Phytochemistry xxx (2016) xxx–xxx

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

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 i n f o a b s t r a c t

Article history: Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding
Received 4 October 2015 tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally charac-
Received in revised form 1 March 2016 terised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to
Accepted 8 March 2016
tropinone reductase I from TAs-producing herbal plant species. The purified 29 kDa recombinant BaTRI
Available online xxxx
exhibited maximum reduction activity at pH 6.8–8.0 when tropinone was used as substrate; it also exhib-
ited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat
Keywords:
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,
Brugmansia arborea
Solanaceae
Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18 mM,
Tropane alkaloids 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
Tropinone reductase I than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were
Enzymatic kinetics also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tro-
Tissue profile pine 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 pro-
files of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all exam-
ined 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 antagonist exhibiting a spectrum of pharmacological effects simi-

Solanaceae plants produce pharmaceutical tropane alkaloids
(TAs), including hyoscyamine (7), anisodamine (8) and scopo-
lamine (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: zhliao@swu.edu.cn, zhihualiao@163.com (Z. Liao).
lar to that of hyoscyamine (7). Although less potent, anisodamine
(8) is less toxic than hyoscyamine (7) and has consequently gar-
nered increasing research interest (Poupko et al., 2007). Moreover,
scopolamine (9) exhibits a higher commercial value than the afore-
mentioned 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

http://dx.doi.org/10.1016/j.phytochem.2016.03.008
0031-9422/Ó 2016 Elsevier Ltd. All rights reserved.

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
2 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx
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 sec-
ondary 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 tro-
pine-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 func-
tionally 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 coag-
ulans (Kushwaha et al., 2013a) and Withania somnifera (Kushwaha
et al., 2013b). Overexpression of the TRI gene in A. belladonna and A.
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
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 biochemi-
cal features of enzymes differ by species (Moyano et al., 2003).
For example, putrescine N-methyltransferase is the committed-
step 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 Solana-
ceae family. This species has very high biomass, and the entire
plant is abundant in alkaloids, although the molecular and bio-
chemical 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 fea-
tures 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
A pair of degenerate primers was designed based on the align-
ment 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 amplifica-
tion 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 gene-
specific 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 reduc-
tases (TRIIs) from other Solanaceous plants, such as A. belladonna
TRII (accession number AGH24753, 63%) and A. luridus TRII (acces-
sion 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.
 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx 3

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 tyro-
sine residue is considered essential for the catalytic activity of TRs
(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 sterospeci-
ficity 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
4 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx

were conserved at analogous positions in BaTRI. Thus, a bioinfor-
matics 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. stra-
monium 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 dif-
fered from those of Solanaceous TRIs (Brock et al., 2008; Chen
et al., 2013).
A 3-dimensional (3D) molecular model of BaTRI was con-
structed by using the DsTRI crystal structure (1ae1. pdb) as a tem-
plate 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 corre-
sponding positions of BaTRI (Fig. S1. 1A and B). An alignment anal-
ysis 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 bacte-
ria was collected. The enzyme assay demonstrated that the super-
natant 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 exhib-
ited 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, Solana-
ceae 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 5

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.

As expected, BaTRI efficiently catalysed the reverse reaction, i.e.,

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 offic-
inalis (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.
the oxidation of tropine (6) to tropinone (5), within a narrow alka-
line pH range. The optimum pH was 9.6, which agreed with that
(pH 9.9) reported for the tropine-oxidation activity of the TRI iso-
lated from D. stramonium (Portsteffen et al., 1994). The high alka-
line 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 opti-
mum pH values were not determined. Unlike TRI from D. stramo-
nium, 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 tro-
pine (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 recapitu-
lated 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 reac-
tion 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 recom-
binant BaTRI was incubated with NADPH and each of the three dif-
ferent 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
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6 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx

and tropine (6) at the optimum pH of 9.6. The Michaelis–Menten
curves of the enzyme for the four substrates were drawn to deter-
mine the Km values and Vmax values of the enzyme for the sub-
strates (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-quinu-
clidinone hydrochloride but significantly slower and weaker than
those of 4-methylcyclohexanone. The Kcat values and Kcat/Km val-
ues 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 val-
ues (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 condi-
tions, 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) exhi-
bit higher reduction velocities for monocyclic ketones than for bi-
cyclic substrates. Solanaceae TRs, particularly pseudotropine-form-
ing reductases (Drager, 2006), and BaTRI also exhibit this property.
At the assay pH of 6.4, the non-ionizable substrate 4-methylcyclo-
hexanone is uncharged, whereas the nitrogen-containing sub-
strates tropinone (5) and 3-quinuclidinone hydrochloride are
positively charged. Thus, as expected, BaTRI exhibited significantly
higher apparent affinity for 4-methylcyclohexanone than tropi-
none (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 rela-
tionship 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. Further-
more, 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 experimen-
tal 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 cat-
alytic 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
 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx 7

