JXB Advance Access originally published online on August 16, 2005
Journal of Experimental Botany 2005 56(420):2611-2618; doi:10.1093/jxb/eri253
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RESEARCH PAPER |
Enhanced secretion of tropane alkaloids in Nicotiana tabacum hairy roots expressing heterologous hyoscyamine-6ß-hydroxylase
1VTT Biotechnology, Tietotie 2, PO Box 1500, FIN-02044 VTT (Espoo), Finland
2Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr Aiguader 80, E-08003 Barcelona, Spain
3Sección de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain
* To whom correspondence should be addressed. Fax: +358 20 722 7071. E-mail: Suvi.Hakkinen{at}vtt.fi
Received 29 April 2005; Accepted 21 June 2005
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Abstract |
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Hyoscyamine-6ß-hydroxylase (H6H; EC 1.14.11.11 [EC] ) catalyses oxidative reactions in the biosynthetic pathway leading from hyoscyamine to the more pharmaceutically valuable tropane alkaloid scopolamine. The h6h gene encoding H6H from Hyoscyamus niger was introduced, under the control of the CaMV 35S promoter, into non-hyoscyamine-producing Nicotiana tabacum and hyoscyamine-producing Hyoscyamus muticus. The transformation was performed using a binary vector system based on Agrobacterium rhizogenes. Production of scopolamine in hairy roots was clearly correlated with the 35S-h6h transcript expression. The engineered N. tabacum and H. muticus hairy roots were studied for the production of scopolamine and other tropane and nicotine alkaloids after feeding the cultures with exogenous hyoscyamine. N. tabacum hairy roots carrying the 35S-h6h transgene showed a more efficient uptake of hyoscyamine from the culture medium and a higher rate of bioconversion of hyoscyamine to scopolamine than those of H. muticus. In particular, the secretion of scopolamine in N. tabacum hairy roots was remarkably high, up to 85% of the total scopolamine being released to the culture medium. Exogenous hyoscyamine also caused changes in nicotine alkaloid accumulation in N. tabacum hairy roots.
Key words: Hairy roots, hyoscyamine-6ß-hydroxylase, Hyoscyamus, Nicotiana, secretion, tropane alkaloids
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Tropane alkaloids, for example, hyoscyamine and scopolamine, have pharmaceutical importance as anticholinergic agents, acting on the parasympathetic nervous system. Of the two, scopolamine is more valuable and is preferred for its higher physiological activity and fewer side-effects. Many plant-derived pharmaceuticals are still isolated from whole plants (e.g. Duboisia, Datura), this often being the only economically feasible production method. Plant cell cultures have been widely studied in order to obtain alternative production systems for compounds such as tropane alkaloids. The major bottleneck, however, is the complexity of the biosynthetic routes of these secondary compounds, involving unknown regulatory genes and enzymes (Oksman-Caldentey and Inzé, 2004
). Only a few functional genes of the tropane alkaloid pathway have been characterized and it is impossible to generalize about how the overexpression of genes involved in tropane alkaloid biosynthesis in different plant species might affect the end-product levels. Increased accumulation of the direct metabolite N-methylputrescine was observed when putrescine N-methyltransferase (pmt) was overexpressed in root cultures of Duboisia hybrids (Moyano et al., 2002), Atropa belladonna roots (Rothe et al., 2003), and Hyoscyamus muticus roots (Biondi et al., 2000), whereas the effect on the alkaloid level was only marginal (Moyano et al., 2003; Rothe et al., 2003). However, regulation of the expression of this gene has been shown to be crucial for alkaloid production in several genera, for example, Datura and Nicotiana (Robins et al., 1991, 1994; Piñol et al., 1999).
