Journal of Experimental Botany, Vol. 54, No. 390, pp. 20652070, September 2003 DOI: 10.

1093/jxb/erg227

RESEARCH PAPER

Alkaloids in plants and root cultures of Atropa belladonna overexpressing putrescine N-methyltransferase
Grit Rothe1, Akira Hachiya2, Yasuyuki Yamada2, Takashi Hashimoto2 and Birgit Drager1,* Institute of Pharmaceutical Biology, Martin-Luther-University Halle-Wittenberg, Hoher Weg 8, D-06120 Halle/Saale, Germany 2 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan
Received 14 February 2003; Accepted 3 June 2003
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1

Abstract
Putrescine N-methyltransferase (PMT) is the rst alkaloid-specic enzyme for nicotine and tropane alkaloid formation. The pmt gene from Nicotiana tabacum was fused to the CaMV 35S promoter and integrated into the Atropa belladonna genome. Transgenic plants and derived root cultures were analysed for gene expression and for levels of alkaloids and their precursors. Scopolamine, hyoscyamine, tropine, pseudotropine, tropinone, and calystegines were found unaltered or somewhat decreased in pmt-overexpressing lines compared to controls. When root cultures were treated with 5% sucrose, calystegine levels were elevated in control roots, but were not affected in pmt-overexpressing roots. 1 mM auxin reduced calystegine levels in control roots, while in pmt-overexpressing roots all alkaloids remained unaltered. Expression level of pmt alone is apparently not limiting for tropane alkaloid formation in A. belladonna.
Key words: Atropa belladonna, calystegine, hyoscyamine, overexpression, putrescine N-methyltransferase, scopolamine, tropane alkaloids.

Introduction Putrescine N-methyltransferase (PMT) is the rst enzyme committed for the biosynthesis of tropane alkaloids and nicotine. The pmt cDNAs were cloned from tobacco (Hibi et al., 1994), Atropa belladonna L., and Hyoscyamus niger L. (Suzuki et al., 1999), and were shown to encode a

protein highly homologous to spermidine synthases of various origins, revealing the evolutionary origin of PMT from spermidine synthase (Hashimoto et al., 1998). In tobacco, pmt expression is well correlated with the level of nicotine formation. For example, induction of pmt transcription by methyl jasmonate and its inhibition by auxin resulted in increased or decreased levels of nicotine, respectively (Imanishi et al., 1998). Nicotine is an insecticide, and its ecological role was postulated to protect tobacco plants against herbivores (Baldwin et al., 1997). Elevated nicotine, however, can bind a considerable portion of total nitrogen, therefore specic induction of nicotine formation by a wound signal appears as an advantage (Baldwin 1998). Nicotine and tropane alkaloids are formed in the roots and transported to the aerial parts of the plant. Accordingly, expression of pmt transcripts is conned to the roots of tobacco and localized in young roots, specically in cortex, endodermis and xylem (Shoji et al., 2000). In A. belladonna, pmt is expressed in the pericycle and a few xylem cells of the root (Suzuki et al., 1999). In transgenic plants and root cultures of A. belladonna, pmt was overexpressed and metabolites in the tropane alkaloid pathway were analysed (Fig. 1). An initial account of this work has been reported briey in an overview on metabolic engineering of plant alkaloid synthesis (Sato et al., 2001). In the previous report, accumulation of polyamines, putrescine, N-methylputrescine, hyoscyamine, and scopolamine, was analysed in T0 or T1 transgenic plants. Other tropane alkaloid producing Solanaceae were transformed with pmt, i.e. Datura metel, Hyoscyamus muticus (Moyano et al., 2003) and a Duboisia hybrid (Moyano et al., 2002). In these studies, root cultures were established, intact plants were not

* To whom correspondence should be addressed. Fax: +49 345 55 27 021. E-mail: draeger@pharmazie.uni-halle.de Journal of Experimental Botany, Vol. 54, No. 390, Society for Experimental Biology 2003; all rights reserved

