Effect of pmt gene overexpression on tropane alkaloid production in ...

Journal of Experimental Botany, Vol. 54, No. 381, pp. 203±211, January 2003 DOI: 10.1093/jxb/erg014 RESEARCH PAPER

Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus Elisabet Moyano1, Katja Jouhikainen2,3,6, PaÈivi Tammela2, Javier PalazoÂn4, Rosa M. CusidoÂ4, M. Teresa PinÄol4, Teemu H. Teeri3,7 and Kirsi-Marja Oksman-Caldentey5,8 1

Departament de CieÁncies Experimentals i de la Salut, Universitat Pompeu Fabra, Avda. Dr Aiguader 80, E-08003 Barcelona, Spain 2 Division of Pharmacognosy, Department of Pharmacy, PO Box 56, FIN-00014 University of Helsinki, Finland 3

Institute of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland SeccioÂn de FisiologõÂa Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal 643, E-08028 Barcelona, Spain 4

5

VTT Biotechnology, PO Box 1500, FIN-02044 VTT (Espoo), Finland

Received 5 March 2002; Accepted 14 August 2002

Abstract In order to increase the production of the pharmaceuticals hyoscyamine and scopolamine in hairy root cultures, a binary vector system was developed to introduce the T-DNA of the Ri plasmid together with the tobacco pmt gene under the control of CaMV 35S promoter, into the genome of Datura metel and Hyoscyamus muticus. This gene codes for putrescine:SAM N-methyltransferase (PMT; EC. 2.1.1.53), which catalyses the ®rst committed step in the tropane alkaloid pathway. Hairy root cultures overexpressing the pmt gene aged faster and accumulated higher amounts of tropane alkaloids than control hairy roots. Both hyoscyamine and scopolamine production were improved in hairy root cultures of D. metel, whereas in H. muticus only hyoscyamine contents were increased by pmt gene overexpression. These roots have a high capacity to synthesize hyoscyamine, but their ability to convert it into scopolamine is very limited. The results indicate that the same biosynthetic pathway in two related plant species can be differently regulated, and overexpression of a given gene does not necessarily lead to a similar accumulation pattern of secondary metabolites. 6

Key words: Datura metel, hairy root cultures, hyoscyamine, Hyoscyamus muticus, 35S-pmt gene, scopolamine, tropane alkaloids.

Introduction Hairy roots result from the transfer and integration of the genes located on the root-inducing plasmid Ri of Agrobacterium rhizogenes into the plant genome and their expression therein (White and Nester, 1980). These types of roots are characterized by fast growth, frequent branching, plagiotropism, and the ability to synthesize the same compounds as the roots of the intact plant (David et al., 1984). Roots of Solanaceae plants are the main site of tropane alkaloid biosynthesis, and hence hairy root cultures are also capable of accumulating high levels of these metabolites (Oksman-Caldentey and Arroo, 2000). On the contrary, since tropane alkaloid production is very low in unorganized in vitro cultures of Solanaceae (Hashimoto and Yamada, 1983; Yamada and Endo, 1984; OksmanCaldentey and Strauss, 1986), it seems that the tropane alkaloid pathway requires root organization in order to be developed completely. This has earlier been demonstrated by PalazoÂn et al. (1995) when Datura stramonium L. root

Deceased. Present address: Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland. 8 To whom correspondence should be addressed. Fax: +358 9 4552103. E-mail: kirsi-marja.oksman@vtt.® 7