Scopolamine is converted from hyoscyamine in a two-step process catalysed by the enzyme hyoscyamine-6ß-hydroxylase (H6H, EC 1.14.11.11
[EC]
). This enzyme catalyses both the hydroxylation of hyoscyamine leading to 6ß-hydroxyhyoscyamine and the epoxidation of the latter leading to scopolamine (Hashimoto and Yamada, 1986). It is localized in the pericycle of the root, where the tropane alkaloids are also synthesized and accumulated (Hashimoto et al., 1986; Matsuda et al., 1991). Furthermore, in studies with H6H isolated from A. belladonna, Suzuki et al. (1999) reported that AbH6H is expressed in root pericycle cells, in the tapetum, and in pollen mother cells. It has been shown that the scopolamine/hyoscyamine ratio can be increased in hairy roots of hyoscyamine-producing plants by overexpressing h6h (Hashimoto et al., 1993b). Yun et al. (1992) reported the successful introduction of the h6h gene from H. niger into a related species A. belladonna, resulting in almost complete conversion of hyoscyamine to scopolamine. Later, Jouhikainen et al. (1999) showed that overexpressing the same gene can also considerably enhance the scopolamine content in H. muticus hairy roots, while the hyoscyamine content remains unaltered. In these examples, the engineering of a single step in the pathway has led to an improved accumulation of the more valuable end-product. Recently, Zhang et al. (2004) simultaneously overexpressed pmt and h6h genes in H. niger, resulting in a scopolamine production of 411 mg l–1, which is the highest level hitherto reported from cultivated hairy roots. Another interesting example of the expression of foreign genes in Solanaceous plants is the formation of resveratrol in Nicotiana as a result of heterologous expression of a gene from groundnut, coding for stilbene synthase (Hain et al., 1990).
Nicotiana and Hyoscyamus, both belonging to the Solanaceae family, produce alkaloids from a common biosynthetic origin (Fig. 1). N-methylputrescine, which is converted from arginine/ornithine-derived putrescine, provides both the pyrroline moiety of nicotine and part of the tropane ring structure of hyoscyamine. Recently, Rocha et al. (2002) introduced both h6h and tropinone reductase I (trI) together into N. tabacum plants and followed the accumulation of alkaloids after feeding the detached leaves with hyoscyamine. In addition to the accumulation of the direct products of these enzymes, they also reported elevated nicotine alkaloid levels in these leaves. However, based on these results it is difficult to evaluate the effects of the individual genes alone, because they were introduced simultaneously. Furthermore, since nicotine and tropane alkaloids are mainly produced in the roots and further transported to the leaves of the intact plant, it would be necessary to examine the effects of the exogenously applied substrate in culture systems, where the actual biosynthesis of these alkaloids takes place.
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In this work, the potential of using Nicotiana tabacum hairy roots for the bioconversion of exogenously supplied hyoscyamine to more valuable scopolamine was assessed by overexpressing the h6h from H. niger in Nicotiana. Secretion of the alkaloids into the culture medium was studied and compared with that of the h6h overexpressing H. muticus hairy roots which naturally produce hyoscyamine. In addition, in this study the effect and function of h6h alone was more closely determined, and the overall alkaloid accumulation in both Nicotiana and Hyoscyamus hairy roots was investigated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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Transformation of Nicotiana tabacum
Leaves of Nicotiana tabacum cv. Xanthi plantlets grown in vitro on MS medium (Murashige and Skoog, 1962
) were used for transformation with Agrobacterium rhizogenes LBA9402 wild type and A. rhizogenes LBA9402 pLAL21 carrying the 35S-h6h and the nptII gene as a marker, constructed by Jouhikainen et al. (1999). The hairy roots appeared 2–4 weeks after the infection and they were excised and cultured individually on MS medium supplemented with 30 g l–1 sucrose and 500 ppm cefotaxime to eliminate the bacteria. For hairy roots carrying the 35S-h6h transgene, kanamycin (50 ppm) was used for selection. Hairy root clones were kept in the dark at 25 °C and routinely subcultured every 25–30 d. After several subcultures, the root clones were transferred to MS liquid medium and kept on a rotary shaker at 100 rpm, 25 °C in the dark. Subculturing was performed every four weeks in fresh medium.
Hairy roots of Hyoscyamus muticus
The hairy roots of Hyoscyamus muticus (strain Cairo) were transformed with A. rhizogenes strains LBA9402 (control) and LBA9402 pLAL21 (carrying 35S-h6h) as described by Jouhikainen et al. (1999). The hairy roots were routinely grown and subcultured in modified B50 medium, as described by Oksman-Caldentey et al. (1991). The best scopolamine-producing clone KB7 (Jouhikainen et al., 1999) was chosen for feeding studies with hyoscyamine.