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) 6.4 2.65 ± 0.19 88.30 ± 1.81 2.93 ± 0.06 1105.66
4-methylcyclohexanone 6.4 0.46 ± 0.04 140.91 ± 7.23 4.67 ± 0.24 10152.17
3-quinuclidinone hydrochloride 6.4 5.17 ± 0.29 16.26 ± 0.54 0.54 ± 0.02 104.45
Tropine (6) 9.6 0.56 ± 0.04 171.62 ± 5.42 5.69 ± 0.18 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 Km (mM) Vmax Kcat (s
1) Kcat/Km

assay (nkat mg
1 (s
1 M
1)
protein)
Tropinone 6.4 4.18 ± 0.51 81.20 ± 3.61 2.40 ± 0.11 574.16
(5)
Tropine 9.6 0.34 ± 0.05 111.90 ± 5.41 3.30 ± 0.16 9705.88
(6)

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

tissues of B. arborea.

Herbal plant species, such as A. belladona, D. metel and H. niger, bio-

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 hyos-
cyamine (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 differ-
ent organs, including primary roots, secondary roots, mature
stems, young stems, mature leaves and young leaves, using real-
time 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 pri-
mary 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.
logically 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 inde-
pendent. 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 synthe-
sized 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). Scopo-
lamine (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), fol-
lowed 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
8 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx
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) con-
tent 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 con-
centration 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 hyoscya-
mine (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 pre-
cursor 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)/hyoscya-
mine (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 tis-
sue-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 biosynthe-
sis 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
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
cells of A. belladonna (Suzuki et al., 1999). Our results are in con-
trast 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 hyoscya-
mine (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 catal-
ysed from hyoscyamine that has accumulated in aerial parts of the
plant, especially the leaves.
The biosynthesis of tropane alkaloids in the roots and subse-
quent 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 biosyn-
thetic 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 sug-
gested 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 indepen-
dent 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 syn-
thesize 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 puta-
tive 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 Solana-
ceae 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 encod-
ing 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 hyoscya-
mine (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).
 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx
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, includ-
ing 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 liq-
uid 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 single-
stranded 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 -ATGGAAGAATCAAAAGTGTCC-
30 ) 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 -GCCACCAGTAAC-
TAGGGCT-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 -CYTTTCTTTGCTTCCCTGCT-
30 ) for the nested amplification; reverse, the universal primers
(UPM and NUP) provided by the kit. The first and nested PCR pro-
cedures 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 -CCCATCCCAAAA-
TAGTTG-30 ) as the forward primer, R-BaTRI-PRO (50 -CGAGCTCGT-
GATGATAATAACAGAGAAC-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 con-
structed 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 -CGGATCCATGGAAGAAT-
CAAAAGTGTCC-30 ) with BamH I and R-BaTRI-PRO (50 -CGAGCTCGT-
GATGATAATAACAGAGAAC-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,
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
9
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 (Trans-
Gen Biotech, Beijing, China). The enzyme activity of eluted frac-
tions was screened, and catalytically active fractions were
pooled. To remove imidazole, dialysis was performed using a dial-
ysis 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 -CGGATCCATGGAA-
GAATCAAAAGTGTCC-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 recombi-
nation 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 pro-
tein 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 sys-
tem, 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 tro-
pinone 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 neg-
ative 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 calcu-
lated with a non-linear regression of the Michaelis–Menten equa-
tion 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 spectrophotome-
ter, 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 fol-
lowing 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,
10 W. Qiang et al. / Phytochemistry xxx (2016) xxx–xxx
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 -CAGATACCGTCC-
TAGTCTCAAC-30 ) and 18SR (50 -CAGCCTTGCGACCATACTC-30 ) were
used as primers; for PGK, F-PGK (50 -TCGCTCTTGGAGAAGGTTGAC-
30 ) 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), aniso-
damine (8) and scopolamine (9) were purchased from Sigma–
Aldrich (Sigma, LA, USA). The HPLC system was a Shimadzu LC-
20A instrument, and the detector was the photo-diode array. The
detecting wavelength was 226 nm. The temperature of the CTP-
ODS 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 Pro-
gram for New Century Excellent Talents in University (NCET-12-
0930) and the Fundamental Research Funds for the Central Univer-
sities (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.
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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