Biosynthesis of cocaine in Erythroxylum coca

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The first step in the biosynthesis of cocaine in Erythroxylum coca: The
characterization of arginine and ornithine decarboxylases
Article  in  Plant Molecular Biology · February 2012
DOI: 10.1007/s11103-012-9886-1 · Source: PubMed
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The first step in the biosynthesis of cocaine in Erythroxylum coca:
the characterization of arginine and ornithine decarboxylases
Teresa Docimo • Michael Reichelt • Bernd Schneider •
Marco Kai • Grit Kunert • Jonathan Gershenzon •
John C. D’Auria
Received: 24 June 2011 / Accepted: 19 January 2012 / Published online: 7 February 2012
� Springer Science+Business Media B.V. 2012
Abstract Despite the long history of cocaine use among
humans and its social and economic significance today, little
information is available about the biochemical and molecular
aspects of cocaine biosynthesis in coca (Erythroxylum coca) in
comparison to what is known about the formation of other
pharmacologically-important tropane alkaloids in species of
the Solanaceae. In this work, we investigated the site of cocaine
biosynthesis in E. coca and the nature of the first step. The two
principal tropane alkaloids of E. coca, cocaine and cinnamoyl
cocaine, were present in highest concentrations in buds and
rolled leaves. These are also the organs in which the rate of
alkaloid biosynthesis was the highest based on the incorpora-
tion of 13CO2. In contrast, tropane alkaloids in the Solanaceae
are biosynthesized in the roots and translocated to the leaves. A
collection of EST sequences from a cDNA library made from
young E. coca leaves was employed to search for genes
encoding thefirst step in tropane alkaloid biosynthesis. Full-
length cDNA clones were identified encoding two candidate
enzymes, ornithine decarboxylase (ODC) and arginine
decarboxylase (ADC), and the enzymatic activities of the
corresponding proteins confirmed by heterologous expression
in E. coli and complementation of a yeast mutant. The tran-
script levels of both ODC and ADC genes were highest in buds
and rolled leaves and lower in other organs. The levels of both
ornithine and arginine themselves showed a similar pattern, so
it was not possible to assign a preferential role in cocaine
biosynthesis to one of these proteins.
Keywords Erythroxylum coca � Tropane alkaloids �
Cocaine � Ornithine and arginine decarboxylases
Abbreviations
qRT–PCR Quantitative reverse transcriptase–
polymerase chain reaction
LC–MS Liquid chromatography–mass
spectrometry
ESTs Expressed sequence tags
NMR Nuclear magnetic resonance
GC–MS Gas chromatography–mass
spectrometry
HPLC–UV–DAD High performance liquid
chromatography–ultraviolet–diode
array detection
HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid)
ESI Electrospray ionization
Introduction
Tropane alkaloids (TAs) are pharmacologically-active
plant metabolites that are widely distributed among
higher plants. Most often described from the Solanaceae,
they are also known in the Euphorbiaceae, Proteaceae,
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-012-9886-1) contains supplementary
material, which is available to authorized users.
T. Docimo � M. Reichelt � G. Kunert � J. Gershenzon �
J. C. D’Auria (&)
Department of Biochemistry, Max Planck Institute for Chemical
Ecology, 07745 Jena, Germany
e-mail: dauria@ice.mpg.de
B. Schneider
NMR Research Group, Max Planck Institute for Chemical
Ecology, Jena, Germany
M. Kai
Mass Spectrometry Research Group, Max Planck Institute for
Chemical Ecology, Beutenberg Campus Hans-Knöll-Straße 8,
07745 Jena, Germany
123
Plant Mol Biol (2012) 78:599–615
DOI 10.1007/s11103-012-9886-1
http://dx.doi.org/10.1007/s11103-012-9886-1
Convolvulaceae, Brassicaceae, Rhizophoraceae and Ery-
throxylaceae (Griffin and Lin 2000). All TAs are derived
from L-ornithine or L-arginine (Dewick 2002) and share an
8-methyl-8-azabicyclo[3.2.1]octane nucleus, widely refer-
red to as a tropane ring. A diverse array of variations
occurs on the core ring structure, such as alkylation,
acylation and hydroxylation. Currently, almost 150 differ-
ent tropane alkaloids (TA) have been identified (Bruce
2008) and examples of well known TAs include atropine
and the calystegines. Atropine and the structurally similar
hyoscyamine and scopolamine are anticholinergic sub-
stances used widely in pharmacy. They occur in several
species of Atropa, Hyoscyamus, and Datura. The calyste-
gines are a class of nortropane alkaloids which are more
widespread than either hyoscyamine or scopolamine and
are found predominantly in the Solanaceae and Convol-
vulaceae. Structurally similar to monosaccharides, caly-
stegines are glycosidase inhibitors that may have potential
for treating diseases such as HIV (Asano 2003).