PCR analysis
Total DNA was isolated from hairy root clones according to Edwards et al. (1991). PCR analysis was performed using the preformulated, predispensed single dose reaction beads ‘Ready to GoTM’ (Pharmacia Biotech). The complete mixture contained 200 ng of total DNA, 12.5 pmol ml–1 of each oligonucleotide primer, 200 µM dNTPs, 1.5 µl Taq polymerase, and buffer supplied by the enzyme manufacturer (1/10V) in a total volume of 25 µl. The oligonucleotide primers used for amplification of the h6h gene were 5'-CCG GAA TTC GGA TCC CAA CGT ATA GAT TCT TC-3 and 5'-CGG GAA TTC GGA TCC CAA ACC ATC ACT GCA AT-3', according to the sequence of the h6h gene from Hyoscyamus niger (Matsuda et al., 1991), and produced a DNA fragment of 1153 bp. On the other hand, the primers used for amplification of the rolC gene (from A. rhizogenes T-DNA) were 5'-TAA CAT GGC TGA AGA CGA CC-3' and 5'-AAA CTT GCA CTC GCC ATG CC-3', according to the sequence of the gene described by Slightom et al. (1986), and produced a DNA fragment of 534 bp. PCR amplifications of h6h and rolC were: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 53 °C for 1 min, extension at 72 °C for 1.5 min, with a final extension at 72 °C for 5 min. The PCR reaction mixtures (10 µl) were then loaded directly onto 1.5% agarose/TBE gel for electrophoretic analysis. A 100 Base-Pair Ladder (Pharmacia Biotech) was used as a molecular weight marker for the PCR-amplified double-stranded DNA fragment.
Alkaloid analysis
Nicotine alkaloids were extracted as described in Häkkinen et al. (2004) using 2,4'-dipyridyl as an internal standard. Alkaloids were determined using a 6890 GC system fitted with a 5973 Mass Selective Detector (MSD) (Agilent Technologies) operating at an ionization voltage of 70 eV (EI mode). The analyses were performed on an NB-54 fused silica capillary column (15 m, 0.20 mm i.d., HNU Nordion) by using a split sampling mode (50:1) and a programmed oven temperature from 70 °C to 100 °C (10 °C min–1) and from 100 °C to 235 °C (15 °C min–1). Injector and detector temperatures were kept at 250 °C. Aliquots of 2 µl were injected by a 7683 AutoSampler (Agilent Technologies). Identification of the nicotine alkaloids was performed based on the mass fragmentation and the retention order (Häkkinen et al., 2004). The tropane alkaloids scopolamine and hyoscyamine were extracted and analysed from N. tabacum cultured hairy roots and culture medium with the method described by Plank and Wagner (1986), and alkaloids from H. muticus hairy roots as follows. Lyophilized roots were weighed (50 mg) and the lipids were removed with 2 ml petroleum ether. After vortexing and centrifugation (3000 rpm, 10 min) the solvent phase was discarded and the sample residue was dried under nitrogen flow. After adding 50 µg internal standard homatropine (Sigma), the samples were made alkaline (pH 9) and the alkaloids were extracted twice with Cl2CH2. Before GC-MS analysis, the dried residue was dissolved in 40 µl Cl2CH2 and derivatized with MSTFA (N-methyl-N-trifluoroacetamide, Pierce). The tropane alkaloids from the culture medium were extracted correspondingly from 2 ml medium. The GC-MS conditions were as desribed in Goossens et al. (2003a) and alkaloids and alkaloid derivatives were identified based on the GC-MS spectral data (Hartmann et al., 1986; Witte et al., 1987).
Northern blot analysis
Extraction of total RNA from N. tabacum hairy roots after 2 weeks of culture in MS medium was performed using the RNeasy kit (Qiagen). Ten micrograms of total RNA were loaded per lane of a denaturating formaldehyde gel (Sambrook et al., 1989). Northern blots were made on Hybond-N+ nylon transfer membrane (Amersham) and hybridized with 32P-labelled probe specific for h6h (full cDNA). Hybridization was performed at 42 °C in the presence of 50% formamide. The blots were washed at 65 °C with saline sodium citrate buffer (SSC), 0.1% SDS (30 min) and with 1x SSC, 0.1% SDS (15 min). The radioactivity on the filter was imaged using HyperfilmTM-MP (Amersham).
Feeding assays
Hairy root cultures of N. tabacum and H. muticus were fed with hyoscyamine added to the culture medium. The roots, weighing 500±5 mg and 100±5 mg from N. tabacum and H. muticus, respectively, were grown in 40 ml liquid MS and 20 ml B50 medium, respectively. The hyoscyamine hydrochloride (Sigma) was dissolved in water and the stock was added to a final concentration of 100 mg l–1 or 200 mg l–1. In control samples, only water was added. The hairy roots were grown as described above. Alkaloid levels were determined both in the roots and in the culture medium after a culturing period of 28 d.
