containing 20 g/l glucose [Fig. 9, d]; 30 g/l glucose [Fig. 9, f]) were performed. Both Aj and Sp were produced in the highest concentrations in the fed-batch culture (Fig. 9, c). The Aj concentration was about 18 mg/l and the amount of Sp was increased about 11-fold compared to the control. Figure 10 shows the change in the HPLC chromatogram of TIAs after 18 d of cultivation. The peaks of Aj (not detected at 0 d) and Sp were particularly increased; the composition and amount of other TIAs were changed, for ex-
DISCUSSION In this study, we attempted to develop a novel method for TIA production by culturing differentiated intact C. roseus leaves directly. Consequently, we have succeeded in stimulating Aj and Sp production and accumulation in the cultured leaves. It has been reported that both compounds are accumulated mainly in the root of intact plants. Therefore, Aj is obtained by extraction from field-grown C. roseus roots in practical applications (23). The Aj content of the C. roseus roots used in this study (grown for about 1 year) was about 1.1 mg/g D.C.W., while in the cultured leaves reported here it was about 1.6 mg/g D.C.W. after 18 d of cultivation. In general, many plants accumulate useful metabolites in the roots of the intact plant. Thus, it is interesting that Aj and Sp were produced in the cultured leaves. It was also shown that the gene expression for key enzymes in the TIA pathway was promoted in this culture system (Fig. 7). These results indicated that leaves separated from intact plants were able to survive in liquid culture conditions, and could maintain both primary and secondary metabolism, and produce secondary metabolites actively. Fukuda and Komamine (24) reported that adjustment of the osmotic pressure in the medium was necessary for the tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans and they found that the cells showed hardly any growth unless the osmotic pressure was adjusted to 0.3 to 0.4 M (about 743 to 990 kPa at 25°C) by the addition of mannitol. There seemed to be a close relationship between osmotic pressure and the mesophyll cell growth and/or differentiation. Light irradiation (60 mmol m–2 s–1) was also an important factor to promote the production of Aj and Sp. The light irradiation increased both the transcription level of TIA enzyme genes (Fig. 7, especially G10h, Str and Sgd) and the production levels of Aj and Sp. It was considered that the
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light acted as a type of signal for activating TIA metabolism. The effect of light irradiation on TIA production in C. roseus cultured cells and tissue culture has been reported previously (reviewed in Ref. 25). It is also well known that light irradiation often restricts secondary metabolite production in many plants, for example, menthol production by Menta arvensis shoots (26). The increase in the D.C.W. of leaves (Fig. 3) might be caused by cell elongation or expansion with consumption of glucose (Fig. 4). In the case of the medium without glucose, the D.C.W. of leaves did not increase during cultivation (data not shown). The addition of glucose to the medium was necessary for promoting the production of Aj and Sp in this system. Therefore, it was considered that cultured leaves used the glucose for producing primary metabolites (cell wall components etc.) and secondary metabolites (TIAs etc.). Aj and Sp production was enhanced by the addition of adequate glucose to the medium. By feeding glucose (10 g/l) at 10 d (Fig. 9, c), Aj was produced at a concentration of about 18 mg/l. Sp was produced in an amount about 11-fold that of the control. However, the feeding of additional glucose (10 g/l) at 18 d caused a decrease in Aj and Sp accumulation (Fig. 9, e). The consumption rate of glucose by the cultured leaves decreased after 21 d of cultivation, and the consumption stopped at 28 d (data not shown). It was considered that the viabilities of the cells in the leaves decreased with the long cultivation time, and the intracellular TIAs were degraded. The same trends were also observed between the results shown in conditions d and f in Fig. 9, which were those of the batch cultures with increased glucose concentration at the start of cultivation. In these cases, the osmotic pressure in the medium might have changed markedly in association with the glucose consumption. The changes in the contents of four TIAs (vindoline, chatharanthine, Aj and Sp) in C. roseus leaf disks during callus induction using phytohormones on solid medium have been reported (27). The contents of vindoline and chatharanthine were markedly decreased, while those of Aj and Sp were continuously increased during 40 d of cultivation. In the system described here, the vindoline content was decreased, while that of Aj and Sp was increased during cultivation using liquid medium without phytohormones. As vindoline is accumulated in the leaf of C. roseus and produced under light irradiation (28), it was assumed that the production of vindoline was promoted in this culture system. However, the area on the HPLC chromatogram