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transgenic plants of the T2 generation. Cultivation was performed as described (Rothe and Drager, 2002). RNA gel blot analysis Expression of pmt in the leaves was analyses as follows. Total RNA (20 mg) isolated from the leaves was separated by electrophoresis on 1.2% formaldehyde agarose gel and blotted onto a Hybond-N membrane (Amersham). The blots were hybridized with tobacco pmt cDNA (1128 bp) or 18S rRNA which were 32P-labelled by using a High Prime DNA Labeling Kit (Roche) in 50% formamide, 5Q SSC, 5Q Denhardt's solution, 1% SDS, 10% dextran sulphate, and 50 mg ml1 herring sperm DNA at 42 C for 16 h. Washing was performed as follows: (1) 5 min at room temperature in 2Q SSC and 0.1% SDS, (2) 30 min at 58 C in 1Q SSC and 0.1% SDS, (3) 30 min at 58 C in 0.2% SSC and 0.1% SDS, and then (4) 15 min at 60 C in 0.1% SSC and 0.1% SDS. In roots, pmt expression was analysed using an RNA probe as described (Sato et al., 2001). PMT activity assay 2 g cultured roots (4-week-old) were blotted dry and homogenized in 2.5 ml buffer (KH2PO4/K2HPO4 100 mM pH 7; 0.25 mM sucrose, 5 mM EDTA, 0.5% (m/v) sodium ascorbate, 3 mM DTT, pepstatin 1 mg ml1) containing sea sand and insoluble PVP (10% m/v). After 10 min centrifugation at 13 000 rpm, the supernatant was applied to a PD-10 column (Pharmacia) previously equilibrated with TRIS-HCl 100 mM pH 9. For elution 3.5 ml of TRIS-HCl were used. Protein concentration in this preparation was determined by the Bradford dye method (Bradford, 1976). The assay mixture contained protein preparation 1 mg, putrescine 3.6 mM and S-adenosyl-L-methionine 0.6 mM in TRIS-HCl pH 9.0. After 30 min at 37 C, 100 ml of the assay mixture were mixed with 100 ml saturated sodium carbonate solution and 400 ml dansylchloride solution (400 ml, 7.5 mg ml1 in acetone). After 1 h incubation at 60 C in the dark, 100 ml of proline solution (100 mg ml1) were added and kept at 37 C for 30 min in the dark. After extraction with 700 ml toluene and evaporation at 37 C, the sample was dissolved in 1 ml acetonitrile. 20 ml of this sample was directly analysed by HPLC on a Hypersil ODS reverse phase C18 column (5 mm particle size, 4 mm diameter, 250 mm length) with a programmed acetonitrile/water gradient from 65% to 90% acetonitrile over 22 min at a ow rate of 1 ml min1 at 40 C. Detection was done by uorescence detector at 365 nm excitation and 510 nm emission. Retention times were 6.1 min, 7.8 min, 10.4 min, and 15.4 min for the putrescine, N-methylputrescine, spermidine, and spermine derivatives. Metabolite analysis Tropane alkaloids and calystegines were extracted and analysed as described (Rothe and Drager, 2002). Polyamines were extracted from plant tissue by 5% perchloric acid (PCA) and separated into free and conjugated polyamines. PCA-insoluble polyamines were hydrolysed in the pellet by 5 M NaOH according to published methods (Tiburcio et al., 1986). The dansylated products were analysed as indicated above for the reaction products of the PMT activity. Statistical treatment of data Data were calculated as averages from 36 independent samples. Standard deviations are given as bars in the graphs.

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Fig. 1. Biosynthetic steps for tropane alkaloids. PMT, putrescine Nmethyltransferase; TR I, tropine forming tropinone reductase; TR II, pseudotropine forming tropinone reductase; H6H, hyoscyamine-6hydroxylase.

investigated. In all preceding studies, alkaloid production was measured as scopolamine and hyoscyamine only. Calystegines, pseudotropine-derived alkaloids that are also dependent on PMT activity (Fig. 1), were not considered, although they are known to be formed in Hyoscyamus muticus (Asano et al., 2001; Sevon et al., 1997), Duboisia and Datura species (Asano et al., 2001; Draeger et al., 1995; Nash et al., 1993). Since A. belladonna contains calystegines in high concentrations (Rothe and Drager, 2002), it is important to follow metabolic changes in calystegines as well as central intermediates in the pathway, such as tropinone, tropine, and pseudotropine. In this study, these and other alkaloids were analysed mainly in the subsequent T2 generation of transgenic plants and root cultures derived from them. Effects of high sucrose and auxin addition on alkaloid formation were also studied in these transgenic samples. Materials and methods
Chemicals Solvents and other chemicals were purchased from Merck Darmstadt or Sigma-Aldrich Taufkirchen. Transgenic plants and root cultures Tobacco pmt cDNA was placed under the control of CaMV 35S promoter with a duplicated enhancer, and cloned into a binary vector for plant transformation as described (Sato et al., 2001). Transgenic A. belladonna plants were regenerated from leaf discs, transferred to soil, and selfed as described (Sato et al., 2001). T2 progeny plants derived from a single T1 plant were mainly used in this study. Root cultures were established by excising the roots of control and