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Results and discussion |
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Characterization and capacity for hyoscyamine bioconversion of N. tabacum transgenic hairy root clones
Ten (10) hairy root clones carrying the 35S-h6h transgene (named H6H) and three (3) control clones, showing good growth and typical hairy root morphology were analysed for their transformed nature. All the H6H clones gave two bands of 1.15 and 0.53 kb after PCR amplification, the former corresponding to the h6h fragment and the latter to the rolC fragment, whereas the control clones only gave the band corresponding to the rolC gene (data not shown).
The hyoscyamine uptake percentage and the biomass production of the control hairy root clones and the clones carrying the 35S-h6h transgene are shown in Table 1. The uptake of the added hyoscyamine was calculated from the amount of hyoscyamine measured from the culture medium at day 28. The degradation of hyoscyamine over 28 d in the culture conditions was found to be negligible. The uptake was, on average, 95% (84–99%) with 100 mg l–1 hyoscyamine, and 94% (78–100%) with 200 mg l–1 hyoscyamine. The biomass accumulation measured after 28 d varied between 0.14 and 0.51 g DW in 40 ml MS medium. The addition of 100 mg l–1 hyoscyamine had no significant effect on the growth, but hyoscyamine added at a higher concentration (200 mg l–1) caused a statistically significant reduction in the growth of all clones (ANOVA F2,36=18.532, P <0.001, for normalized values). Hyoscyamine concentrations of 300 mg l–1 and above had clear toxic effects on N. tabacum hairy roots and were therefore not included in the studies.
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In order to establish a possible relationship between the 35S-h6h transgene expression and the capacity of the transgenic hairy root clones to produce scopolamine, i.e. to carry out the bioconversion of exogenously applied hyoscyamine, the expression levels of the 35S-h6h transgene in the H6H clones were assayed by northern blot analysis. As shown in Fig. 2, the transgene expression was observed in H6H 1, H6H 14, and H6H 101, which were the only clones to produce scopolamine. This correlation is in accordance with earlier results that confirm h6h gene expression to be essential for scopolamine production in plant systems (Hashimoto and Yamada, 1986
; Jouhikainen et al., 1999). The bioconversion to scopolamine was highest when 100 mg l–1 hyoscyamine was used, the best clones H6H 1 and H6H 14 having conversion rates of 45% and 40%, respectively (Table 2).
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The total (medium and root-associated) scopolamine content was again highest in the clones H6H 1 and H6H 14 (Table 2). When the amount of added hyoscyamine was doubled (from 100 mg l–1 to 200 mg l–1), the production of scopolamine increased by 11% and 53% (calculated from Table 2) in the root clones H6H 1 and H6H 14, respectively. Thus, the most efficient absolute conversion rate was obtained with 100 mg l–1 hyoscyamine. The overall scopolamine production was high, even up to 64.1 mg l–1 (clone H6H 14). The low amounts of scopolamine produced by clone H6H 101 were most probably linked to the weak h6h expression (Fig. 2).
Production of tropane alkaloids in transgenic H. muticus hairy roots after feeding with hyoscyamine
Exogenous hyoscyamine was effectively taken up by both control and KB7 (with the 35S-h6h transgene) clones; the residual hyoscyamine found in the medium was less than 3% and less than 7% when hyoscyamine was added in concentrations of 100 mg l–1 and 200 mg l–1, respectively (Table 3). However, by contrast with the result observed for hairy root cultures of N. tabacum, exogenous hyoscyamine addition in concentrations even up to 500 mg l–1 did not affect the growth of H. muticus hairy roots (data not shown). Regarding the alkaloid production, although the root-associated hyoscyamine content in the control clone (without the 35S-h6h transgene) increased linearly with an increasing amount of added substrate, the control clone produced only trace amounts of total (medium and root associated) 6ß-hydroxyhyoscyamine and no scopolamine 28 d after feeding (Table 3). Compared with non-fed clones, the concentration of total hyoscyamine found in fed control clones was 3-fold (with 200 mg l–1). The total hyoscyamine content in hyoscyamine-fed roots was less than expected (135 mg l–1 and 218 mg l–1 after feeding 100 mg l–1 and 200 mg l–1, respectively), taking into account the endogenous production of 77 mg l–1 (Table 3). There are several possibilities causing the lower hyoscyamine content than expected, for instance feedback inhibition of certain biosynthetic steps by high concentration of this alkaloid, or degradation of the added precursor that can be used as nutrients by the hairy roots (Hashimoto and Yamada, 1983).
