Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids

Phytochemistry xxx (2016) xxx–xxx

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Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids Wei Qiang a, Ke Xia a, Qiaozhuo Zhang a, Junlan Zeng a, Yuanshe Huang b, Chunxian Yang a, Min Chen c, Xiaoqiang Liu a, Xiaozhong Lan d, Zhihua Liao a,⇑ a Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China b College of Agronomy, Anshun University, Anshun 561000, China c SWU-TAAHC Medicinal Plant Joint R&D Centre, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China d TAAHC-SWU Medicinal Plant Joint R&D Centre, Agricultural and Animal Husbandry College, Tibet University, Nyingchi of Tibet 860000, China

a r t i c l e

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Article history: Received 4 October 2015 Received in revised form 1 March 2016 Accepted 8 March 2016 Available online xxxx Keywords: Brugmansia arborea Solanaceae Tropane alkaloids Tropinone reductase I Enzymatic kinetics Tissue profile

a b s t r a c t Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29 kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8–8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65 mM, 88.3 nkat mg
1 and 2.93 S
1, respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18 mM, 81.20 nkat mg
1 and 2.40 S
1 at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56 mM, 171.62 nkat.mg
1 and 5.69 S
1, respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34 mM, 111.90 nkat mg
1 and 3.30 S
1, respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Solanaceae plants produce pharmaceutical tropane alkaloids (TAs), including hyoscyamine (7), anisodamine (8) and scopolamine (9) (Fig. 1). Hyoscyamine (7) and scopolamine (9) (Fig. 1) are widely known for their anticholinergic properties and are widely used for pain relief, anaesthesia, and for the treatment of drug addiction, motion sickness and Parkinson’s disease (Wang et al., 2011). Anisodamine (8) is also a non-specific cholinergic ⇑ Corresponding author at: School of Life Sciences, Southwest University, Chongqing 400715, China. E-mail addresses: [email protected], [email protected] (Z. Liao).

antagonist exhibiting a spectrum of pharmacological effects similar to that of hyoscyamine (7). Although less potent, anisodamine (8) is less toxic than hyoscyamine (7) and has consequently garnered increasing research interest (Poupko et al., 2007). Moreover, scopolamine (9) exhibits a higher commercial value than the aforementioned compounds and has approximately a 10 times greater market demand due to its higher pharmacological activity and fewer side effects (Hashimoto et al., 1993; Oksman-Caldentey, 2007). However, scopolamine (9) yield from plants is commonly lower than that of hyoscyamine (7). Due to difficulties and high cost of industrial synthesis, these alkaloids continue to be extracted from several species of Solanaceae, including those from the genera Datura, Duboisia, Hyoscyamus and Atropa. Given the

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Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx

acutangulus significantly enhanced the content of hyoscyamine (7) and greatly improved the production of scopolamine (9), indicating that TRI is a promising target for genetic engineering to increase TAs production in plants or in vitro cultures (Kai et al., 2009; Richter et al., 2005). The biosynthesis of TAs has been extensively and intensively studied at the molecular and biochemical levels in many herbal plant species, including in D. stramonium (Nakajima et al., 1998; O’Hagan and Robins, 1998), H. niger (Li et al., 2006; Nakajima and Hashimoto, 1999) and A. belladonna (Bedewitz et al., 2014). However, TAs biosynthesis has not yet been studied in arborous plant species. Although all TAs-producing plants rely on the same TAs biosynthetic pathway, the regulation of genes and biochemical features of enzymes differ by species (Moyano et al., 2003). For example, putrescine N-methyltransferase is the committedstep enzyme involved in TAs biosynthesis in H. muticus (Moyano et al., 2003) but not A. belladonna (Rothe et al., 2003). The biosynthesis, accumulation and regulation of TAs in arborous plants may be distinct and constitute an interesting subject of study. Brugmansia arborea, previously named Datura arborea according to Flora Reipublicae Popularis Sinicae, is the perennial arborous TA-producing plant species and belongs to the Solanaceae family. This species has very high biomass, and the entire plant is abundant in alkaloids, although the molecular and biochemical identification of TAs biosynthetic genes in B. arborea have rarely been studied. In the present study, the cDNA of TRI from B. arborea (namely BaTRI) was isolated, the tissue profiles of BaTRI gene and TAs were determined, and the biochemical features of this enzyme were functionally characterised based on the purified recombinant protein from E. coli. This research will improve our understanding of the biosynthesis and regulation of TAs in different botanical systems, provide a useful candidate for the metabolic engineering of TAs biosynthesis in this plant species, and simultaneously help to exploit new resources to meet the increasing demand for scopolamine (9).

2. Results and discussion 2.1. Gene cloning and sequence analysis

Fig. 1. The biosynthetic pathway to tropane alkaloids in the Solanaceae and flowering plants of Brugmansia arborea (Photographed by Min Li, Plant Photo Bank of China). TRI, Tropinone reductase I; TRII, Tropinone reductase II; PMT, Putrescine N-methyltransferase; H6H, Hyoscyamine-6b-hydroxylase.

increasing demand for herbal medicine, new resources for secondary metabolite extraction need to be investigated (Dehghan et al., 2013). Two tropinone reductases (TRs) constitute an important branch point in the TA biosynthetic pathway. Tropinone reductase I or tropine-forming reductase (TRI; EC 1.1.1.206) reduces tropinone (5) to tropine (6) during TAs biosynthesis, whereas tropinone reductase II (TRII; EC 1.1.1.236) reduces tropinone (5) to pseudotropine (10), diverging metabolic flux to nortropane calystegine A3 (11) (Nakajima et al., 1993) (Fig. 1). TRI belongs to the family of short chain dehydrogenases/reductases (SDRs) that catalyse NAD(P) (H)-dependent redox reactions and TRI activity controls metabolic flux towards hyoscyamine (7) and downstream TAs biosynthesis (Drager, 2006). The genes encoding TRI have been cloned and functionally identified in several herbal plant species, including Datura stramonium (Nakajima et al., 1993), Hyoscyamus niger (Nakajima et al., 1999b), Anisodus acutangulus (Kai et al., 2009), Withania coagulans (Kushwaha et al., 2013a) and Withania somnifera (Kushwaha et al., 2013b). Overexpression of the TRI gene in A. belladonna and A.