The narcotic cocaine is the main tropane alkaloid found
in Erythroxylum coca (coca) plants. Coca is an ancient
medicinal plant, the leaves of which have been chewed by
South American Indians for thousands of years during
times of great physical strain or in the case of illness. Coca
leaves are still widely used by indigenous peoples (Plow-
man 1982). As a TA, cocaine is a catecholamine re-uptake
inhibitor and after its discovery was quickly adopted for
use as an anesthetic, especially in odontoiatry (Ruetsch
et al. 2001). However, dramatic side effects, such as itch-
ing, tachycardia and hallucinations, accompanied by a
strong physical and mental addiction led to the replacement
of cocaine with safer semi-synthetic drugs. Today, cocaine
is notorious as being one of the most ‘‘abused’’ recreational
drugs of the modern era. This molecule is the primary
alkaloid produced in cultivated Erythroxylum species, such
as E. coca and E. novogranatense and seems to be
restricted to members of the genus. There are approxi-
mately 230 species described for Erythroxylum (family
Erythroxylaceae) which range throughout the tropics,
including South America and Madagascar (Griffin and Lin
2000). The cocaine content of cultivated Erythroxylum
species ranges from 0.2 to 1% of the leaf dry weight,
whereas closely related wild species contain no more than
0.01% (Plowman and Rivier 1983).
The biosynthesis of cocaine and other TAs has been the
subject of much study. Most of the early investigations
utilized in vivo feeding of radiolabeled precursors in the
biosynthesis of the core tropane ring. These studies were
generally performed by injection of TA-producing plants
with precursors or immersion in solution. In the first suc-
cessful experiments, labeled compounds were fed to plants
such as Datura stramonium and Atropa belladonna
(Ahmad and Leete 1970; Mizusaki et al. 1968). However,
these feeding strategies were not effective in E. coca plants
suggesting that TA biosynthesis might be operating in a
different way in Erythroxylum than in Solanaceae species
(Leete 1980). Alternatively, by painting leaves with radio-
labeled precursors instead of feeding through cuttings or
through the roots, Leete showed that the tropane backbone
in E. coca is most likely formed according to the same
scheme as had been previously described for plants of the
Solanaceae, although this has not yet been confirmed by
any modern enzymological or molecular investigations.
The hypothetical pathway leading to cocaine as sug-
gested by Leete and others (Hoye et al. 2000; Leete 1980)
begins from the polyamine putrescine, which is formed
from the amino acids L-arginine or L-ornithine (Fig. 1).
Putrescine is mono-methylated to afford N-methylputres-
cine. Then, oxidation of the primary amino group yields
4-(methyl-1-amino)butanal, the cyclic form of which is the
N-methyl D1-pyrrolinium salt. Condensation with aceto-
acetate (possibly as the thioester with coenzyme A) leads to
a thioester of 4-(l-methyl-2-pyrrolidinyl)-3-oxobutanoic
acid, then oxidation yields a new iminium salt which then
undergoes another aldol condensation to yield a product
with a bicyclic tropane ring system. After CoA hydrolysis
and methyl ester formation, the compound methylecgonone
(2-carbomethoxy-3-tropinone) is produced, which is a
direct precursor of cocaine (Leete et al. 1991). Subsequent
steps presumably include the reduction of methylecgonone
to methylecgonine and then benzoylation via benzoyl-CoA
to yield cocaine (Bjorklund and Leete 1992) (Fig. 1).
To investigate cocaine biosynthesis in Erythroxylum, we
have begun by focusing our attention on the early steps of
the pathway since these may be especially important in
regulating the rate of this process. According to Leete
(Leete 1982), the amino acid L-ornithine is the direct pre-
cursor of putrescine and the incorporation of this poly-
amine into the tropane ring in E. coca proceeds via a
symmetrical intermediate. While L-arginine potentially
meets these same requirements, experimental evidence
obtained by feeding radio-labeled [5-14C]ornithine to
E. coca plants suggested that arginine does not play a
critical role in TA biosynthesis (Leete 1980; Leete et al.
1954) contrary to what is known for plants in the Solana-
ceae (Robins et al. 1991; Tiburcio and Galston 1986).