ã Society for Experimental Biology 2003

204 Moyano et al.

cultures treated with auxin lost their organization and were not able to synthesize tropane alkaloids. Similarly, hairy root cultures of several Solanaceae plants displaying callus-like morphology achieved very low tropane alkaloid contents (Moyano et al., 1999; Jouhikainen et al., 1999). Until very recently the lack of understanding of the regulation of secondary metabolite pathways has limited the general use of metabolic engineering in medicinal plants. Hyoscyamine is usually the main alkaloid in transgenic root cultures of many Solanaceae plants including Hyoscyamus, while scopolamine is only produced in small amounts, except Datura metel L. and Duboisia sp. which accumulate this alkaloid at high levels (Muranaka et al., 1993; Celma et al., 2001). However, recently it has been shown that an increase in the expression of the hyoscyamine-6b-hydroxylase (h6h) gene coding for the last enzyme involved in the tropane alkaloid biosynthesis, can considerably enhance the production of scopolamine in hairy root cultures of Hyoscyamus muticus L. (Jouhikainen et al., 1999). Putrescine-N-methyltransferase (PMT) is the enzyme catalysing the ®rst committed step in the tropane alkaloid pathway converting putrescine into N-methylputrescine. Although PMT occurs at higher activity levels in D. stramonium hairy root cultures than either arginine decarboxylase or ornithine decarboxylase, and in this sense does not appear to be rate-limiting, this enzyme can limit the ¯ux of putrescine into tropane alkaloids since its expression is very sensitive to the environment where the roots are growing (PinÄol et al., 1999). Previously Robins et al. (1991) reported that hairy root cultures of Datura supplemented with auxin lost PMT activity before showing any morphological alteration. Moreover, Biondi et al. (2002) have documented a considerable increase of PMT activity as well as an increase in polyamine contents and tropane alkaloids after methyljasmonate treatment in transformed roots of H. muticus. However, it has recently been shown that overexpression of PMT in Atropa belladonna L. does not affect tropane alkaloid levels either in transgenic plants or in hairy roots (Sato et al., 2001). In the present work, the gene from Nicotiana tabacum L. coding for PMT has been inserted into hairy roots of D. metel and H. muticus under the control of the constitutive CaMV 35S promoter, in order to in¯uence tropane alkaloid production. Moreover, the morphology and alkaloid production capacity of these engineered hairy roots have been compared with the respective ones of control hairy roots. As far as it is known, this is the ®rst time that the overexpression of the tobacco pmt gene has been demonstrated to improve tropane alkaloid production in hairy root cultures in a plant speciesdependent manner.

Materials and methods Bacterial strains and cultures The Escherichia coli strain DH5a and Agrobacterium tumefaciens strain C58C1(pRiA4) were used. The latter is a cured derivative of the nopaline strain C58 (Van Larebeke et al., 1974) containing the pRi from A. rhizogenes A4. The E. coli strain was grown at 37 °C in LB medium (Miller, 1972) and the Agrobacterium strain at 28 °C in YEB medium (Maniatis et al., 1982). Construction of the plasmid pBMI The pTVPMT plasmid carrying the cDNA which codes for putrescine-N-methyltransferase from tobacco was a gift from Professor T Hashimoto of the Nara Institute of Science and Technology (Kyoto, Japan). In order to construct the binary vector known as pBMI, the NcoIBamHI fragment of 1400 bp, corresponding to the cDNA for PMT was isolated from pTV-PMT and then subcloned in pET-3d (Novogen), after separating the NcoI-BamHI fragment from this plasmid. The plasmid pET-3d was used because the strong expression vector for plants, pRoc 2275-C does not have a NcoI site. The resulting plasmid pET-3d-PMT was introduced into competent cells of E. coli DH5a strain following the method described by Hanahan (1983). Transformants were screened for ampicillin resistance and subsequently characterized by restriction endonuclease analysis. The 1439 bp XbaI-BamHI fragment from the plasmidic cDNA of the ampicillin-resistant E. coli cells was subcloned into the plasmid pRoc-2275-C, after separating the same fragment from this plasmid. In the subcloning steps carried out for constructing the pBMI plasmid, ligations were cohesive ended. The T-DNA region of the resulting binary vector (¯anked by the 25 bp right and left border sequences), in addition to the cDNA for tobacco PMT subcloned between the 35S promoter and the terminal sequence of the gene for nopaline synthesis, also carried the gene for neomycine phosphotransferase (NPTII) under the control of the nopaline synthesis promoter (Nos-Pro) and the terminal sequence of the enzyme itself (Nos-Ter). This plasmid, known as pBMI was kept in E. coli DH5a and then transferred to the A. tumefaciens C58C1(pRiA4) strain as described by Mozo and Hooykaas (1991). The recombinant clones of A. rhizogenes were selected following Kado and Liu (1981). Establishment and culture of transformed roots of Datura metel Sterile leaf sections of D. metel L. strain metel were inoculated with A. rhizogenes strain A4 or A. tumefaciens strain C58C1(pRiA4, pBMI) carrying the pmt gene (Cusido et al., 1999). Roots which appeared 4±6 weeks after the inoculation, were cultured separately on solid Gamborg B5 medium (Gamborg et al., 1986) supplemented with carbenicillin (500 mg l±1) to eliminate the bacteria and, in the case of transformed roots carrying the pmt gene, with kanamycin 50 mg l±1. Rapidly growing clones with no bacterial contamination were used to establish the cultures of hairy root clones. After several subcultures, transformed roots were transferred to half-strength B5 solid medium (B5/2). Each hairy root clone was also cultured in liquid medium (40 ml of B5/2 hormone-free medium in Erlenmeyer ¯asks), maintained on an orbital shaker at 100 rpm at 25 °C in the dark, and subcultured every 4 weeks. Establishment and culture of transformed roots of Hyoscyamus muticus Young plants of H. muticus L. strain Cairo were used for transformation with Agrobacterium as for D. metel (Vanhala et al., 1995). Each induced hairy root was cultivated separately in liquid culture (5±10 ml), ®rst in the presence of cefotaxime (500 mg l±1) to remove excess bacteria and then in modi®ed Gamborg's B50