decreased to half that of the control for 18 d of cultivation (Fig. 10). In this study, we established adequate culture conditions for maximizing the ability of intact plant leaves of C. roseus to produce secondary metabolites (Aj and Sp) and thus developed a novel system. These results represent the first step in the development of a novel production system for plant secondary metabolites. The improvement of alkaloid productivities and simplification of the preparation of leaves are necessary for the use of this method in practical applications. In order to enhance the productivities of Aj and Sp, and to produce other TIAs in the system described here, it is necessary to investigate many factors affecting TIA metabolism. A number of approaches for increasing TIA productiv-
ity in calli and tissue culture have been investigated (25, 29, 30). These approaches might be useful to enhance TIA production in this system. This methodology could be generally applied to any plant, even plants for which it is difficult to induce callus formation or cause infection with Agrobacterium, and may also be useful for the production of various metabolites using many other plants. Application of this methodology to other plant leaves is currently being investigated. ACKNOWLEDGMENTS The authors are grateful to Dr. Shinichi Miyamura, University of Tsukuba, Japan, for instructions for using SEM. We would like to thank Prof. Frank DiCosmo, University of Toronto, Canada, for his linguistic advice. This study was supported in part by the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
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15. Yamada, Y., Kobayashi, S., Watanabe, K., and Hayashi, U.: Production of horseradish peroxidase by plant cell culture. J. Chem. Technol. Biotechnol., 38, 31–39 (1987). 16. Aoyagi, H., Sakamoto, Y., Asada, M., and Tanaka, H.: Indole alkaloids production by Catharanthus roseus protoplasts with artificial cell walls containing of guluronic acid rich alginate gel. J. Ferment. Bioeng., 85, 306–311 (1998). 17. Jung, K. H., Kwak, S. S., Kim, S. W., Lee, H., Choi, C. Y., and Liu, J. R.: Improvement of the catharanthine productivity in hairy root cultures of Catharanthus roseus by using monosaccharides as a carbon source. Biotechnol. Lett., 14, 695–700 (1992). 18. Farnsworth, N. R., Blomster, R. N., Damratoski, D., Meer, W. A., and Cammarato, L. V.: Studies on Catharanthus alkaloids. VI. Evaluation by means of thin-layer chromatography and ceric ammonium sulfate spray reagent. Lloydia, 27, 302– 314 (1964). 19. Morris, P., Scragg, A. H., Smart, N. J., and Stafford, A.: T.l.c. separation of Catharanthus alkaloids, p. 141–143. In Dixon, R.A. (ed.), Plant cell culture: a practical approach. IRL Press, Eynsham (1985). 20. Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M.: Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant. J., 34, 733–739 (2003). 21. Mohr, H. and Schopfer, P.: Concept of water potential in the water relations of cells, p. 45–51. In Mohr, H. and Schopfer, P. (ed.), Plant physiology. Springer-Verlag, Berlin, Heidelberg, New York (1994). 22. Blom, T. J. M., Sierra, M., Vanvliet, T. B., Franke-van Dijk, M. E. I., De Koning, P., van Iren, F., Verpoorte, R., and Libbenga, K. R.: Uptake and accumulation of ajmalicine into isolated vacuoles of cultured cell of Catharanthus
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roseus (L.) G. Don. and its conversion into serpentine. Planta, 183, 170–177 (1991). Drapeau, D., Blanch, H. W., and Wilke, C. R.: Economic assessment of plant cell culture for the production of ajmalicine. Biotechnol. Bioeng., 30, 946–953 (1987). Fukuda, H. and Komamine, A.: Establishment of an experimental system for the study of trachery element differentiation from single cells isolated from the mesophyll of Zinnia elegans. Plant. Physiol., 65, 57–60 (1980). Moreno, P. R. H., van der Heijden, R., and Verpoorte, R.: Cell and tissue cultures of Catharanthus roseus (L.) G. Don: a literature survey II. Updating from 1988 to 1993. Plant Cell Tissue Organ Cult., 42, 1–25 (1995). Asai, I., Yoshihira, K., Omoto, T., Sakui, N., and Shimomura, K.: Growth and monoterpene production in shoot culture and regenerates of Mentha arvensis. Plant Tissue Cult. Lett., 11, 218–225 (1994). Morris, P.: Regulation of product synthesis in cell cultures of Catharanthus roseus. III. Alkaloid metabolism in cultured leaf tissue and primary callus. Planta Med., 52, 127–132 (1986). Balsevich, J., De Luca, V., and Kurz, W. G. W.: Altered alkaloid pattern in dark grown seedlings of Catharanthus roseus. The isolation and characterization of 4-desacetoxyvindoline: a novel indole alkaloid and proposed precursor of vindoline. Heterocycles, 24, 2415–2421 (1986). van der Heijden, R., Verpoorte, R., and Ten Hoopen, H. J. G.: Cell and tissue cultures of Catharanthus roseus (L.) G. Don: a literature survey. Plant Cell Tissue Organ Cult., 18, 231–280 (1989). Asada, M. and Shuler, M. L.: Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ, elicitors and alginate immobilization. Appl. Microbiol. Biotechnol., 30, 475–481 (1989).