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In clone KB7 (carrying the 35S-h6h transgene) the endogenous hyoscyamine production was higher than that of the control clone (Table 3). The addition of hyoscyamine resulted in a higher production of the intermediate 6ß-hydroxyhyoscyamine, whereas the accumulation of scopolamine increased only slightly with 100 mg l–1 hyoscyamine (Table 3). Earlier, it was shown that, in the system in which hyoscyamine is fed, the H6H activity becomes limiting and causes the accumulation of 6ß-hydroxyhyoscyamine (Hashimoto et al., 1993a
).
When 100 mg l–1 hyoscyamine was added to the cultures, the conversion rate to scopolamine was 15% (Table 3). However, since the uptake of hyoscyamine did not increase linearly with the increasing concentrations of exogenous hyoscyamine, the conversion rate to scopolamine was clearly lower in 200 mg l–1 feeding, being only 2% (Table 3). In another study, Hashimoto and Yamada (1983) reported approximately 20% conversion from hyoscyamine to scopolamine in adventitious roots of H. niger after feeding with 0.1 mM (29 mg l–1) hyoscyamine. However, with 1 mM (290 mg l–1) hyoscyamine the conversion decreased to around 10%.
Compared with the control clone, the KB7 clone also produced slightly higher amounts of other minor alkaloids, for example pseudotropine, apoatropine, and 6ß-hydroxy-apoatropine (data not shown). The latter was not detected in the control clone. The amounts of apoatropine and 6ß-hydroxy-apoatropine increased with the increasing concentrations of added hyoscyamine, whereas the accumulation of pseudotropine remained stable.
Secretion of tropane alkaloids
In N. tabacum hairy root clones carrying the 35S-h6h transgene, scopolamine was efficiently secreted extracellularly; up to 85% of the produced scopolamine was detected in the culture medium (Table 2). This secretion rate is high compared with other reported values, when only up to 20% of the produced scopolamine has been found in the culture medium of Hyoscyamus or Atropa sp. (Hashimoto et al., 1986, 1993b; Jouhikainen et al., 1999). However, after feeding with hyoscyamine (1 mM), Hashimoto and Yamada (1983) reported that approximately 60–70% of the total scopolamine produced was detected in the extracellular medium. It was found that, compared with transgenic N. tabacum hairy root clones, in H. muticus carrying the 35S-h6h transgene, the scopolamine production was lower and, in particular, the secretion of scopolamine into the medium (7–12%) decreased considerably (Table 3). It is known that some secondary compounds, even though produced by the plant itself, may cause intrinsic toxicity to the cells. Scopolamine is a foreign substance for N. tabacum, in contrast to Hyoscyamus in which scopolamine is a compound endogenously produced. It may become toxic to N. tabacum cells in high concentrations, and is therefore efficiently transported to the surrounding medium. Recently, it has been shown with N. tabacum cell suspension cultures, that the secretion of nicotine alkaloids can be stimulated and the tolerance to exogenously supplied alkaloids (hyoscyamine and scopolamine) can be enhanced by using PDR-type ATP binding cassette transporters (Goossens et al., 2003b). Considering large-scale production systems, extracellular secretion of the product is desirable, as it reduces the costs during both production and downstream processing, allowing the use of a continuous culturing system and direct recovery of the metabolites from the medium.