pmt gene overexpression and tropane alkaloid biosynthesis 205 medium (20 ml) in 100 ml conical ¯asks (Oksman-Caldentey et al., 1991). The hairy root clones were routinely subcultured every 3 weeks as described above. Extraction and determination of alkaloids The extraction and determination of scopolamine and hyoscyamine in the transformed roots of D. metel were carried out as reported earlier (PinÄol et al., 1996). In H. muticus, quantitative tropane alkaloid determinations were performed on methanol extracts of hairy root clones and on samples of the medium using enzyme immunoassay (Vanhala et al., 1998) and radioimmunoassay (Oksman-Caldentey et al., 1987) for scopolamine and hyoscyamine, respectively. Polymerase chain reaction analysis The presence of the transferred Agrobacterium rol and pmt genes was tested by PCR as described previously SevoÂn et al. (1995) and Moyano et al. (1999). Genomic DNA was extracted from the putative engineered hairy root clones and wild-type hairy root clones according to Edwards et al. (1991). The oligonucleotide primers used for ampli®cation of the pmt gene were 5¢-GCCATTCCCATGAACGGCC-3¢ (position 108±127 nt) and 5¢-CCTCCGCCGATGATCAAAACC-3¢ (position 569±549 nt), according to the sequence data of the pmt gene from Nicotiana tabacum L. (Hibi et al., 1994). The complete PCR mixture contained 200 ng total DNA, 12.5 pmol ml±1 of each oligonucleotide primer, 200 mM dNTPs, 1.5 U Taq Polymerase (Pharmacia Biotech.) and buffer supplied by the enzyme manufacturer (1/10 V) in a total volume of 25 ml. PCR was carried out in the following conditions: 1 cycle of 95 °C, 5 min; 30 cycles of 94 °C, 1 min; 62 °C, 1 min; 72 °C 1.5 min; 1 cycle of 72 °C, 5 min. Products (10 ml) were analysed on 1.5% agarose/TBE gel where the expected size was 450 bp. Putrescine N-methyltransferase (PMT) activity assay PMT activity in transgenic root cultures of H. muticus was measured principally with the method previously employed by Feth et al. (1985) and Hibi et al. (1992). All extraction and puri®cation procedures were performed at +4 °C. First, root material (2 g FW) was washed with tap water and crushed in 2.5 ml of potassiumphosphate buffer A (100 mM, pH 7.5) containing 0.25 M sucrose, 5 mM EDTA, 0.5% (w/v) sodium ascorbate, 3 mM dithiothreitol (DTT), and protease inhibitor mix (CompleteÔ Mini, Boehringer Mannheim). This suspension was homogenized with a pinch of sea sand and mixed with 10% (w/v) insoluble polyvinylpolypyrrolidone (PVPP). After 10 min centrifugation at 13 000 rpm, the supernatant was diluted with potassium-phosphate buffer B (50 mM, pH 7.5, containing 1 mM EDTA and 1 mM DTT), and added to a PD-10 column (Pharmacia) previously equilibrated with the same buffer. Proteins were eluted with 3.5 ml of buffer B and collected into four fractions (2 mM DTT and protease inhibitor mix added). Protein concentration of each fraction was determined according to Bradford (1976). The protein-richest fraction was then selected for PMT activity testing performed as in Feth et al. (1985). Brie¯y, the enzyme reaction was performed in buffer B with putrescine and SAM concentrations of 3.6 mM and 0.6 mM, respectively. After 30 min incubation at 30 °C, the reaction was stopped by 1 min heating at 100 °C. Samples were stored at ±20 °C. N-methylputrescine concentrations of the samples were determined with HPLC analysis after dansylation. The HPLC system consisted of an Agilent Hypersil BDS-C18 column (5 mm, 4.63150 mm), Waters 600 Pump and Waters 2487 Dual l Absorbance Detector (detection wavelength 217 nm). The isocratic mobile phase consisted of an aqueous solution of 2% H3PO4 adjusted to pH 5.2 by triethylamine and acetonitrile (40:60, ¯ow rate 1 ml min±1).