Putrescine is ubiquitous in plants as a critical component
of primary metabolism (Tiburcio et al. 1997). In most
plants, putrescine is synthesized from ornithine via orni-
thine decarboxylase (ODC) except for the model plant
A. thaliana which does not seem to contain a gene
encoding ODC in its genome (Hanfrey et al. 2001). An
alternate route to putrescine which involves arginine
decarboxylation via arginine decarboxylase (ADC) has
been identified(Kakkar and Sawhney 2002; Malmberg
et al. 1998). Biochemical studies have suggested that the
600 Plant Mol Biol (2012) 78:599–615
123
ODC-mediated route to putrescine is particularly important
in providing polyamines for normal cellular division, dif-
ferentiation, and development. On the other hand, putres-
cine synthesized via the ADC-route is considered to be
necessary for cell expansion and environmental stress
responses (Malmberg et al. 1998). Whether arginine or
ornithine is the preferential route to putrescine in tobacco
and other alkaloid-producing plants, has been the subject of
debate (Chintapakorn and Hamill 2007). Controversial
results about the function of ODC and ADC in root cultures
have been reported using difluoromethylornithine (DFMO)
and difluoromethylarginine (DFMA) as suicide inhibitors
of ODC and ADC, respectively. The biochemical inter-
conversion of ornithine into arginine and vice versa via the
urea cycle makes the interpretation of some of these results
unclear. Experiments with Heliotropium angiospermum
shoots (Birecka et al. 1987) and Senecio vulgaris root
cultures (Hartmann et al. 1988) have shown that L-arginine
is the only source of putrescine for pyrrolizidine alkaloid
formation. Consistent with this, DFMO and DFMA
experiments on transgenic D. stramonium root culture
confirmed that the L-arginine/agmatine/N-carbamoylpu-
trescine/putrescine pathway (involving ADC) is more
important than the L-ornithine/putrescine pathway
(involving ODC) for putrescine formation (Robins et al.
1990). Using suicide inhibitors combined with labeling
experiments, Tiburcio and co-workers (Tiburcio et al.
1991) demonstrated that in Nicotiana tabacum cultures
L-arginine was also the principal source of putrescine for
tobacco alkaloids. However, more recently, the antisense
mediated reduction of arginine decarboxylase activity in
N. tabacum was shown to cause only minimal variation in the
overall alkaloid profile (Chintapakorn and Hamill 2007).
The tissue localization of ornithine and arginine decar-
boxylase suggested that ODC may be the main enzyme
responsible for the synthesis of putrescine in plant roots,
whereas in photosynthetic tissues, such as leaves, putres-
cine is mainly synthesized via ADC (Borrell et al. 1995).
Since to date all precursors for tropane alkaloids have been
reported to be synthesized in the root and then translocated
to the aerial part of the plants (Flores and Martin-Tanguy
1991), it may be surmised that ODC and not ADC is likely
to play the major role in the biosynthesis of putrescine-
derived alkaloids. However, this issue requires further
investigation.
Considering that tropane alkaloids are scattered in
diverse families of flowering plants, they could well be
biosynthesized in different ways. The last common
ancestor shared between tropane producing plant families
(Brassicaceae, Convolvulaceae, Erythroxylaceae, Euphor-
biaceae, Proteaceae, Rhizophoraceae and Solanaceae) is
believed to have lived between 108 and 146 million years
ago (Soltis et al. 2008). Since this ancestor also gave rise to
many plant families that are devoid of tropane alkaloids,
two theories on the evolution of the tropane alkaloid
pathway in different families can be proposed. One possi-
bility is that tropane alkaloid biosynthesis evolved once
and was subsequently lost many times. The second
Fig. 1 Hypothetical pathway of tropane alkaloid biosynthesis in
Erythroxylum leading to the production of cocaine, based on previous
findings predominantly from Solanaceae species. ODC ornithine
decarboxylase, ADC arginine decarboxylase, PMT putrescine meth-
yltransferase, DAO diamino oxidase
Plant Mol Biol (2012) 78:599–615 601
123
hypothesis is that tropane biosynthesis evolved repeatedly
and independently. Since the Erythroxylaceae, Brassica-
ceae and Rhizophoraceae share a common ancestor which
gave rise to the Malvids and Fabids about 83–107 million
years ago, a third option may exist in which tropane
alkaloid biosynthesis may have evolved more than once,
but not independently in each family in which it occurs
(Wang et al. 2009). Which of these theories is correct can
only be shown once the molecular mechanisms and
enzymes involved in tropane biosynthesis have been elu-
cidated in all of these families. So far, research on tropane
biosynthesis on an enzymatic level has been performed
mostly in the Solanaceae with a few reports having recently
been reported from members of the Brassicaceae. There-
fore, a major motivation of this research was not only to
determine the pathway of cocaine biosynthesis in E. coca,
but also to understand whether the TA biosynthetic path-
way is similar in different lineages.