Results
Stable overexpression of the pmt transgene

Atropa belladonna plants regenerated directly from leaf discs were designated as the T0 generation, and their selfed

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progeny as the T1 and T2 generations. Many transgenic lines showed kanamycin segregation ratios of higher than three resistant to one sensitive plants at the T1 population, indicating more than one transgene insertion locus in the genome. Transgenic plants were phenotypically normal, and the T2 progeny originating from a single T1 plant with a high pmt expression level was analysed to see whether the pmt transgene was still overexpressed. High level of pmt expression was observed in leaves of all transgenic plants transformed with the 35S-pmt vector, whereas leaves of non-transformed wild-type plants and vectortransformed control plants gave no signal (representative data are shown in Fig. 2). The expression level of the 35S-pmt transgene varied among T2 siblings, as shown for two T2 sibling lines, Nos 11/1 and 11/2, in Fig. 2. Cultured roots were established from several transgenic plants of T1 and T2 generation. These cultured roots as well as young branch roots from whole plants were found to express several-fold higher levels of the pmt transcript compared to wild-type and control roots, which showed rather weak expression of endogenous pmt (representative data are shown in Fig. 3). The results are consistent with the nding that pmt is normally transcribed only in root tissue of Atropa belladonna plants (Suzuki et al., 1999). The detection of elevated pmt transcripts in the leaves and roots of plants from the T0 to the T2 generations proves the stable expression of the transgene.
Accumulation of alkaloids

Tropane alkaloids were measured in young leaves, fully expanded leaves, stems, and roots of T2 plants (Fig. 4). In pmt-overexpressing plants, the concentrations of hyoscyamine (which is the major alkaloid in A. belladonna leaves) and of scopolamine generally decreased to slight or moderate degrees compared to control plants. The vector-transformed plants appeared to have decreased alkaloid

levels, but their growth and development were slower and owering was reduced and delayed compared with both control and pmt-transformed plants, which showed equally good growth. Calystegine A3 was the major calystegine component, followed by much smaller amounts of A5 and B2. These calystegines accumulated at similar or somewhat lower levels in the 35S-pmt plants, compared to the vector control. The pathway intermediates tropinone, tropine, and pseudotropine were slightly lower in roots of the control plant (0.3 mmol g1 dry mass for both tropine and pseudotropine, and 0.2 mmol g1 for tropinone) than in the pmt-overexpressing roots (0.35 mmol g1 dry mass for tropine, 0.5 mmol g1 dry mass for pseudotropine, and 0.3 mmol g1 for tropinone). Overall, the alkaloid composition remained similar in the 35S-pmt plants and the control plants. Next, cultured roots were analysed. The accumulation of hyoscyamine and scopolamine was not different in pmtoverexpressing roots and wild-type and vector-transformed roots (Fig. 5). In A. belladonna root cultures, calystegine A3 accumulates at a much higher level than hyoscyamine on a molar basis. There was not a large difference in the accumulation of calystegines between 35S-pmt and control cultures. The intermediates of alkaloid biosynthesis, tropine, pseudotropine, and tropinone showed no signicant differences between control and pmt-overexpressing root cultures. The level of N-methylputrescine was higher in transgenic roots. While pmt-transformed lines contained 0.60.95 mmol g1 dry mass total N-methylputrescine (free, conjugated and bound), the levels in control and vector control roots were lower, 0.3 mmol g1 dry mass. Levels of putrescine, spermine and spermidine were comparable in all root cultures, 69 mmol g1 dry mass total putrescine, and 23 mmol g1 dry mass each of total spermidine and total

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Fig. 2. Accumulation of pmt transcript in leaves of Atropa belladonna T2 plants and of tobacco. 20 mg total RNA isolated from leaves of wild type (WT) plant, vector control (VC) plant, pmt-overexpressing lines 11/1 and 11/2, and Nicotiana tabacum (Nt) as negative control. The probe was a 1128 bp fragment of tobacco pmt. Loading control with a probe of 18S rRNA from Lycopersicon esculentum.

Fig. 3. Accumulation of pmt transcript in Atropa belladonna roots. 10 mg total RNA isolated from young plant roots (PR), cultured roots (CR) of wild type (WT), pmt-overexpressing lines 32 and 11. The probe was full-length tobacco pmt. The size of pmt mRNA is 1.4 kb. Equal RNA loading in each lane is shown by ethidium bromide staining of total RNA.

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Fig. 4. Alkaloids in T2 plants of A. belladonna. WT, wild type; VC, vector control; 11/1 and 11/2, pmt-overexpressing plants.