Northern blot analysis and quanti®cation of pmt gene product Northern blot analysis was carried out with seven randomly selected transformed clones of D. metel and with all the transformed root clones of H. muticus. Total RNA was isolated from the hairy roots after 2 weeks of cultivation using the RNeasy kit (Qiagen). Ten micrograms total RNA were loaded per lane of a denaturating formaldehyde gel. Northern blots were made on Hybond-N+ nylon transfer membranes (Amersham) and hybridized with a 32P-labelled probe speci®c for pmt (full cDNA). Hybridization was performed at 42 °C in the presence of 50% formamide. The blots were washed at 42 °C with 23 saline sodium citrate buffer (SSC), 0.1% SDS (30 min) and with 13 SSC, 0.1% SDS (15 min). The radioactivity on the ®lter was imaged using Hyper®lmÔ-MP (Amersham). The band of 1.4 kb that appears in the Northern blot corresponded to the mRNA of pmt gene. The intensity of this band was quanti®ed using Phoretix software as described by PalazoÂn et al. (1998).

Results Growth and root morphology Transformed root cultures of D. metel and H. muticus overexpressing the pmt gene appeared 4±6 weeks after the Agrobacterium inoculation in 80±90% of the leaf explants. Several phenotypic characteristics, e.g. branching, plagiotropism, colour, and callus formation of the hairy root clones, were followed during their growth in order to compare the morphology and the alkaloid production of the clones. Transgenic root cultures of Datura were preselected by culturing them in a medium supplemented with kanamycin. Later, the presence of the pmt gene in the root genome was con®rmed in 100% of root clones tested by PCR. In Hyoscyamus, 15 hairy root clones out of 20 (75%) were found to be positive for the pmt transgene in the PCR studies. The phenotype and morphology of the Datura and Hyoscyamus root clones carrying the pmt gene differed from control clones (carrying Agrobacterium rol genes but not the 35S-pmt transgene). At the start of growth on solid medium, purple pigment formation, which later turned brown, most probably indicating the presence of phenolic compunds such as anthocyanins, was observed in the roots of Hyoscyamus (Fig. 1). There was also slight purple pigmentation in the control roots induced by strain C58C1(pRiA4); thus this peculiar colour formation in the hairy roots might be due to the pRiA4 plasmid in this plant species. This phenomenon has never been found in Hyoscyamus hairy root clones transformed with other plasmids. In the case of Datura root cultures, a very high percentage (80%) of the pmt transformed root cultures established, aged faster and turned brown when cultured on solid medium. These root clones were discarded and only root cultures showing a normal growth capacity were considered for further experiments. In Hyoscyamus the majority of the clones were brownish, but otherwise their appearance and growth was normal. Regarding the biomass production of Datura root cultures overexpressing the tobacco pmt gene, these results

206 Moyano et al.

Fig. 1. A typical purple pigment formation in a Hyoscyamus muticus hairy root clone induced by C58C1 (pRiA4, pBMI) 7 d after subculturing.