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Development of a Novel System for Producing Ajmalicine and Serpentine Using Direct Culture of Leaves in Catharanthus roseus Intact Plant AKIRA IWASE,1 HIDEKI AOYAGI,1 MASARU OHME-TAKAGI,2 AND HIDEO TANAKA1* Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan1 and Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, Tsukuba, Ibaraki 305-8562, Japan2 Received 10 December 2004/Accepted 11 January 2005
Due to problems of production instability, the production of plant secondary metabolites using dedifferentiated cells (callus) is not always feasible on an industrial scale. To propose a new methodology, which does not use dedifferentiated cells, a novel system for producing useful secondary metabolites using the direct culture of intact plant leaves was developed. Catharanthus roseus was used as a model medicinal plant to produce terpenoid indole alkaloids (TIAs) by suspension culture of the leaves in the phytohormone-free MS liquid medium. Adjustment of the osmotic pressure (993 kPa at 25°C) in the medium, light irradiation (60 mmol m–2 s–1) and addition of glucose (10 g/l) were effective to promote the production of TIAs such as ajmalicine (Aj) and serpentine (Sp). On the basis of semi-quantitative RT-PCR analyses, it was revealed that the culture conditions promoted gene expression of enzymes in the TIA pathway in the cultured leaves. By feeding glucose (10 g/l) on day 10 of the culture period, Aj was produced at a concentration of about 18 mg/l and Sp was produced at a concentration about 11-fold that of the control. These results represent the first step in the development of a novel production system for plant secondary metabolites. [Key words: Catharanthus roseus, cell culture, terpenoid indole alkaloid, secondary metabolite, biosynthesis]
sion of transgenes in calli (9). Moreover, it has been reported that the production instability occurred in a transgenic callus even though the transgenes were stably expressed (10). Thus, useful metabolite production using a callus is a risky technology. Much attention therefore has been focused on the development of a useful metabolite production system using genetically stable cells instead of a callus. The biosynthesis and accumulation of secondary metabolites in higher plants take place in highly differentiated cells during their developmental processes (11, 12). Therefore, many approaches using differentiated cells, such as those of multiple shoots, adventitious roots and hairy roots, have been proposed. However, establishment of cultured organs is not always feasible for all plants. Moreover, the procedure to select cultured organs with high productivity is timeconsuming and involves the induction of a callus using adequate phytohormones or Agrobacterium infection. Cells that constitute an organ of an intact plant have the ability to produce secondary metabolites. In other words, the organ is considered to represent a state of immobilization with differentiated cells at high density with the ability to stably produce secondary metabolites due to the cell to cell communication.
From the end of the 1960s, the production of useful metabolites (mainly secondary metabolites) using plant cell culture (callus) technology has been widely investigated (1). As a callus can grow indefinitely and culture conditions for its growth can be easily controlled, the technology has great potential for producing useful metabolites on an industrial scale. However, there are only a few cases of practical applications, such as the production of shikonin (2), tuberose polysaccharides (3), and paclitaxel (reviewed in Ref. 4). One of the main problems is instability associated with calli for useful metabolite production (5). In general, the ability of a callus to produce secondary metabolites is lost during the induction of callus (being in a dedifferentiated state) formation from an intact plant (being in a differentiated state) using phytohormones. Even if a cell line with high productivity is screened using various selection techniques, a loss in productivity frequently occurs over time during subculturing (6). Such callus instability might be due to genetic and epigenetic changes such as chromosome breakage (7) and DNA methylation (8). The phenomenon of DNA methylation has also been reported to be involved in the expres* Corresponding author. e-mail: [email protected] phone: +81-(0)29-853-7297; 7212 fax: +81-(0)29-853-4605 208
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We considered that if the optimal culture conditions for maximizing the secondary metabolite production ability of intact plant organs are established, a simple and stable system for producing secondary metabolites might be developed. On the basis of this idea, the direct culture of intact plant organs was investigated using phytohormone-free liquid medium. Catharanthus roseus (L.) G. Don, which has been widely employed for the production of useful metabolites (13), was used in this study as a model medicinal plant. C. roseus biosynthesizes terpenoid indole alkaloids (TIAs) such as dimeric alkaloid vinblastine (anticancer activity), monomeric ajmalicine (anti-hypertension activity) and its oxidized form, serpentine (tranquillizer activity). In this paper, we propose a novel methodology, which does not use a callus (dedifferentiated cells), for producing useful secondary metabolites using intact plant organs. As a model, a system for the production of ajmalicine (Aj) and serpentine (Sp) by the direct culture of leaves of C. roseus was developed.