A pair of degenerate primers was designed based on the alignment of the TRI nucleotide sequences of D. stramonium and D. innoxia, two herbaceous species belonging to the same genus of Datura, which also includes B. arborea, to carry out PCR amplification using a cDNA library of B. arborea roots as a template. This reaction resulted in an 819-bp fragment with high sequence homology to D. stramonium TRI. 50 RACE and 30 RACE with genespecific primers yielded two 133-bp and 364-bp PCR products, respectively. The three PCR product sequences were assembled to generate a 1138-bp cDNA that was physically confirmed by two gene-specific primers, F-BaTRI-full and R-BaTRI-PRO. The full-length BaTRI cDNA contained a 819-bp coding sequence, which encodes a polypeptide of 272 amino acid residues with a calculated molecular mass of 29.70 kDa and theoretical isoelectric point (pI) of 5.79. The deduced protein sequence exhibited the highest sequence identity with TRIs from D. stramonium (95%, accession number P50162) and D. innoxia (95%, accession number KJ676865), followed by TRI from A. belladonna (90%, accession number AFP55030). As expected, it exhibited evidently lower sequence identity with pseudotropinone-forming tropione reductases (TRIIs) from other Solanaceous plants, such as A. belladonna TRII (accession number AGH24753, 63%) and A. luridus TRII (accession number AGL76990, 63%). Thus, the cloned gene was named B. arborea tropinone reductase I (BaTRI) and deposited in the NCBI database with accession number KJ676866.

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx

Multiple alignments of BaTRI amino acid sequence with other characterised TRIs are shown in Fig. 2. Like other members of the SDR superfamily, BaTRI shared a conserved TGXXXGXG motif involved in NADPH binding (Oppermann et al., 2003), the NNAG motif of SDRs (Oppermann et al., 2003) and the catalytic tetrad motif (N-S-Y-K) of TRs (Filling et al., 2002), among which the tyrosine residue is considered essential for the catalytic activity of TRs

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(Nakajima et al., 1999a). Eleven amino acids (Val110, His112, Ile159, Ala160, Leu165, Val168, Val203, Leu208, Val209, Ile223 and Phe226) were predicted to be in contact with the tropinone (5) substrate (Nakajima et al., 1998), and site-directed mutagenesis of various residues of TRs established that five residues (His112, Ala160, Val168, Ile223 and Phe226) contributed to the sterospecificity of the respective TRIs (Nakajima et al., 1999a). All residues

Fig. 2. Comparison of tropinone reductase-I from B. arborea (BaTRI) with other known tropinone reductases via a multiple sequence alignment. Identical amino acids are shown in white on a black background, and the conserved amino acids are shown in black on a grey background. Blue-decorated residues in blue boxes are involved in NADPH binding. Red highlights denote residues conserved in SDRs. Eleven yellow highlighted residues participate in tropinone (5) binding. A red box designates the signature YXXXK motif of short chain dehydrogenases/reductases (SDR), and sequences highlighted in green denote a catalytic tetrad. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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were conserved at analogous positions in BaTRI. Thus, a bioinformatics analysis suggested that the cloned BaTRI was a functional TRI enzyme that catalyses the reduction of tropinone (5) to tropine (6). The phylogenetic analysis revealed that BaTRI and other TRIs from Solanaceous plants were grouped in the same TRI cluster (group I). BaTRI exhibited close phylogenetic proximity to D. stramonium TRI and D. innoxia TRI, which coincided with the fact that these species have a closer relationship (Fig. 3). Naturally, all TRIIs from Solanaceous plants were assembled into the TRII cluster (group II). Putative TRs or SDR-like proteins from plants outside of Solanaceae, such as AtTRI, VvTRI and CoTRI, constituted a third cluster (group III). CoTRI and DnTRI were the only members of group III that had been functionally validated to exhibit tropinone (5) reduction activity, but their characteristics significantly differed from those of Solanaceous TRIs (Brock et al., 2008; Chen et al., 2013). A 3-dimensional (3D) molecular model of BaTRI was constructed by using the DsTRI crystal structure (1ae1. pdb) as a template to comparatively analyse the substrate-binding pocket of BaTRI with that of DsTRI, which shared 95% sequence identity with BaTRI. The predicted structure had a deep cleft between the core structure and a small lobe, which constituted a binding pocket involved in contacting tropinone (5) (Nakajima et al., 1998). Among the pocket, five amino acids critical for the stereospecificity of the reaction mentioned above were conserved at the corresponding positions of BaTRI (Fig. S1. 1A and B). An alignment analysis of the active site environments of BaTRI, DsTRI (1ae1.pdb) and DsTRII (1ipf.pdb) showed strong homology between BaTRI and DsTRI and significant variation between BaTRI and DsTRII (Fig. S1. 1C). BaTRI could be perfectly superimposed onto DsTRI,