did not show any clear effect of the transgene on the root growth ability after 4 week's cultivation (Fig. 2), since the fresh and dry weight values achieved by the transgenic roots were very similar to the average of those obtained by the three control root lines studied. However, considerable variation in growth capacity between the individual clones was observed, for example, clone PMT22 achieved twofold higher fresh weight at the end of the growth period than clone PMT15. As previously reported (SevoÂn et al., 1998; Moyano et al., 1999), this fact is very frequent in transformed root cultures, since each clone arises from an independent transformation event. Depending on the presence of certain T-DNA genes, mainly the aux1 gene, in the genome of the root clone obtained, growth can be very different. Growth capacity, measured both as fresh and dry weight, of Hyoscyamus roots was very much higher when compared to Datura clones (Fig. 2). This fact revealed that transformed roots of Hyoscyamus constitute a very effective system to produce biomass of hairy roots in vitro. On the other hand, all Hyoscyamus root clones carrying the 35S-pmt gene reached signi®cantly (P >0.01) lower fresh and dry weight than control roots (three control clones) after 4 weeks of culture, showing a negative effect of the transgene on the growth capacity.

Tropane alkaloid production The pmt transformed root clones of Datura displayed an alkaloid spectrum similar to that of the transformed control roots. As also mentioned in a previous report (Cusido et al., 1999), scopolamine was the main alkaloid obtained in all the clones (Fig. 3). Clone PMT10 was the most productive among all the Datura root clones established. Great differences in alkaloid contents among the clones were observed, but the majority of PMT clones reached higher scopolamine and hyoscyamine levels than the control ones after 4 weeks of growth, with only few clones (e.g. PMT22 and PMT31) presenting lower alkaloid contents than control ones at the end of the culture period (Fig. 3). Transformed roots of Hyoscyamus accumulated much higher hyoscyamine amounts than the ones of Datura (approximately 10-fold), whereas scopolamine contents were very low (lower than 0.2 mg l±1). This hyoscyaminerich genotype is typical for hairy root cultures of H. muticus (Vanhala et al., 1995; SevoÂn et al., 1998) indicating very low activity of the h6h gene in this plant species (Jouhikainen et al., 1999). The contents of hyoscyamine in the pmt transformed hairy roots were on average 2±3fold higher (Fig. 3), but the scopolamine contents were similar to the controls.

pmt gene overexpression and tropane alkaloid biosynthesis 207

Fig. 2. Fresh and dry weight (g l±1) of transformed root culture clones at the end of the culture period (28 d). Control values represent the mean of three individual control lines. Measurements are based on 3±6 replicates. Standard deviations were 8±10% of the values represented.

Fig. 3. Tropane alkaloid production (mg l±1) of transformed root culture clones at the end of the culture period (28 d). Control values represent the mean of three individual control lines. Measurements are based on 3±6 replicates. Standard deviations were 3±10% of the values represented.

These results, in accordance with this study's predictions, showed that the overexpression of the tobacco pmt gene under the control of the 35S promoter enhanced

alkaloid production of D. metel-transformed roots, and this improvement affected both hyoscyamine and scopolamine contents. Furthermore, the results indicated the capacity of

208 Moyano et al.

Datura roots partially to convert the additional amount of hyoscyamine into scopolamine and revealed that the H6H activity in these roots is not low compared to the ones of Hyoscyamus.

Fig. 4. Northern blot results corresponding to Datura clones carrying 35S-pmt gene. Each line was charged with the same quantity of total RNA.

The pmt gene expression

In order to establish a possible relationship between transgene expression and the capacity of root cultures to synthesize tropane alkaloids, the levels of the tobacco pmt gene product in the transformed roots were studied. For this reason Datura and Hyoscyamus root clones with differing capacities to biosynthesize tropane alkaloids were selected. RNA gel blot results showed (Fig. 4) a clear band of 1.4 kb corresponding to cDNA of pmt in the selected transgenic root clones, whereas in the control ones no band of 1.4 kb was visible. The same quantity of total RNA was charged in each lane of the gel and the intensity of the pmt band was analysed photodensitometrically by Phoretix software. These experiments were carried out in triplicate although only one is shown. Figure 5 represents the results of densitometrical analysis, showing in both types of roots, a relationship between pmt gene expression and the capacity of the culture to biosynthesize alkaloids. In all cases, roots with a high intensity of the pmt band