MATERIALS AND METHODS Plant material C. roseus (L.) G. Don was propagated from cuttings, which were obtained from naturally grown plants in Okinawa Prefecture, Japan. It was grown in a growth cabinet (temperature, 25°C; photoperiod, 16 h; dark period, 8 h; light intensity, 60 mmol m–2 s–1). The fully expanded leaves, third to the sixth from the shoot apical meristem, were used for the experiments. Cultivation and analysis Opposite leaves were separated from the stem on the left and right as shown in Fig. 1, a and b. The leaves were washed with distilled water (D.W.) containing 0.005% (v/v) Polyoxyethylene sorbitan monooleate (Wako Pure Chemical Industries, Osaka) and then, their surfaces were sterilized using
FIG. 1. Schematic diagram of the method used for symmetric cutting and cultivation of C. roseus leaves. a, Left side leaf; b, right side leaf; c or e, left side of a leaf; d or f, right side of a leaf.
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both 70% EtOH for 1 min and 2% chlorine sodium hypochlorite solution (Wako) for 15 min. Finally, the leaves were washed three times with sterile D.W. After the surfaces of the leaves were wiped with sterile paper, these leaves were cut and separated at the midrib into left and right sections (Fig. 1, c–f). Suspension culture was carried out in 200-ml Erlenmeyer flasks on a rotary shaker (120 rpm) at 25°C. One gram of the fresh leaves was used to inoculate 50 ml of phytohormonefree MS medium (14) containing 10 g/l glucose. The pH of the medium was adjusted to 5.7 before autoclaving at 121°C for 20 min. After the cultivation, the total medium volume was measured and the leaves were separated from the medium using a 32-mm stainless mesh. The leaves were washed with a large amount of D.W. and then frozen in liquid nitrogen and freeze-dried for 72 h. The weight of leaves was measured and the dry cell concentration was calculated. The residual glucose concentration was measured with the Glucose test kit Wako (Wako). Peroxidase activity was measured according to the procedure described previously (15). Osmotic pressure measurement Osmotic pressure of the medium was measured by a Digital Micro Osmometer OM801 (Vogel, Giessen, Germany) based on the freezing point depression system. In order to measure the osmotic pressure of C. roseus leaves, sufficient water was supplied to the intact plant 24 h before the measurement. The leaves, third to the sixth from the shoot apical meristem, were frozen in the liquid nitrogen and mashed, centrifuged at 9000´g for 5 min, and then the supernatant was used for the osmotic pressure measurement. TIAs extraction and analysis Both intracellular and extracellular alkaloid levels were determined. The extraction of TIAs was performed using the method reported previously (16). To quantify the TIAs, HPLC analysis (17) was performed with the LC-VP HPLC system (Shimadzu, Kyoto). Amount of serpentine was expressed as its HPLC chromatogram area and the total TIAs was expressed as the total area on the HPLC chromatogram. To identify the alkaloids, TLC (Silica Gel 60 F254 Plates; Merck & Co., Frankfurt, Germany) analysis was performed by the method reported previously (18, 19). The peak fractions containing TIA were isolated by the HPLC system, and were identified on the basis of RF values, fluorescence under UV irradiation, and chromogenic reaction with 1% ceric ammonium sulfate and Dragendorff’s reagents. Scanning electron microscopy Leaves were fixed in FAA solution (2.5% formaldehyde, 2.5% acetic acid, 45% ethanol and 50% D.W.) under vacuum for 30 min. They were then dehydrated in an ethanol series (50% to 100%), and put into 100% isoamyl acetate. They were dried at the critical point (WCP-2 Critical Point Dryer; Hitachi, Tokyo). After the samples were sputtered with PtPd for 2 min (Ion Sputter; Hitachi), they were examined by scanning electron microscopy (JSM-6330F Field Emission Scanning Electron Microscope; JEOL, Tokyo) at 5 kV. RNA analyses RT-PCR was performed by the method reported previously (20). RNA was isolated from cultured or uncultured leaves using the TRIzol RNA isolation method. First-strand cDNA was prepared from 5 mg of total RNA with the Ready-ToGo T-primed First-Strand Kit (Amersham Bioscience, Piscataway, NJ, USA) according to the manufacturer’s instructions. Gene expression of five key enzymes in the TIA pathway, namely geraniol 10-hydroxylase (G10H), cytochrome P450-reductase (CPR), tryptophan decarboxylase (TDC), strictosidine synthase (STD) and strictosidine-D-glucosidase (SGD), was checked (Fig. 2). The gene of C. roseus putative 60-kDa chaperonin beta subunit (CHAP) was used as an internal control. This gene was expressed constitutively in cultured leaves under our culture conditions. PCR was performed using Taq DNA polymerase (Takara Bio, Shiga) with genespecific primers under the following conditions: 95°C for 1 min, followed by 35 cycles of the next three steps, 95°C for 1 min, 55°C
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TABLE. 1. Relative amounts (left : right) of F.C.W., D.C.W. and total HPLC peak area in the opposite leaves of C. roseus Relative amount (left side : right side) F.C.W. D.C.W. HPLC area a :b 1.008 ± 0.050 0.999 ± 0.042 1.001 ± 0.090 c :d 1.011 ± 0.046 0.990 ± 0.047 1.016 ± 0.137 Opposite leaves means left (a) and right (b) side leaves and in the left (c) and right (d) side of each leaf as shown in Fig. 1. Data were expressed as mean ± S.D. (a:b, n = 6; c:d, n = 9). Opposite leaf sections
FIG. 2. Schematic diagram of the main TIA biosynthetic pathway and enzymes. Solid arrows, Single enzymatic conversions. Dotted arrows, Multiple enzymatic conversions. Abbreviations of enzymes in the figure are as follows: CPR, cytochrome P450-reductase; G10H, geraniol 10-hydroxylase; TDC, tryptophan decarboxylase; STR, strictosidine synthase; SGD, strictosidine-D-glucosidase.