whereas seven residues of BaTRI and DsTRI, i.e., His112, Ile159, Ala160, Leu165, Val168, Ile223 and Phe226, were substituted by Tyr100, Val147, Ser148, Val153, Glu156, Leu210 and Leu213, respectively, in DsTRII. These structural features implied again that BaTRI may represent a functional form of TRI. 2.2. Purification of the recombinant BaTRI The coding region of BaTRI was subcloned into the protein expression vector pET28a and then overexpressed in E. coli Rosetta. After induction for 6 h by IPTG, the soluble fraction of lysed bacteria was collected. The enzyme assay demonstrated that the supernatant exhibited tropinone reductase I activity, suggesting that recombinant BaTRI was functionally expressed. Thus, a Ni+–NTA affinity chromatography column was adopted to purify His-tagged recombinant BaTRI. On the SDS–PAGE gel, the molecular weight of the purified recombinant BaTRI was approximately 29 kDa, which was consistent with the calculated molecular weight of BaTRI. The molecular weight of the recombinant BaTRI was similar to those of DsTRI (Nakajima et al., 1999a) and HnTRI (Nakajima and Hashimoto, 1999) (Fig. 4). 2.3. Enzymatic activities of BaTRI at different pH conditions The optimal pH value for the activity of BaTRI was screened (Fig. 5). For the reduction activity or forward reaction, BaTRI exhibited optimal catalytic velocities over a relatively broad pH range of 6.8–8.0, and its activity was highest at pH 8.0. By contrast, Solanaceae TRIs were previously reported to exhibit significant catalytic activity over a relatively narrow pH range, although the optimum pH values for HnTRI (Hashimoto et al., 1992), StTRI (Kaiser et al.,

Fig. 3. Phylogenetic relationship of BaTRI with other plant TRs. The tree was constructed with maximum likelihood method in MEGA4.1 using sequences from Datura innoxia TRI (KJ676865), Datura stramonium TRI (AAA33281.1), Hyoscyamus niger TRI (BAA85844.1), Anisodus acutangulus TRI (ACB71202.1), Withania coagulans TRI (AGB56644.1), Solanum tuberosum TRI (CAC34420.1), Datura stramonium TRII (AAA33282.1), Hyoscyamus niger TRII (AAB09776.1), Solanum tuberosum TRII (CAB52307.1), Anisodus acutangulus TRII (ACB71203.1), Dendrobium nobile TRI (AFD23287.1), Hordeum vulgare (BAJ87177.1), Oryza sativa (ABF95192.1), Zea mays (ACG34080.1), Dendrobium nobile TRII (AFD23289.1), Glycine max (XP_003552795.1), Populus trichocarpa (EEE95485.1), Vitis vinifera (XP_002282554.1), Arabidopsis lyrata (ABW81053.1), Arabidopsis cebennensis (ABW81184.1), Boechera divaricarpa (ABW74581.1), Arabidopsis thaliana (AAM10204.1), and Cochlearia officinalis (CAO02390.1) along with the BaTRI sequence.

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx

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Fig. 4. SDS–PAGE analysis of affinity-purified His-tagged recombinant BaTRI. IPTG-induced cells were sonicated and then subjected to centrifugation. The supernatant (soluble fraction) was collected and loaded on a Ni++–NTA affinity column to purify the His-tagged recombinant BaTRI. The fraction flowing through the affinity column and the elutes of imidazole gradient elution (concentration gradient of 50 mM, 100 mM, 150 mM, and 200 mM) were resolved on a 12% SDS–PAGE gel. The gel was stained with Coomassie brilliant blue R-250. M, protein molecular weight marker. Arrows indicate the band corresponding to recombinant BaTRI protein.

Fig. 5. Effect of pH on catalytic activity of BaTRI for both reduction and oxidation reaction of tropinone (5) and tropine (6) using NADPH and NADP+, respectively. Each point is the mean of triplicate assays.

2006), WcTRI (Kushwaha et al., 2013a), WsTRI (Kushwaha et al., 2013b) and DsTRI (Nakajima et al., 1999a), tended to be acidic or neutral, i.e., 6.1, 6.4, 6.6, 6.7 and 7.0, respectively. Interestingly, the optimum pH of BaTRI, i.e., pH 8.0, was identical to that of another TR, namely CoTR, which was isolated from Cochlaeria officinalis (Brock et al., 2008), a cruciferous plant that produces tropane esters. CoTR is a non-specific TR that can act both as a TRI and TRII and is consequently considered a possible ancestor of specialised TRs (TRI and TRII). In fact, only uncharged substrates have been suggested to be accepted by TRIs (Portsteffen et al., 1994). Thus, for tropinone (5), with pKa 8.9 (Drager, 2002), a high pH close to 8.9, where more tropinone (5) was uncharged, could confer TRI high affinity and possible high activity. In brief, a wide range of environments, from weakly acidic to weakly alkaline, could be suitable for the optimal reduction activity of TRI.

As expected, BaTRI efficiently catalysed the reverse reaction, i.e., the oxidation of tropine (6) to tropinone (5), within a narrow alkaline pH range. The optimum pH was 9.6, which agreed with that (pH 9.9) reported for the tropine-oxidation activity of the TRI isolated from D. stramonium (Portsteffen et al., 1994). The high alkaline pH optima favour the formation of the uncharged substrate, tropine (6), the pKa of which is 10.8 (Drager, 2002). However, TRI from H. niger exhibited maximum activity at a lower pH of 7.6 for tropine (6) oxidation (Hashimoto et al., 1992). Recently, the reverse reaction activities of TRIs have also been reported for two other Solanaceae plants, Withania coagulans (Kushwaha et al., 2013a) and W. somnifera (Kushwaha et al., 2013b), but the optimum pH values were not determined. Unlike TRI from D. stramonium, which exhibited similar reduction and oxidation activities (the Vmax of tropine (6) oxidation at pH 9.9 was 6.2 nkat mg
1 protein, and the Vmax of tropinone (5) reduction at pH 6.4 was 7.1 nkat mg -1 protein), BaTRI exhibited a significantly higher tropine (6) oxidation activity (121 nkat mg
1 protein) at pH 8.0 than tropinone (5) reduction activity (87 nkat mg
1 protein) at pH 9.6. Notably, the high oxidation activity in vitro may not be recapitulated in vivo in the cytoplasm, where the physiological pH is weakly acidic and outside the pH range of tropinone (5) oxidation activity. Thus, in planta, BaTRI is highly oriented in favour of the forward reaction (tropine (6) formation). Thus, the enzymatic reaction is likely irreversible, as observed in W. coagulans (Kushwaha et al., 2013a) and W. somnifera (Kushwaha et al., 2013b). 2.4. Enzymatic kinetics of BaTRI To understand the enzymatic kinetics of BaTRI, purified recombinant BaTRI was incubated with NADPH and each of the three different substrates, the natural substrate tropinone (5) and two structural analogues of tropinone (3-quinuclidinone hydrochloride and 4-methylcyclohexanone), and the forward reaction was allowed to proceed at pH 6.4. The assay pH was set to 6.4 to enable a direct comparison with the reported DsTRI of D. stramonium. The reverse reaction was also studied by incubating BaTRI with NAD+