Fig. 5. Comparative study of alkaloid production (mg g±1 DW) and densitometrical analysis of Northern blot results corresponding to selected root clones of Datura and Hyoscyamus. Experiments were carried out three times.

pmt gene overexpression and tropane alkaloid biosynthesis 209

Fig. 6. Comparison of the PMT activity and hyoscyamine content in transgenic (®lled diamonds) and control (®lled triangles) hairy root clones of Hyoscyamus muticus.

reached a high alkaloid content (Fig. 5). For example, the two best producing clones of Hyoscyamus KC2 and KC17 also showed the strongest pmt expression in the Northern blot experiments. In addition, the PMT enzymatic activity and the alkaloid content in transgenic Hyoscyamus hairy roots were measured simultaneously (Fig. 6). The PMT activity was the highest after 7 d of cultivation when the alkaloid contents were still low, and gradually decreased with the time. However, the ratio of the PMT activity of individual clones remained the same through the whole cultivation period. The highest PMT activity was observed in clone KC17, which had the strongest pmt expression according to northern blot as well. This clone also presented the second highest hyoscyamine content among all the clones studied. However, in some transgenic clones this kind of positive correlation seems to be absent suggesting that PMT activity is not the sole factor contributing to elevated alkaloid contents. Discussion A binary vector was constructed to introduce the tobacco pmt gene and the T-DNA of pRiA4 of Agrobacterium rhizogenes, into D. metel and H. muticus explants. Results of agroinfection revealed an excellent capacity of the construction to transfer the T-DNA of pRiA4 into the plant genome and to develop the hairy root syndrome. The majority of the established transformed root cultures, also overexpressed the pmt gene under the control of the 35S promoter and showed several altered metabolic traits, such as rapid ageing, reduction of growth in Hyoscyamus cultures and modi®cations in secondary metabolite pathways. According to Kholodenko et al. (1998) an undesirable effect of metabolic engineering is the promotion of metabolic ¯ux alterations that can induce cell death. This fact produces metabolic changes not directly related to transgene presence. For this reason, brownish root clones of Datura were rejected and the remaining ones were

maintained for further studies. As previously mentioned, in Hyoscyamus this kind of selection was not performed and transgenic roots carrying the pmt gene grew less rapidly than the control roots. Rapid ageing of transformed roots has previously been connected with high alkaloid production (Jouhikainen et al., 1999), but the altered phenotype obtained in this study might be due to the formation of other compounds. On the other hand, due to the role of polyamines in plant development (Martin-Tanguy, 1997) premature senescence of root cultures in both plant species and slower growth capacity in Hyoscyamus can be caused by pmt overexpression in polyamine metabolism, because the substrate of PMT enzyme (putrescine) is shared with polyamine metabolism (Sato et al., 2001). On the other hand, alkaloid content was closely related to the presence of the 35S-pmt transgene, showing that ectopic expression of tobacco pmt increased the biosynthetic ¯ux towards the tropane alkaloids. Consequently, tropane alkaloid contents of transformed roots in both D. metel and H. muticus were enhanced up to 5-fold. In H. muticus, the enhancement concerned only hyoscyamine, which may be due in this species to a limiting amount of H6H activity that would convert hyoscyamine to scopolamine (Jouhikainen et al., 1999). In H. muticus transgenic hairy roots, both PMT enzymatic activity and alkaloid content were measured. Although all lines transgenic for 35S-pmt contained elevated levels of hyoscyamine, there was not always correlation between the amount of extractable PMT and the amount of alkaloids. The biosynthesis of tropane alkaloids in Hyoscyamus takes place in speci®c cells of the pericycle (Kanegae et al., 1994). In A. belladonna, it has been shown that regulation of the plant's endogenous pmt is also pericycle speci®c (Suzuki et al., 1999). It is evident that the transcriptional control by the 35S promoter of the transgenic pmt gene in the hairy root lines analysed here is not cell type speci®c. The fact that the altered expression pattern of pmt rather than the amount of PMT enzyme increases tropane alkaloid production in H. muticus hairy roots, shows that tropane alkaloid biosynthesis is very complex and might be slightly different in various plant species. Furthermore, it may also indicate that the transgene allows bypassing of the endogenous control of metabolic ¯ux to the alkaloids that would take place at the level of the ®rst committed enzymatic step in their biosynthesis. Acknowledgements This work has been partially supported by a grant from the Boehringer Ingelheim Industry and the Spanish CICYT (PB-1243). The work of KJ was supported by a grant from the Pharmacy Graduate School Program of the University of Helsinki. The authors are very grateful to Professor T Hashimoto of the Nara Institute of Science and Technology (Kyoto, Japan) for supplying the pTVPMT