for 1 min and 72°C for 1 min, then 72°C for 4 min. The DNA products of the RT-PCR were cloned using the TA cloning vector (pGEM-T Easy Vector System; Promega, Fitchburg, WI, USA), and then sequenced (3100-Avant Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). Primers used were as follows, 5¢-ATGGATTCTAGCTCGGA GAAGTTG-3¢ and 5¢-TCACCAGACATCTCGGAGATACC-3¢ for Cpr (GenBank accession no. X69791), 5¢-ATGGATTACCTTAC CATAATATT-3¢ and 5¢-TTAAAGGGTGCTTGGTACAGCAC-3¢ for G10h (AJ251269), 5¢-ATGGGCAGCATTGATTCAACAAAT3¢ and 5¢-TCATCAAGCTTCTTTGAGCAAATC-3¢ for Tdc (X67662), 5¢-ATGGCAAACTTTTCTGAATCTAAA-3¢ and 5¢TAGCTAGAAACATAAGAATTTCCC-3¢ for Str (X61932), 5¢ATGGGATCTAAAGATGATCAGTCC-3¢ and 5¢-TTAGTATTTTT GCTTCTTGACTAAC-3¢ for Sgd (AF112888), and 5¢-ACTCTTG TGGTGAATAAACTTC-3¢ and 5¢-TTTGAGCTCCGTTTCAGTT TGC-3¢ for Chap (AF329435).
RESULTS Development of a method for culturing the leaves of C. roseus In this study, the leaf was selected as the optimal organ because it is a renewable resource, and is considered to be appropriate for practical use, i.e., separation of roots or stems from intact plants causes fatal damage. Moreover, using the leaf had advantages for setting up the experimental control as described below in detail. In general, the productivity of secondary metabolites in the leaves of higher plants is different based on position of leaves, age, each individual plant and environmental conditions. In order to evaluate the cultivation of leaves of an in-
tact plant, it is necessary to set appropriate experimental controls. We focused on the symmetrical feature of C. roseus leaves (Fig. 1). The relative ratios (left leaf : right leaf) of fresh cell weight (F.C.W.), dry cell weight (D.C.W.) and TIAs in opposite leaves were approximately one, suggesting that there were no marked differences between the leaves (Table 1). HPLC chromatograms showed that the compositions of the TIAs in the left and right side leaves of C. roseus were almost the same (data not shown). These tendencies were also observed between the left and right side of each leaf. On the basis of these results, we constructed a new culture system using the leaves (Fig. 1). After cutting and separating opposite leaves (Fig. 1, a and b), the surfaces of the leaves were sterilized, and then these leaves were cut symmetrically as shown in Fig. 1, c, d, e and f. One piece of leaf (Fig. 1, c) was used for the control without cultivation. The F.C.W., D.C.W., residual glucose concentration, peroxidase activity and TIA amount were measured during cultivation and these were compared to those of the control. It was thus possible to evaluate the cultivation using the leaves of an intact plant. One gram of fresh C. roseus leaves was cultivated in 50 ml of phytohormone-free MS10G medium (MS liquid medium containing 10 g/l glucose) using 200-ml Erlenmeyer flasks on a rotary shaker (120 rpm) under dark conditions. The leaves consumed glucose completely over 12 d of cultivation (Fig. 3A). The D.C.W. of the leaves was increased about twofold over 12 d of cultivation (Fig. 3B). The mesophyll cells in a cross section of a leaf were elongated and the thickness of the section increased from approximately 100 mm to 160 mm over 12 d of cultivation (Fig. 4A, C). The epidermal cells of the leaf were also expanded (Fig. 4B, D). It was found that the size of stomata was not changed but the epidermal cells around stomata were expanded. The leaf cells could consume glucose and grow, suggesting that leaves separated from the intact plant maintain primary metabolism even in liquid culture conditions. However, the total amount of TIAs (including both intracellular and extracellular TIA) was decreased to less than half of that in control leaves (0 d). Culture conditions for producing TIAs To produce TIAs in cultured leaves, various culture conditions were examined. First, the effect of osmotic pressure in the medium on TIA production was investigated. The leaves of intact C. roseus are aerial, so the leaves suspended in liquid medium are exposed to considerable osmotic stress. The leaves cultivated in liquid culture under isotonic conditions (namely, the osmotic pressure in the medium is equal to that found intracellularly) were assumed to be damaged little by osmotic stress. The osmotic pressure in the vacuoles of vari-
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FIG. 5. Effects of osmotic pressure (mannitol addition) on total TIA amount (relative HPLC area) of C. roseus cultured leaves after cultivation for 14 d. The relative amount was calculated from the value of peak area after cultivation divided by the value of peak area before cultivation. Dotted line, Relative amount = 1. Data are expressed as mean± SD (n = 3).