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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and tropine (6) at the optimum pH of 9.6. The Michaelis–Menten curves of the enzyme for the four substrates were drawn to determine the Km values and Vmax values of the enzyme for the substrates (Fig. 6), and turnover rate (Kcat values) and catalytic efficiency (Kcat/Km values) were deduced from the Km values and Vmax values. All enzymatic kinetics parameters are listed in Table 1. The reduction velocity (Vmax) and affinity (Km) of BaTRI to tropinone were 88.3 ± 1.81 nkat mg
1 and 2.65 ± 0.19 mM, respectively, notably faster and stronger than those of 3-quinuclidinone hydrochloride but significantly slower and weaker than those of 4-methylcyclohexanone. The Kcat values and Kcat/Km values corroborated these findings. Noticeably, compared with the reduction of tropinone (5), BaTRI exhibited a faster oxidation velocity (171.62 ± 5.42 nkat mg
1) and stronger affinity (0.56 ± 0.04 mM) to tropine (6), with 10-fold higher Kcat/Km values (10160.71 s
1 M
1) than that (1105.66 s
1 M
1) of tropinone at pH 9.6. This result confirms that BaTRI is a functional enzyme that catalyses a reversible reaction in vitro, but the real enzyme activity should be considered to depend on physiological pH conditions, as mentioned above. 3-Quinuclidinone hydrochloride and 4-methylcyclohexanone are typical structural analogues of tropinone (5) representing monocyclic and bi-cyclic substrates (Fig. S2), respectively, and these compounds are widely used to test the substrate specificity of TRs (Brock et al., 2008; Chen et al., 2013). TRs from C. officinalis (Brock et al., 2008) and Dendrobium nobile (Chen et al., 2013) exhibit higher reduction velocities for monocyclic ketones than for bicyclic substrates. Solanaceae TRs, particularly pseudotropine-forming reductases (Drager, 2006), and BaTRI also exhibit this property. At the assay pH of 6.4, the non-ionizable substrate 4-methylcyclohexanone is uncharged, whereas the nitrogen-containing substrates tropinone (5) and 3-quinuclidinone hydrochloride are positively charged. Thus, as expected, BaTRI exhibited significantly higher apparent affinity for 4-methylcyclohexanone than tropinone (5) and 3-quinuclidinone hydrochloride, of which only the

deprotonated fractions can be bound by BaTRI, as is discussed above with respect to the optimum pH. A comparison of the enzymatic kinetics of BaTRI with DsTRI, a counterpart isolated from D. stramonium which has a close relationship with B. arborea, established that the affinity of BaTRI for tropinone (5) was twice as low (Km = 2.65 mM) as that of DsTRI, but the turnover was twice as high as that of native DsTRI (Vmax = 88.3 nkat mg
1) (Km = 1.3 mM, Vmax = 43 nkat mg
1) (Portsteffen et al., 1994), indicating the same catalytic efficiency at pH 6.4. In fact, the pH optimum of 6.4 for DsTRI activities was below the optimal pH range of 6.8 to 8.0 for BaTRI activity. Furthermore, for Solanaceae TRIs, the Km values for substrates containing proton-accepting nitrogen considerably decreased at less acidic conditions (Kaiser et al., 2006; Portsteffen et al., 1994), which was also discussed above. To experimentally compare the catalytic efficiency between BaTRI and DsTRI at pH 6.4 and validate that BaTRI might have a higher activities at pH above 6.4 than DsTRI, the recombinant TRI from D. stramonium was purified (Fig. S3) and the enzyme kinetics of DsTRI for tropinone (5) reduction and tropine (6) oxidation were determined under the same experimental condition as BaTRI. Michaelis–Menten curves of the enzyme for the two substrates (tropinone (5)/tropine (6)) were shown in Fig. S4 and enzymatic kinetics parameters were listed in Table 2. For tropinone (5) reduction at pH 6.4, both its affinity (Km = 4.18 mM) and reduction velocity (Vmax = 81.20 nkat mg
1) of DsTRI were lower and slower than those of BaTRI, so the catalytic efficiency of DsTRI (574 s
1 M
1) was half of that of BaTRI. For tropine (6) oxidation at pH 9.6, the affinity of DsTRI for tropine (6) was higher (Km = 0.34 mM) but the oxidation velocity (Vmax = 111.9 nkat mg
1) was slower than those of BaTRI, showing similar catalytic efficiency (9705.88 s
1 M
1) to that of BaTRI. Enzyme activities of tropinone (5) reduction with tropinone (5) concentration of 5 mM at pH 6.4, 6.8 and 7.0 indicated that BaTRI had significantly higher activity than DsTRI (Fig. 7). DsTRI has been reported to be an effective gene to enhance TAs synthesis, since its

Fig. 6. Michaelis–Menten curves for the NADPH-dependent reduction reaction of tropinone (5) (A), 3-quinuclidinone hydrochloride (B), 4-methylcyolohexanone (C) and NADP+-dependent oxidation reaction of tropine (6) (D) catalysed by BaTRI. The buffer for the reduction reaction was potassium phosphate (0.1 M, pH 6.4), and the buffer for the oxidation was glycine–NaOH (0.1 M, pH 9.6). Each point is the mean of triplicate assays.