Fig. 5. Alkaloids in 28-d-old root cultures of A. belladonna. WT, wild type; VC, vector control; 11/1 and 11/2, pmt-overexpressing roots; IBA indole butyric acid.

spermine. Enzyme activity of PMT was enhanced in pmtoverexpressing root cultures. In vector control roots 911 pkat mg1 protein were measured, while from

pmt-overexpressing roots 56-fold of the activity could be measured in the assay. The results indicate that overexpression of pmt leads to enhanced transcription, more

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active PMT enzyme and enhanced N-methylputrescine levels, but the subsequent alkaloid metabolites are not affected. Carbohydrate and hormone treatments have been shown to alter alkaloid formation in root cultures of A. belladonna (Rothe et al., 2001). Elevated carbohydrate supply in combination with reduced mineral salts had both a nutritional and a specic alkaloid-enhancing effect in the root cultures (Rothe and Drager, 2002). The effect of 5% sucrose and reduced mineral salts can be seen with the wild-type roots and vector-transformed roots, but pmtoverexpressing roots did not signicantly respond to the elevated sucrose level (Fig. 5). When the synthetic auxin, indole butyric acid, was supplied at 1 mM to root cultures of the wild type and the vector-transformed line, growth was enhanced, but the formation of tropane alkaloids, particularly of calystegines, was inhibited, as reported previously. The auxin treatment, however, did not signicantly inhibit alkaloid formation in 35S-pmt roots (Fig. 5). Such unresponsiveness of 35S-pmt lines to sucrose and auxin treatments may be attributable to the fact that the pmt transgene is driven by the constitutive CaMV 35S promoter, and that the nutritional and hormonal signals probably control the endogenous pmt gene expression via its own promoter elements. Discussion It is concluded that increased pmt expression alone is not sufcient for increased alkaloid production in A. belladonna plants and in root cultures. Comparable results were shown for a root culture from a Duboisia hybrid transformed with a similar pmt-overexpressing vector (Moyano et al., 2002). In Hyoscyamus muticus root culture, the N-methylputrescine level was enhanced by a jasmonate treatment, but without signicant effect on the accumulation of hyoscyamine (Biondi et al., 2000). When H. muticus and Datura metel roots cultures that overexpressed pmt were analysed, the individual clones showed both elevated and decreased hyoscyamine and scopolamine content (Moyano et al., 2003). The authors state that two related plant species may display different regulation in alkaloid metabolism. Intact plants were not regenerated in those studies. It is demonstrated that constitutive overexpression of pmt maintains the continued formation of tropane alkaloids even when root cultures were treated with auxin at the levels inhibitory to the alkaloid formation in wild-type roots. Inhibition of alkaloid formation by auxin has been reported several times for tropane alkaloid-forming roots (Hashimoto et al., 1986), and PMT activity was found to be elevated after auxin reduction in tobacco callus (Feth et al., 1986). Expression of tobacco pmt genes has been shown to be inhibited by auxin treatment to the roots (Hibi et al., 1994). Possibly, pmt is not the only gene affected by

these treatments, but other enzyme genes in the pathway are also down-regulated. Under these adverse conditions when the rate of alkaloid formation is slow, increased PMT levels enhance alkaloid formation, in particular, calystegine formation. In the pmt-overexpressing A. belladonna plants, N-methylputrescine was overproduced by the immediate action of the introduced gene product (Sato et al., 2001). It is shown in the present study that tropinone and its downstream pathway metabolites, tropine and pseudotropine, did not accumulate in the transgenic tissues in considerably higher quantities than in control tissues. Thus, one or more steps in the pathway between N-methylputrescine and tropinone are further rate-limiting in the transgenic A. belladonna. The conversion involves a diamine oxidase which preferentially acts on N-methylputrescine to produce the 1-methyl-D1-pyrrolinium cation (Hashimoto et al., 1990), but the enzymes responsible for further conversion of this cation to tropinone are not known. Apparently there are multiple steps that limit the rate of product synthesis in the tropane alkaloid pathway. The nicotine biosynthesis pathway in tobacco consists of many fewer steps than the tropane alkaloid pathway. This may have resulted in a moderate increase in nicotine accumulation by a similar overexpression of pmt in transgenic tobacco plants (Sato et al., 2001). For overproduction of tropane alkaloids, multiple enzymes in the pathway should be simultaneously overexpressed in transgenic plants. Acknowledgements
GR thanks the Deutsche Forschungsgemeinschaft and the Japan Society for the Promotion of Science for a grant to work at NAIST in 1999, co-operative project `Molecular mechanisms of secondary product biosynthesis in plants' 446 JAP 113/190/0-1. The work in Halle was nancially supported by a grant to BD from the Deutsche Forschungsgemeinschaft (Dr 227/2-2). The work in NAIST was supported in part by a grant (RFTF program 00L01605) to TH from the Ministry of Education, Science, Sports and Culture of Japan. In the Halle laboratory, Ursula Kodel helped with numerous alkaloid analysis, Dr Bettina Rahfeld gave valuable hints for gel blot analysis, and Olaf Stenzel established the polyamine measurement procedure.