FIG. 3. (A) Time course of glucose concentration during the cultivation of C. roseus leaves (1 g fresh weight) in MS phytohormone-free medium with 10 g/l glucose. (B) Initial (0 d) and final (14 d) D.C.W. of C. roseus leaves after cultivation in the medium. Data are expressed as mean±SD (n = 10).
FIG. 6. Effects of light irradiation and glucose addition on total TIA amount of C. roseus leaves after cultivation for 14 d. a, Continuous dark; b, 7 d light and 7 d dark; c, 7 d dark and 7 d light; d, continuous light; e, continuous light without glucose addition. For experiments a–d, 10 g/l glucose and 40 g/l mannitol were added to the MS medium without phytohormones. However, for experiment e, glucose was not added and the mannitol concentration was increased to 50 g/l. Dotted line, Relative amount = 1. Data are expressed as mean ± SD (n = 3).
FIG. 4. Scanning electron micrographs of a cross-section of C. roseus leaves (A, C) and surface (B, D). (A, B) Control leaf (0 d). (C, D) Cultured leaf (12 d). Scale bar: 50 mm.
ous plants is ordinarily from 500 to 1500 kPa (21). As a result of our experiments, the average value of the osmotic pressure in C. roseus leaf cells was found to be approximately 993 kPa at 25°C. Therefore, MS10G + 40M (addition of 40 g/l mannitol to the medium; the osmotic pressure was 993 kPa at 25°C) was prepared as the isotonic medium. The leaves cultivated in the isotonic medium did not exhibit a
decrease in the total amount of TIAs (Fig. 5). However, in the case of both the low osmotic pressure medium (D.W.; the osmotic pressure is almost 0 kPa at 25°C and MS10G + 0M, no addition of mannitol; the osmotic pressure was about 375 kPa at 25°C) and high osmotic pressure medium (MS10G + 70M, addition of 70 g/l mannitol to the medium; the osmotic pressure was about 1439 kPa at 25°C), the total amount of TIAs in the leaves was decreased. Leaves may retain their secondary metabolism ability in liquid culture under optimal osmotic conditions. Adequate control of the osmotic pressure in the medium was necessary for producing TIAs in this culture system. Secondly, the effect of light irradiation on the production of TIAs was investigated. As light irradiation is known to promote secondary metabolism in many plants, it was also
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FIG. 7. Expression profiles of genes encoding enzymes in the TIA biosynthetic pathway (Fig. 2) were analyzed by semi-quantitative RT-PCR. Dark (Fig. 6, a) and light (Fig. 6, d) culture conditions were compared. Chap was used as an internal control of gene expression. Chap, Catharanthus roseus putative 60 kDa chaperonin beta subunit.
FIG. 8. Time courses of glucose and dry cell concentration (A), ajmalicine concentration (B), relative amount of serpentine (C) and peroxidase activity (D) during the cultivation of C. roseus leaves in MS + glucose (10 g/l) + mannitol (40 g/l) medium without phytohormones under continuous light irradiation. Open bars show extracellular, while closed bars show intracellular concentration, relative amount and activity. n.d. Not detected. Data are expressed as mean±SD (n=3).