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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Table 1 Kinetic parameters of BaTRI-catalysed reaction. The data represent means of three independent measurements ± SD. The Km and Vmax were calculated from the Michaelis– Menten equation with a non-linear regression. The Kcat value was calculated by dividing Vmax by Et (the number of enzymes in each assay). Substrate

pH assay

Km (mM)

Vmax (nkat mg
1 protein)

Kcat (s
1)

Kcat/Km (s
1 M
1)

Tropinone (5) 4-methylcyclohexanone 3-quinuclidinone hydrochloride Tropine (6)

6.4 6.4 6.4 9.6

2.65 ± 0.19 0.46 ± 0.04 5.17 ± 0.29 0.56 ± 0.04

88.30 ± 1.81 140.91 ± 7.23 16.26 ± 0.54 171.62 ± 5.42

2.93 ± 0.06 4.67 ± 0.24 0.54 ± 0.02 5.69 ± 0.18

1105.66 10152.17 104.45 10160.71

Table 2 Kinetic parameters of DsTRI-catalysed reaction. The data represent means of three independent measurements ± SD. The Km and Vmax were calculated from the Michaelis–Menten equation with a non-linear regression. The Kcat value was calculated by dividing Vmax by Et (the number of enzymes in each assay). Substrate

pH assay

Km (mM)

Tropinone (5) Tropine (6)

6.4

4.18 ± 0.51

9.6

0.34 ± 0.05

Kcat (s
1)

Kcat/Km (s
1 M
1)

81.20 ± 3.61

2.40 ± 0.11

574.16

111.90 ± 5.41

3.30 ± 0.16

9705.88

Vmax (nkat mg
1 protein)

Fig. 8. Real-time PCR-based comparison of the expression of BaTRI in different tissues of B. arborea.

Fig. 7. Comparison of the catalytic activities between BaTRI and DsTRI for tropinone (5) reduction at pH 6.4, pH 6.8 and pH 7.0. Concentration of tropinone (5) was 5 mM. The data represent means of three independent measurements. Bars with asterisks are significantly different (t test, P < 0.01).

overexpression in A. belladonna increased the production of hyoscyamine (7) and scopolamine (9) by 3- and 5-fold, respectively (Richter et al., 2005). Thus, BaTRI may be a promising target for genetic engineering to increase TAs synthesis in plants, and we will attempt to over-express this gene in TAs-producing plants in our future work to verify its function. 2.5. Tissue profile of BaTRI and TAs accumulation The relative expression levels of BaTRI were detected in different organs, including primary roots, secondary roots, mature stems, young stems, mature leaves and young leaves, using realtime quantitative PCR. As shown in Fig. 8, the tissue profile analysis showed that BaTRI was expressed in all examined organs, but at different levels. Secondary roots exhibited the highest BaTRI expression of all measured organs, and the expression level of BaTRI in the secondary roots was twofold higher than that in primary roots and mature stems and approximately 3–5 times higher than that in young stems, mature leaves and young leaves. The biosynthesis of plant secondary metabolites is tightly controlled due to the spatial and temporal expression of biosynthetic genes.

Herbal plant species, such as A. belladona, D. metel and H. niger, biologically synthesize tropane alkaloids in the secondary roots, in which TAs-biosynthetic genes, such as TRI and H6H, are specifically expressed (Pramod et al., 2010; Suzuki et al., 1999). The tissue expression profile showed that expression of BaTRI was organ independent. Although this expression pattern significantly differed from tissue profiles of TRI genes from other TAs-producing herbal plant species, such as in H. niger (Nakajima and Hashimoto, 1999) and A. belladonna (our unpublished data), the constitutive expression of TRI transcripts has become a developing concept given several very recent similar reports (Chen et al., 2013; Cheng et al., 2013; Dehghan et al., 2013; Kai et al., 2009). Higher BaTRI transcript levels in the secondary roots and lower differential expression in other organs suggested that TAs are mainly synthesized in the secondary roots of B. arborea, and other plant organs might also contribute to TAs biosynthesis. The reduction of tropinone (5) catalysed by TRI is a key step at the midpoint of TAs biosynthesis that competes for substrates with pseudotropine-forming TRII and regulates the metabolic flux into downstream hyoscyamine (7), anisodamine (8) and scopolamine (9) biosynthesis pathways. Thus, the accumulation profile of the three alkaloids corresponding to the BaTRI expression profile in the corresponding organs of B. arborea was detected (Fig. 9). Scopolamine (9) was consistently the predominant alkaloid in all the tested organs, with highest contents in mature leaves (1.55 mg g
1), young leaves (1.47 mg g
1) and mature stems (1.43 mg g
1) (these three levels did not significantly differ), followed by the young stems (0.78 mg g
1) and primary roots (0.76 mg g
1), and the lowest contents in the secondary roots (0.14 mg g
1). Similar results have been reported for A. belladonna (Qiang et al., 2014), D. stramonium (Miraldi et al., 2001), D. metal (Pramod et al., 2010), H. senecionis (Dehghan et al., 2013) and H. muticus (Dehghan et al., 2013), for which the scopolamine (9)

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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Fig. 9. Contents of tropane alkaloids in different tissues of B. arborea.