Nicotine alkaloids
Nicotine alkaloids were determined from two control clones (LBA 1 and LBA 2) and two 35S-h6h transgenic clones (H6H 14 and H6H 101), exhibiting the highest and the lowest capacities for scopolamine production, respectively. N. tabacum hairy root clones produced nicotine as the major alkaloid, followed by anatabine, anatalline, anabasine, and nornicotine. The total root-associated nicotine alkaloid content varied in the range 0.63–5.10 mg g–1 DW and 2.17–5.43 mg g–1 DW in control and transgenic clones, respectively (Table 4). Interestingly, when hyoscyamine was fed to the cultures, the nicotine alkaloid levels were altered. The total alkaloid content in the control hairy root clones was increased 2–6-fold, mainly contributing to the increment in nicotine levels. It is doubtful whether hyoscyamine could be used as a substrate for nicotine biosynthesis. More probably, the resulting increased biosynthetic activity could be caused by recognition of hyoscyamine as a foreign substance, thus causing an elicitory effect in N. tabacum. Several nicotine alkaloids in Nicotiana have previously been shown to be induced by elicitors, for example, methyl jasmonate (Imanishi et al., 1998; Goossens et al., 2003a). In the clones carrying the 35-h6h transgene, the exposure to hyoscyamine did not cause such a remarkable increase in nicotine alkaloid contents, and clone H6H 101 even showed lower levels of all alkaloids with a hyoscyamine concentration of 200 mg l–1 than that observed in the case of the non-fed clone (Table 4). Since the transgenic clones had the capacity of converting hyoscyamine into scopolamine, the activity of the secondary metabolite biosynthesis was directed towards the tropane alkaloid branch putatively at the expense of endogenous nicotine alkaloid production. Earlier, Rocha et al. (2002) have also reported 3–13-fold higher concentrations of nicotine in the leaves of h6h- and tropinone reductase I-transformed N. tabacum clones. It was considered that these changes in the nicotine metabolism arose from perturbation of the normal biosynthesis due to the genetic engineering of the biosynthetic pathway. When the nicotine alkaloid levels of seven individual 35S-h6h transformed root clones were analysed, a slight increment in alkaloid levels of transgenic clones compared with the alkaloid levels of the control clones was observed, mainly accounting for the increase in nicotine and nornicotine (Table 5). However, taking into account the high variation in the total nicotine alkaloid levels in individual clones (0.6–5.1 mg g–1 DW in control clones and 0.3–6.8 mg g–1 DW in h6h-transformed clones), this increase was not considered significant. In addition, no correlation between the alkaloid levels and H6H expression (Fig. 2) was observed. Therefore, these observations support the role of exogenously applied foreign phytoalexin, hyoscyamine, rather than that of the transformation of the 35S-h6h gene alone. The variation in the alkaloid production capacity of H. muticus hairy roots has been extensively studied before (Sevón et al., 1998). Although the long-term hyoscyamine production of root clones was shown to be stable for up to 50 passages of subculturing, the production between individual protoplast-derived hairy root clones varied up to 40-fold, between 0.04% and 1.55% DW (n=171).
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Recently, Palazón et al. (2003)
obtained hairy roots of Duboisia overexpressing the h6h gene from H. niger and reported elevated total alkaloid contents for this strain, which endogenously produces both hyoscyamine and scopolamine. In these roots, the feedback inhibition caused by the accumulated hyoscyamine diminished, thus resulting in elevated overall alkaloid levels. In this study it was shown that hyoscyamine was efficiently taken up by the N. tabacum roots, and it is unclear whether hyoscyamine could result in lowered feedback inhibition in nicotine alkaloid biosynthesis, for example, affecting intracellular signalling mechanisms, and further lead to elevated nicotine alkaloid accumulation in hyoscyamine-fed roots.
No nicotine alkaloids were observed in H. muticus hairy roots, either in control or hyoscyamine-fed roots. This was an expected result, as there are no previous reports indicating nicotine alkaloid biosynthesis in Hyoscyamus sp. Hitherto, only some Solanaceae plants such as Duboisia sp. as well as Anthocercis, Lycopersicon, and Solanum sp. are known to be capable of producing both tropane and nicotine alkaloids (Evans and Ramsey, 1979; Siegmund et al., 1999).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionReferences |
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It has been shown that the hairy root cultures of Nicotiana tabacum expressing the h6h gene from H. niger have a high capacity for converting exogenously applied hyoscyamine into pharmaceutically valuable scopolamine. Furthermore, the scopolamine was efficiently secreted to the medium. Exogenous application of the foreign substrate hyoscyamine resulted in enhanced production of various nicotine alkaloids, suggesting that the regulation of the production of these defence-related compounds is probably more complex than presently known. The mechanism by which hyoscyamine functions at the cellular level requires further studies, for example, whether exogenous hyoscyamine affects the expression levels of individual genes or enzymes, or whether it can trigger the overall up-regulation of the whole metabolic pathway. The use of transformed hairy root cultures offers the great advantages of combining metabolic engineering approaches in systems that are genetically and biochemically stable and which possess the capacity for high secondary metabolite production. Thus, the application of fast-growing plant cell culture systems, such as hairy roots, will offer new insights and possibilities in improving the production efficiencies of known or novel high-value compounds of plant origin.
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Acknowledgements |
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The authors thank Dr Tuulikki Seppänen-Laakso and Dr Into Laakso for help in GC-MS analysis and Kari Kammiovirta for skilful technical assistance. STH is a recipient of a predoctoral fellowship of the Finnish Graduate School on Applied Bioscience. This work was supported by a grant from the Spanish MCYT (BIO2002-03614 and BIO2002-02328) and from the National Technology Agency of Finland (Tekes) programme ‘NeoBio’ to KMOC.
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References |
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