expected to be effective to promote TIA production in this culture system. TIA production in the leaves was promoted by light irradiation (60 mmol m–2 s–1) (Fig. 6, b–d). Under continuous light conditions (Fig. 6, d), the total amount of TIAs was increased about 2.8-fold compared to that in the control. Moreover, it was clear from the HPLC chromatogram that the production of Aj and Sp was enhanced significantly by light irradiation (data not shown). Thirdly, the effect of the addition of glucose under light irradiation on the production of TIAs was investigated. TIAs were produced under the conditions listed for Fig. 6, d. However, in the absence of glucose (containing only 50 g/l mannitol), the total amount of TIA did not increase (Fig. 6, e) and their composition did not change (data not shown). This result suggested that the addition of glucose was needed for TIA production. Expression of enzyme genes in the TIA pathway The changes in the expression of enzyme genes in the TIA biosynthetic pathway were analyzed and compared under light and dark conditions by semi-quantitative RT-PCR analysis (Fig. 7). Under light conditions, Aj and Sp production was promoted (Fig. 6, d), whereas it was not in the dark (Fig. 6, a). G10h was expressed only on 0 d (control) under dark conditions during 14 d of cultivation. In the case of light conditions, G10h was continuously expressed at high level until 12 d of cultivation. The transcript levels of Cpr during cultivation was higher under the light conditions than in the dark. The transcript level of Tdc (not detected at 0 d) was also higher under the light conditions than in the dark. Expression of the Str gene was detected on 0 d (control), but it was not detected during the cultivation as in the case of G10h. The Sgd gene was only induced under light conditions. From these results, it was clear that the culture conditions (MS10G + 40M under light irradiation) promoted the expression of genes encoding enzymes in the TIA pathway. Effect of the addition of glucose to the medium on TIA production Figure 8 shows the time courses of glucose concentration, dry cell concentration, Aj concentration, and relative amount of Sp and peroxidase activity during the cultivation of C. roseus leaves in MS10G + 40M medium under continuous light irradiation.
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FIG. 10. HPLC chromatogram of alkaloids in cultured leaves. The culture conditions were as described in Fig. 9, c. The arrows show peaks of TIAs. Vd, Vindoline; Aj, ajmalicine; Ca, catharanthine; Sp, serpentine.
ample, the concentration of vindoline was decreased slightly and catharanthine was produced in small amount. FIG. 9. Effects of glucose feeding method on (A) ajmalicine and (B) serpentine production. a, Control leaves; b, MS + glucose (10 g/l) + mannitol (40 g/l) (12-d batch culture); c, MS + glucose (10 g/l) + mannitol (40 g/l) (18-d fed-batch culture). Glucose (10 g/l) was added on day 10; d, MS+glucose (20 g/l)+mannitol (30 g/l) (22-d batch culture); e, MS + glucose (10 g/l) + mannitol (40 g/l) (28-d fed-batch culture). Glucose (10 g/l) was added on days 10 and 18; f, MS+glucose (30 g/l)+ mannitol (10 g/l) (28-d batch culture). n.d., Not detected. Data are expressed as mean ± SD (n = 3).
The dry cell concentration was gradually increased during 7 d of cultivation and then leveled off. Glucose was consumed completely by 12 d (Fig. 8A). Aj was produced and reached the maximum concentration (7 mg/l) at 7 d, when the cell growth stopped, and then decreased (Fig. 8B). The Sp amount started to increase after 7 d, and reached the maximum value (about 7-fold that of the control) at 12 d, when the glucose was consumed completely (Fig. 8C). Both alkaloids were mainly accumulated within the cells. As peroxidase is considered to convert Aj into Sp in C. roseus (22), the peroxidase activity was measured during cultivation (Fig. 8D). The change in peroxidase activity was correlated with Sp production. As the lack of glucose in the medium was likely to decrease the amounts of Aj and Sp (Fig. 8), the effect of the addition of glucose to the medium on the production of Aj and Sp in cultured leaves was investigated. Two types of fed-batch cultures (glucose was fed at 10 d [Fig. 9, c]; fed at 10 d and 18 d [Fig. 9, e]) and batch cultures (using medium containing 20 g/l glucose [Fig. 9, d]; 30 g/l glucose [Fig. 9, f]) were performed. Both Aj and Sp were produced in the highest concentrations in the fed-batch culture (Fig. 9, c). The Aj concentration was about 18 mg/l and the amount of Sp was increased about 11-fold compared to the control. Figure 10 shows the change in the HPLC chromatogram of TIAs after 18 d of cultivation. The peaks of Aj (not detected at 0 d) and Sp were particularly increased; the composition and amount of other TIAs were changed, for ex-