contents of leaves and (or) stems were higher than those of roots. Thus, the aerial parts of TAs-producing plants tend to accumulate significantly higher amounts of scopolamine (9) compared with the root. In contrast to scopolamine (9), the anisodamine (8) content was highest in the primary roots (0.62 mg g
1) followed by mature stems (0.22 mg g
1). A small amount of anisodamine (8) was also detected in the young stems and secondary roots (0.13 mg g
1 and 0.09 mg g
1, respectively), but only trace amounts of anisodamine (8) (0.01 mg g
1) were present in both the mature leaves and young leaves, to approximately the same level as reported in the leaves of H. senecionis and H. muticus (Dehghan et al., 2013). Similarly, the concentration of hyoscyamine (7) was highest in the primary roots (0.60 mg g
1), and this concentration was approximately four to ten times higher than that of the aerial parts of the plant. However, hyoscyamine (7) was not detected in secondary roots. The higher contents of hyoscyamine (7) and anisodamine (8) in the roots than in the aerial parts may be related to the fact that the root is a major organ for the biosynthesis of TAs. Furthermore, expression of BaTRI was highest in the root, which is consistent with the slightly higher total TAs contents in the primary roots. The profile of TAs accumulation demonstrates that B. arborea is a rare scopolamine (9)-predominant TAs resource plant. The scopolamine (9)/hyoscyamine (7) ratio was the highest ever reported in the young leaves, as high as 24.7, and the secondary roots did not contain hyoscyamine (7). The latter is the direct precursor of BaH6H, which catalyses scopolamine (9) biosynthesis. Thus, a higher scopolamine (9)/hyoscyamine (7) ratio implies a higher enzymatic activity of BaH6H (Dehghan et al., 2013). To the best of our knowledge, the higher scopolamine (9)/hyoscyamine (7) ratio in the entire plant, especially in the aerial parts, has never been observed and raises questions about the role of BaH6H in the production of scopolamine (9) in various tissues. Interestingly, the tissue profile showed that BaH6H (accession number KR006981) was expressed in all tissues but exhibited tissue-specific variations (Fig. 8) (Qiang et al., 2015). Specifically, the expression of BaH6H was highest in the mature leaves, which also contained the highest levels of scopolamine (9), followed by the secondary roots, young leaves, young stems and mature stems. However, BaH6H was not expressed in the primary roots. H6H is a molecularly and histochemically well characterised TAs biosynthesis pathway enzyme, the expression of which is restricted to the root pericycle cells of H. niger (Kanegae et al., 1994) and D. metel (Pramod et al., 2010) and the pericycle, tapetum and pollen mother

cells of A. belladonna (Suzuki et al., 1999). Our results are in contrast to those obtained for common herbal plants but in agreement with the organ-independent expression of H6Hs observed in A. acutangulus (Kai et al., 2007) and H. senecionis (Dehghan et al., 2013). Transcripts of BaH6H were detected in the aerial parts of B. arborea, and the positive correlation between BaH6H expression and the scopolamine (9) content in leaves suggest that hyoscyamine (7) is converted to scopolamine (9) in the secondary roots and subsequently translocated to the primary roots and aerial parts. Scopolamine (9) biosynthesis may also be continually catalysed from hyoscyamine that has accumulated in aerial parts of the plant, especially the leaves. The biosynthesis of tropane alkaloids in the roots and subsequent translocation to the aerial parts of the plant has been well established in Datura, Hyoscyamus and Atropa plants based on grafting experiments. The reported catalytic activities of biosynthetic pathway genes detected only in the roots and in situ hybridization, immunohistochemistry and immuno-localisation of related genes in H. niger and A. belladonna corroborate that specific cells in the root are the synthetic sites and that TAs undergo acropetal transport, possibly through the xylem (De Luca and St Pierre, 2000; Ziegler and Facchini, 2008). Recently, the extensive exploitation of TAs resource plants revealed a series of pathway genes, including PMT, TRI and H6H that are expressed in the aerial parts of A. acutangulus, H. senecionis, and W. coagulans, challenging previous understanding. In fact, a recent study suggested that tropane alkaloids might have evolved independently in plants, at least in the Solanaceae and Erythroxylaceae (Jirschitzka et al., 2012), and a report published shortly thereafter provided solid evidence that unambiguously identified independent de novo tropane alkaloid biosynthesis in the aerial parts of W. somnifera (Kushwaha et al., 2013b). Herein, the constitutive expression of BaTRI in all tissues agrees with the findings of W. somnifera TRI. Yet only gene expression data does not establish independent de novo synthetic competence of these tissues to synthesize TAs, so other in vivo experiments must be conducted as the next work to provide more evidences.

3. Conclusions In the present study, a TAs biosynthesis gene encoding the putative tropine (6)-forming tropinone reductase was isolated from a woody resource plant, B. arborea, then heterologously expressed and biochemically characterised for the first time. The deduced protein, BaTRI, contained the same specific motifs and conserved active amino acids as other reported TRIs from herbaceous Solanaceae plants and had the closest genetic relationship with TRIs from D. stramonium and D. innoxia, which belong to the same genus of Datura. The pH optimum of BaTRI for the forward reaction differed (6.8–8.0) from that of DsTRI, and its enzymatic activity for the reverse reaction at pH 9.6 was much higher. BaTRI exhibited higher catalytic efficiency than DsTRI at pH 6.4. Interestingly, the encoding BaTRI and the scopolamine (9)-biosynthetic gene BaH6H were constitutively expressed in the root and aerial parts of the plant. The tropane alkaloids accumulation profile demonstrated that B. arborea is a scopolamine (9)-predominant TAs resource plant with considerably high scopolamine (9) contents in the leaves and mature stem. Together with expression profiles of BaTRI and BaH6H, this finding suggested that the biosynthesis of hyoscyamine (7), and especially scopolamine (9), took place not only in roots but also in the aerial parts of B. arborea. Because scopolamine (9) exhibits higher pharmaceutical value and worldwide demand than hyoscyamine (7), the high scopolamine (9) content in the leaves indicates that B. arborea farming may be a promising approach for the economic production of scopolamine (9).

Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008

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4. Experimental 4.1. Plant materials Thirty-year-old B. arborea plants grew locally in the Garden of Southwest University in Chongqing, China. Various organs, including the young leaves, mature leaves, young stems, mature stems, primary roots and secondary roots were collected in October 2013. After collection, all samples were immediately placed in liquid N2 for further study. 4.2. Gene cloning Total RNAs from the different tissues of B. arborea were isolated using a RNAplant plus kit (Tiangen, Beijing, China). A SMARTTM RACE cDNA Amplification Kit was used to synthesize singlestranded cDNAs as templates for PCR amplification of the core cDNA fragment, the 30 -RACE and 50 -RACE of the gene of interest, according to the manufacturer’s protocol (ClonTech, California, USA). A pair of primers of F-TRI (50 -ATGGAAGAATCAAAAGTGTCC30 ) and R-TRI (50 -AAGCAGCAGGGAAGCAAAG-30 ) was used to clone the core fragment on a MyCycler instrument (BIO-RAD, California, USA). Gene-specific primers were designed to rapidly amplify cDNA ends (RACE) based on the core cDNA fragment sequence information. The following primer pairs were used for each 50 RACE PCR reaction: forward, the universal primers (UPM and NUP) provided by the kit; reverse, R-TRI-5-1 (50 -GCCACCAGTAACTAGGGCT-30 ) for the first 50 -end amplification, R-TRI-5-2 (50 TGGTGCCTTTGAGACTCCATC-30 ) for the nested amplification. The following primer pairs were used for the 30 -RACE PCR: forward, F-TRI-3-1 (50 -GACAATTTTATTGTCAAGACTCC-30 ) for the first 30 end PCR amplification, F-TRI-3–2 (50 -CYTTTCTTTGCTTCCCTGCT30 ) for the nested amplification; reverse, the universal primers (UPM and NUP) provided by the kit. The first and nested PCR procedures were carried out according to the conditions described in the protocol. The following pair of primers was used to confirm the physical cDNA of BaTRI: F-BaTRI-full (50 -CCCATCCCAAAATAGTTG-30 ) as the forward primer, R-BaTRI-PRO (50 -CGAGCTCGTGATGATAATAACAGAGAAC-30 ) as the reverse primer. Each PCR product was subcloned into the pMD-18T vector and sequenced. 4.3. Sequence analysis Comparative and bioinformatics analyses of BaTRI were carried out online at the websites http://www.ncbi.nlm.nih.gov and http:// www.expasy.org. The nucleotide sequence, deduced amino acid sequence and ORF (open reading frame) encoded by BaTRI were analysed, and the sequences were compared based on a database search using the BLAST program. The phylogenetic tree was constructed by MEGA (http://www.megasoftware.net). The structural modelling was performed using Swiss-Modeling (http://www. swissmodel.expasy.org/) and Pymol (http://www.pymol.org). 4.4. Recombination and purification of BaTRI and DsTRI A pair of primers, F-BaTRI-PRO (50 -CGGATCCATGGAAGAATCAAAAGTGTCC-30 ) with BamH I and R-BaTRI-PRO (50 -CGAGCTCGTGATGATAATAACAGAGAAC-30 ) with Sac I, were used to isolate the coding sequence of BaTRI by proofreading DNA polymerase. The BaTRI coding sequence was subcloned into the E. coli expression vector pET28a with BamH I and Sac I, and then introduced into E. coli strain Rosetta (DE3). The overexpression of BaTRI was induced by the addition of IPTG (0.5 mM) at 28 °C for 6 h. To purify the BaTRI recombinant enzyme, cells were collected by centrifugation (10 min at 5000g) and resuspended in lysis buffer (300 mM NaCl,

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50 mM NaH2PO4, 10 mM imidazole, 10 mM Tris base, pH 8.0). The cells were lysed in an ice-cold ultrasound bath (5  5 s) for 30 min. The lysate was centrifuged (12000g for 30 min), and the soluble fraction in the supernatant was applied to a ProteinIsoTM Ni–NTA Resin column according to the manufacturer’s instructions (TransGen Biotech, Beijing, China). The enzyme activity of eluted fractions was screened, and catalytically active fractions were pooled. To remove imidazole, dialysis was performed using a dialysis bag with a molecular weight cut-off of 14,000 Da (Biosharp, Hefei, China) in ice-cold dialysate (0.05 M potassium phosphate, pH 6.4). For heterologous expression of DsTRI (accession number L20473), a pair of primers F-DsTRI-PRO (50 -CGGATCCATGGAAGAATCAAAAGTGTCC-30 ) with BamH I and R-DsTRI-PRO (50 GGAGCTCGTGATGATAACAACTTGGAAC-30 ) with Sac I, designed at the same position as BaTRI, were used, and procedures of recombination and purification are the same as above. Induced expressed protein and affinity purified fractions were detected by SDS–PAGE analysis. The protein concentration was determined using a protein quantitative kit based on Bradford method (TransGen Biotech, Beijing, China). 4.5. Enzymatic assays The enzyme activity was assayed as described previously (Portsteffen et al., 1994). Briefly, the substrate reduction activity was measured based on NADPH + H+ consumption, which was spectrophotometrically followed at 340 nm and 30 °C (U-3010 spectrophotometer, HITACHI, Japan). For a standard reaction system, each 1 ml sample contained 20 lg protein, 200 lM NADPH + H+, 5 mM substrate (0.01–30 mM for the determination of Km values, the concentration range of each substrate needed to be adjusted according to the results of enzymatic assays, 20 mM tropinone for pH optimum assay), and 0.1 M potassium phosphate at pH 6.4. All substrates were commercially available: tropinone 5, tropine 6 and 4-methylcyclohexanone were obtained from Sigma (Sigma–Aldrich, St. Louis, MO,); 3-quinuclidinone hydrochloride was purchased from Aladdin (Aladdin, Shanghai, China). For the reverse reaction, tropinone 5 and NADPH were replaced by tropine 6 (0.01–5 mM for the determination of Km value) and NADP+ (300 lM), respectively in 0.1 M glycine buffer at pH 9.6. As a negative control, boiled proteins were added instead of purified recombinant BaTRI. Data were collected during the initial linear phase of the enzyme reaction to calculate the kinetic parameters, which could be achieved by adjusting the amount of enzyme and 20 lg used here was suitable. The kinetic constants were calculated with a non-linear regression of the Michaelis–Menten equation using OriginPro 8.0. All assays were repeated three times, and mean values with standard deviations are reported. 4.6. Real-time quantitative PCR analysis To detect the expression levels of BaTRI in different organs, including the secondary roots, primary roots, mature stems, young stems, mature leaves and young leaves, real-time quantitative PCR (qPCR) was carried out with a pair of primers for BaTRI (forward primer: 50 -CGTGAGAAGCTTATGCAAACTG-30 ) and reverse primer: 50 -CTTCAATAATGGGTAAGCAATCTGA-30 ). The total RNAs for each sample were extracted using a RNAplant plus kit (Tiangen, Beijing, China). The quality and concentration of the RNAs were assessed via agarose gel and spectrophotometer (U-3010 spectrophotometer, HITACHI, Japan) analyses. First-strand cDNA was synthesized from RNA (1 lg) using an iScriptTM cDNA Synthesis Kit (Bio-Rad, USA). qPCR was performed using iTaqTM Universal SYBRÒ Green Supermix (Bio-Rad, USA) in a final volume (20 ll) under the following conditions: initial denaturation at 94 °C for 2 min, 40 cycles of denaturation at 94 °C for 15 s and annealing at 57.5 °C for 15 s,