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References
Asano N, Yokoyama K, Sakurai M, et al. 2001. Dihydroxynortropane alkaloids from calystegine-producing plants. Phytochemistry 57, 721726. Baldwin IT. 1998. Jasmonate-induced responses are costly but benet plants under attack in native populations. Proceedings of the National Academy of Sciences, USA 95, 81138118. Baldwin IT, Zhang ZP, Diab N, Ohnmeiss TE, McCloud ES, Lynds GY, Schmelz EA. 1997. Quantication, correlations and manipulation of wound-induced changes in jasmonic acid and nicotine in Nicotiana sylvestris. Planta 201, 397404. Biondi S, Fornale S, Oksman-Caldentey KM, Eeva M, Agostani S, Bagni N. 2000. Jasmonates induce over-accumulation of

2070 Rothe et al.

methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures. Plant Cell Reports 19, 691697. 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, 248254. Draeger B, Van-Almssick A, Mrachatz G. 1995. Distribution of calystegines in several Solanaceae. Planta Medica 61, 577579. Feth F, Wagner R, Wagner KG. 1986. Regulation in tobacco callus of enzyme activities of the nicotine pathway. I. The route ornithine to methylpyrroline. Planta 168, 402407. Hashimoto T, Mitani A, Yamada Y. 1990. Diamine oxidase from cultured roots of Hyoscyamus niger: its function in tropane alkaloid biosynthesis. Plant Physiology 93, 216221. Hashimoto T, Tamaki K, Suzuki K, Yamada Y. 1998. Molecular cloning of plant spermidine synthases. Plant and Cell Physiology 39, 7379. Hashimoto T, Yukimune Y, Yamada Y. 1986. Tropane alkaloid production in Hyoscyamus root cultures. Journal of Plant Physiology 124, 6176. Hibi N, Higashiguchi S, Hashimoto T, Yamada Y. 1994. Gene expression in tobacco low-nicotine mutants. The Plant Cell 6, 723735. Imanishi S, Hashizume K, Nakakita M, Kojima H, Matsubayashi Y, Hashimoto T, Sakagami Y, Yamada Y, Nakamura K. 1998. Differential induction by methyl jasmonate of genes encoding ornithine decarboxylase and other enzymes involved in nicotine biosynthesis in tobacco cell cultures. Plant Molecular Biology 38, 11011111. Moyano E, Fornale S, Palazon J, Cusido RM, Bagni N, Pinol MT. 2002. Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene. Phytochemistry 59, 697 702. Moyano E, Jouhikainen K, Tammela P, Palazon J, Cusido RM, Pinol MT, Teeri HT, Oksman-Caldentey KM. 2003. Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus. Journal of Experimental Botany 54, 203211. Nash RJ, Rothschild M, Porter EA, Watson AA, Waigh RD, Waterman PG. 1993. Calystegines in Solanum and Datura species and the death's-head hawk-moth (Acherontia atropus). Phytochemistry 34, 12811283. Rothe G, Drager B. 2002. Tropane alkaloidsmetabolic response to carbohydrate signal in root cultures of Atropa belladonna. Plant Science 163, 979985. Rothe G, Garske U, Drager B. 2001. Calystegines in root cultures of Atropa belladonna respond to sucrose, not to elicitation. Plant Science 160, 10431053. 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, 367372. Sevon N, Draeger B, Hiltunen R, Oksman-Caldentey KM. 1997. Characterization of transgenic plants derived from hairy roots of Hyoscyamus muticus. Plant Cell Reports 16, 605611. Shoji T, Yamada Y, Hashimoto T. 2000. Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant and Cell Physiology 41, 831839. Suzuki K, Yamada Y, Hashimoto T. 1999. Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle. Plant and Cell Physiology 40, 289297. Tiburcio AF, Kaur SR, Galston AW. 1986. Polyamine metabolism ans osmotic stress. II. Improvement of oat protoplasts by an inhibitor of arginine decarboxylase. Plant Physiology 82, 375378.

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