DISCUSSION In this study, we attempted to develop a novel method for TIA production by culturing differentiated intact C. roseus leaves directly. Consequently, we have succeeded in stimulating Aj and Sp production and accumulation in the cultured leaves. It has been reported that both compounds are accumulated mainly in the root of intact plants. Therefore, Aj is obtained by extraction from field-grown C. roseus roots in practical applications (23). The Aj content of the C. roseus roots used in this study (grown for about 1 year) was about 1.1 mg/g D.C.W., while in the cultured leaves reported here it was about 1.6 mg/g D.C.W. after 18 d of cultivation. In general, many plants accumulate useful metabolites in the roots of the intact plant. Thus, it is interesting that Aj and Sp were produced in the cultured leaves. It was also shown that the gene expression for key enzymes in the TIA pathway was promoted in this culture system (Fig. 7). These results indicated that leaves separated from intact plants were able to survive in liquid culture conditions, and could maintain both primary and secondary metabolism, and produce secondary metabolites actively. Fukuda and Komamine (24) reported that adjustment of the osmotic pressure in the medium was necessary for the tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans and they found that the cells showed hardly any growth unless the osmotic pressure was adjusted to 0.3 to 0.4 M (about 743 to 990 kPa at 25°C) by the addition of mannitol. There seemed to be a close relationship between osmotic pressure and the mesophyll cell growth and/or differentiation. Light irradiation (60 mmol m–2 s–1) was also an important factor to promote the production of Aj and Sp. The light irradiation increased both the transcription level of TIA enzyme genes (Fig. 7, especially G10h, Str and Sgd) and the production levels of Aj and Sp. It was considered that the
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light acted as a type of signal for activating TIA metabolism. The effect of light irradiation on TIA production in C. roseus cultured cells and tissue culture has been reported previously (reviewed in Ref. 25). It is also well known that light irradiation often restricts secondary metabolite production in many plants, for example, menthol production by Menta arvensis shoots (26). The increase in the D.C.W. of leaves (Fig. 3) might be caused by cell elongation or expansion with consumption of glucose (Fig. 4). In the case of the medium without glucose, the D.C.W. of leaves did not increase during cultivation (data not shown). The addition of glucose to the medium was necessary for promoting the production of Aj and Sp in this system. Therefore, it was considered that cultured leaves used the glucose for producing primary metabolites (cell wall components etc.) and secondary metabolites (TIAs etc.). Aj and Sp production was enhanced by the addition of adequate glucose to the medium. By feeding glucose (10 g/l) at 10 d (Fig. 9, c), Aj was produced at a concentration of about 18 mg/l. Sp was produced in an amount about 11-fold that of the control. However, the feeding of additional glucose (10 g/l) at 18 d caused a decrease in Aj and Sp accumulation (Fig. 9, e). The consumption rate of glucose by the cultured leaves decreased after 21 d of cultivation, and the consumption stopped at 28 d (data not shown). It was considered that the viabilities of the cells in the leaves decreased with the long cultivation time, and the intracellular TIAs were degraded. The same trends were also observed between the results shown in conditions d and f in Fig. 9, which were those of the batch cultures with increased glucose concentration at the start of cultivation. In these cases, the osmotic pressure in the medium might have changed markedly in association with the glucose consumption. The changes in the contents of four TIAs (vindoline, chatharanthine, Aj and Sp) in C. roseus leaf disks during callus induction using phytohormones on solid medium have been reported (27). The contents of vindoline and chatharanthine were markedly decreased, while those of Aj and Sp were continuously increased during 40 d of cultivation. In the system described here, the vindoline content was decreased, while that of Aj and Sp was increased during cultivation using liquid medium without phytohormones. As vindoline is accumulated in the leaf of C. roseus and produced under light irradiation (28), it was assumed that the production of vindoline was promoted in this culture system. However, the area on the HPLC chromatogram decreased to half that of the control for 18 d of cultivation (Fig. 10). In this study, we established adequate culture conditions for maximizing the ability of intact plant leaves of C. roseus to produce secondary metabolites (Aj and Sp) and thus developed a novel system. These results represent the first step in the development of a novel production system for plant secondary metabolites. The improvement of alkaloid productivities and simplification of the preparation of leaves are necessary for the use of this method in practical applications. In order to enhance the productivities of Aj and Sp, and to produce other TIAs in the system described here, it is necessary to investigate many factors affecting TIA metabolism. A number of approaches for increasing TIA productiv-
ity in calli and tissue culture have been investigated (25, 29, 30). These approaches might be useful to enhance TIA production in this system. This methodology could be generally applied to any plant, even plants for which it is difficult to induce callus formation or cause infection with Agrobacterium, and may also be useful for the production of various metabolites using many other plants. Application of this methodology to other plant leaves is currently being investigated. ACKNOWLEDGMENTS The authors are grateful to Dr. Shinichi Miyamura, University of Tsukuba, Japan, for instructions for using SEM. We would like to thank Prof. Frank DiCosmo, University of Toronto, Canada, for his linguistic advice. This study was supported in part by the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).
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