210 Moyano et al. plasmid carrying the cDNA which codes for putrescine-N-methyltransferase from tobacco.

References Blondi S, Scaramagli S, Oksman-Caldentey K-M, Poli F. 2002. Secondary metabolism in root and callus cultures of Hyoscyamus multicus L.: the relationship between morphological organization and response to methyl jasmonate. Plant Science 163, 563±569. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein±dye binding. Analytical Biochemistry 72, 248±254. Celma RC, PalazoÂn J, Cusido RM, PinÄol MT, Keil M. 2001. Decreased scopolamine yield in ®eld-grown Duboisia plants regenerated from hairy roots. Planta Medica 67, 249±253. Cusido RM, PalazoÂn J, PinÄol MT, Bon®ll M, Morales C. 1999. Datura metel: in vitro production of tropane alkaloids. Planta Medica 65, 144±148. David C, Chilton MD, Tempe J. 1984. Conservation of T-DNA in plants regenerated from hairy root cultures. Bio/Technology 2, 73±76. Edwards E, Johnstone C, Thompson A. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19, 1349. Feth F, Arfmann H-A, Wray V, Wagner KG. 1985. Determination of putrescine N-methyltransferase by high performance liquid chromatography. Phytochemistry 24, 921± 923. Gamborg OL, Miller RA, Ojima K. 1986. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50, 151±158. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166, 557±562. Hashimoto T, Yamada Y. 1983. Scopolamine production in suspension cultures and rediffentiated roots of Hyoscyamus niger. Planta Medica 47, 121±141. Hibi N, Fujita T, Hatano M, Hashimoto T, Yamada Y. 1992. Putrescine N-methyltransferase in cultured roots of Hyoscyamus albus. Plant Physiology 100, 826±835. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y. 1994. Gene expression in tobacco low-nicotine mutants. The Plant Cell 6, 723±735. Jouhikainen K, Lindgren L, Jokelainen T, Hiltunen R, Teeri TH, Oksman-Caldentey K-M. 1999. Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 208, 545±551. Kado CI, Liu ST. 1981. Rapid procedure for detection and isolation of large and small plasmids. Journal of Bacteriology 145, 1365±1373. Kanegae T, Kajiya H, Amano Y, Hashimoto T, Yamada Y. 1994. Species-dependent expression of the hyoscyamine 6bhydroxylase gene in the pericycle. Plant Physiology 105, 483± 490. Kholodenko BN, Cascante M, Hoek JB, Westerhoff HV, Schwaber J. 1998. Desired metabolite concentrations and ¯uxes. Biotechnology and Bioengineering 59, 239±247. Maniatis T, Friotsh EF, Sambrook J. 1982. Molecular cloning, a laboratory manual. New York: Cold Spring Harbor Laboratory. Martin-Tanguy J. 1997. Conjugated polyamines and reproductive development: biochemical, molecular and physiological approaches. Physiologia Plantarum 100, 675±688.