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and a final extension at 72 °C for 20 s. After each run, a melting curve was analysed by heating the samples from 60 to 95 °C to determine the specificity of amplification. The reaction was carried out on an iQTM5 real-time qPCR system (Bio-Rad, USA). All samples were analysed for three times, and data were normalised according to the expression level of 18S rRNA and PGK (Li et al., 2014) as internal reference genes. For 18S rRNA, 18SF (50 -CAGATACCGTCCTAGTCTCAAC-30 ) and 18SR (50 -CAGCCTTGCGACCATACTC-30 ) were used as primers; for PGK, F-PGK (50 -TCGCTCTTGGAGAAGGTTGAC30 ) and R-PGK (50 -CTTGTCCGCAATCACTACATCAG-30 ) were used as primers. The relative abundance of each transcript was quantified following the Pfaffl Method, which was internally programmed into the iQTM5 real-time PCR detection system (Bio-Rad, USA). 4.7. HPLC analysis of tropane alkaloids Fresh plant material was dried at 40 °C and ground into very fine powder. Dry powder (200 mg) was accurately weighed for alkaloid extraction and detection according to previously reported methods (Qin et al., 2014; Wang et al., 2011). The mobile phase consisted of CH3CN: 20 mM NH4OAc (11:89, v/v), and the NH4OAc solution included 0.1% HCOOH (pH 4.0). The sample was eluted at a rate of 1 ml/min. Standard samples of hyoscyamine (7), anisodamine (8) and scopolamine (9) were purchased from Sigma– Aldrich (Sigma, LA, USA). The HPLC system was a Shimadzu LC20A instrument, and the detector was the photo-diode array. The detecting wavelength was 226 nm. The temperature of the CTPODS column (150 mm  4.6 mm) was 40. For each assay, 20 ll of sample was injected, and each sample was analysed three times. Acknowledgements This research was financially supported by the NSFC Projects (31370333), National Hi-Tech Project (2011AA100605), the Program for New Century Excellent Talents in University (NCET-120930) and the Fundamental Research Funds for the Central Universities (XDJK2013A024). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2016. 03.008. References Bedewitz, M.A., Gongora-Castillo, E., Uebler, J.B., Gonzales-Vigil, E., WiegertRininger, K.E., Childs, K.L., Hamilton, J.P., Vaillancourt, B., Yeo, Y.S., Chappell, J., DellaPenna, D., Jones, A.D., Buell, C.R., Barry, C.S., 2014. A root-expressed Lphenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna. Plant Cell 26, 3745–3762. Brock, A., Brandt, W., Drager, B., 2008. The functional divergence of short-chain dehydrogenases involved in tropinone reduction. Plant J. 54, 388–401. Chen, W., Cheng, X.F., Zhou, Z.H., Liu, J.J., Wang, H.Z., 2013. Molecular cloning and characterization of a tropinone reductase from Dendrobium nobile Lindl. Mol. Biol. Rep. 40, 1145–1154. Cheng, X.F., Chen, W., Zhou, Z.H., Liu, J.J., Wang, H.Z., 2013. Functional characterization of a novel tropinone reductase-like gene in Dendrobium nobile Lindl. J. Plant Physiol. 170, 958–964. De Luca, V., St Pierre, B., 2000. The cell and developmental biology of alkaloid biosynthesis. Trends Plant Sci. 5, 168–173. Dehghan, E., Ahmadi, F.S., Ravandi, E.G., Reed, D.W., Covello, P.S., Bahrami, A.R., 2013. An atypical pattern of accumulation of scopolamine and other tropane alkaloids and expression of alkaloid pathway genes in Hyoscyamus senecionis. Plant Physiol. Biochem. 70, 188–194. Drager, B., 2002. Analysis of tropane and related alkaloids. J. Chromatogr. A 978, 1– 35. Drager, B., 2006. Tropinone reductases, enzymes at the branch point of tropane alkaloid metabolism. Phytochemistry 67, 327–337. Filling, C., Berndt, K.D., Benach, J., Knapp, S., Prozorovski, T., Nordling, E., Ladenstein, R., Jornvall, H., Oppermann, U., 2002. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J. Biol. Chem. 277, 25677–25684.

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Please cite this article in press as: Qiang, W., et al. Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids. Phytochemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.03.008