Miller JH. 1972. Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory. Moyano E, Fornale S, PalazoÂn J, Cusido RM, Bon®ll M, Morales C, PinÄol MT. 1999. Effect of Agrobacterium rhizogenes T-DNA on alkaloid production in Solanaceae plants. Phytochemistry 52, 1287±1292. Mozo T, Hooykaas PJJ. 1991. Electroporation of megaplasmids into Agrobacterium. Plant Molecular Biology 16, 917±918. Muranaka T, Ohkawa H, Yamada Y. 1993. Continuous production of scopolamine by a culture of Duboisia leichhardtii hairy root clone in a bioreactor system. Applied Microbiology and Biotechnology 40, 219±223. Oksman-Caldentey K-M, Arroo R. 2000. Regulation of tropane alkaloid metabolism in plants and plant cell cultures. In: Verpoorte R, Alfermann AW, eds. Metabolic engineering of plant secondary metabolism. Dordrecht: Kluwer Academic Publishers, 253±281. Oksman-Caldentey K-M, KivelaÈ O, Hiltunen R. 1991. Spontaneous shoot organogenesis and plant regeneration from hairy root cultures of Hyoscyamus muticus. Plant Science 78, 129±136. Oksman-Caldentey K-M, Strauss A. 1986. Somaclonal variation of scopolamine content in protoplast±derived cell culture clones of Hyoscyamus muticus. Planta Medica 52, 6±12. Oksman-Caldentey K-M, Vuorela H, Strauss A, Hiltunen R. 1987. Variation in the tropane alkaloid content of Hyoscyamus muticus plants and cell culture clones. Planta Medica 53, 349± 354. PalazoÂn J, Altabella T, Cusido RM, Ribo M, PinÄol MT. 1995. Growth and tropane alkaloid production in Agrobacterium transformed roots and derived callus of Datura. Biologia Plantarum 37, 161±168. PalazoÂn J, Cusido RM, Gonzalo J, Bon®ll M, Morales C, PinÄol MT. 1998. Relation between the amount of rolC gene product and indole alkaloid accumulation in Catharanthus roseus transformed root cultures. Journal of Plant Physiology 153, 712±718. PinÄol MT, PalazoÂn J, Cusido RM, Ribo M. 1999. In¯uence of calcium ion-concentration in the medium on tropane alkaloid accumulation in Datura stramonium hairy roots. Plant Science 141, 41±49. PinÄol MT, PalazoÂn J, Cusido RM, Serrano M. 1996. Effects of Ri T-DNA from Agrobacterium rhizogenes on growth and hyoscyamine production in Datura stramonium root cultures. Botanica Acta 109, 133±138. Robins RJ, Bent FG, Rhodes MJC. 1991. Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures. 3. The relationship between morphological integrity and alkaloid biosynthesis. Planta 185, 390±395. Sato F, Hashimoto T, Hachiya A, Tamura K, Choi K-B, Morishige T, Fujimoto H, Yamada Y. 2001. Metabolic engineering of plant alkaloid biosynthesis. Proceedings of the National Academy of Sciences, USA 98, 367±372. SevoÂn N, Oksman-Caldentey K-M, Hiltunen R. 1995. Ef®cient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus. Plant Cell Reports 14, 738±742. SevoÂn N, Hiltunen R, Oksman-Caldentey K-M. 1998. Somaclonal variation in transformed root cultures and protoplast-derived hairy root cultures of Hyoscyamus muticus. Planta Medica 64, 37±41. Suzuki K, Yamada Y, Hashimoto T. 1999. Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant Cell Physiology 40, 289±297. Van Larebeke N, Engler G, Holsters M, Van den Esacker S, Schilperoort RA, Schell J. 1974. Large plasmid in

pmt gene overexpression and tropane alkaloid biosynthesis 211 Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252, 169±170. Vanhala L, Eeva M, Lapinjoki S, Hiltunen R, OksmanCaldentey K-M. 1998. Effect of growth regulators on transformed root cultures of Hyoscyamus muticus. Journal of Plant Physiology 153, 475±481. Vanhala L, Hiltunen R, Oksman-Caldentey K-M. 1995. Virulence of different Agrobacterium strains on hairy root

formation of Hyoscyamus muticus. Plant Cell Reports 14, 236± 240. White F, Nester EW. 1980. Hairy root: plasmid encodes virulence in Agrobacterium rhizogenes. Journal of Bacteriology 141, 1134± 1141. Yamada Y, Endo T. 1984. Tropane alkaloid production in cultured cells of Duboisia leichhardtii. Plant Cell Reports 3, 186±188.