In the present study, we report the distribution of
cocaine and some of its possible precursors in E. coca in
different organs and different stages of leaf development to
determine the site of its biosynthesis. In addition, we per-
formed a biochemical and molecular study of the two
decarboxylase enzymes, ODC and ADC, proposed to form
the first biosynthetic intermediate, putrescine. These results
suggest that tropane alkaloid biosynthesis may indeed have
evolved on multiple occasions.
Results
Content of tropane alkaloids and potential precursors
varies with organ and leaf development
In order to learn about the sites of tropane alkaloid biosyn-
thesis in E. coca and which substrates might be involved, we
determined the distribution of cocaine and potential precur-
sors in different organs, including buds, leaves, stems and
roots. Leaves were sampled at three developmental stages
(Supplementary Material Fig 1). Analysis was conducted on
6 month old E. coca plants grown in a greenhouse, and the
distribution and the accumulation of tropane alkaloids, amino
acids, and polyamines were measured by HPLC. In addition,
the metabolite composition of stems and roots was also
determined. An HPLC–UV quantitative analysis of the major
tropane alkaloids revealed that the amount of cocaine (ben-
zoylmethylecgonine) increased during leaf development,
reaching a maximum concentration of 32.53 nmol mg-1 of
dry weight in the mature leaves. Since cocaine is accompa-
nied by cinnamoyl cocaine (cinnamoylmethylecgonine) in
most of the cultivated coca species (Johnson and Emche
1994; Plowman 1982), we also measured the concentration
of the (E)- and (Z)-cinnamoyl cocaine isomers. The
concentration of (E)-cinnamoyl cocaine is highest in the buds
(146.89 nmol mg-1 Dw), where the amount is approxi-
mately 10 times higher than cocaine (15.25 nmol mg-1 Dw)
while a maximun of 10.48 nmol mg-1 Dw (Z)-cinnamoyl
cocaine was detected in stage 2 leaves. Neither cocaine nor
(E)-cinnamoyl cocaine were found in the roots (Fig. 2).
Amino acid distribution was also studied for the same
samples. An LC–MS triple quad (triple quadrupole mass
spectrometer) quantitative analysis showed that there is a
generally higher accumulation of amino acids in the shoot
organs compared to the roots and that accumulation often
decreases with leaf development (Table 1). The two pre-
cursors of putrescine, arginine and ornithine, also followed
this pattern. For example, the amount of arginine decreased
from 15.31 and 23.41 nmol mg-1 dry weight in buds and
youngest leaves, respectively, to 4.08 nmol mg-1 dry
weight in mature leaves. Ornithine levels were very low in
comparison to the other quantified amino acids, being
nearly 100 times lower than arginine.
While putrescine is thought to be the entry point into the
TA biosynthetic pathway, other polyamines derived from
putrescine can in theory also provide the basic structure of
the tropane core (Leete 1985). We tried to quantify
spermine and spermidine, but their levels in the plant were
undetectable in all organs examined. However, putrescine
was detected in all organs with the lowest levels in the buds
(0.47 nmol mg-1 Dw) and over fivefold higher concen-
trationsin the last two leaf stages (Fig. 3). No evidence
was found for the presence of putrescine, spermine or
spermidine conjugates which were sought by both LC–MS
and HPLC coupled with fluorescence detection (see
‘‘Materials and methods’’).
0
20
40
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120
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180
Cocaine 
(E)-cinnamoyl cocaine 
(Z )-cinnamoyl cocaine 
n
m
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l (
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g
D
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1
*
Buds 3 Stem1 2 Roots
 Leaf stage
Fig. 2 HPLC–UV analysis of tropane alkaloids in E. coca tissues.
Cocaine and cinnamoyl cocaine were extracted from 100 mg of fresh
tissue in 50% MeOH and quantified by HPLC–UV. Cocaine was
measured at 235 nm and cinnamoyl cocaine, both (E)- and (Z)-
isomers, were measured at 280 nm. Values presented are
means ± SD (n = 3). HCompounds were not detected
602 Plant Mol Biol (2012) 78:599–615
123
Tropane alkaloid biosynthesis occurs primarily in buds
and young leaves
To determine the site of cocaine biosynthesis, E. coca
plants were pulsed with 13CO2 for 2 days and the distri-
bution of 13C-labeled tropane alkaloids was monitored in
different organs after 3 days of chase with 12CO2. Incor-
poration of 13C-label into methylecgonine and derivatives
(cocaine and cinnamoyl cocaine), as measured by their
molecular ion clusters in LC–MS spectra, was predomi-
nantly observed in the buds, younger leaf stages and in the
stem, but not in mature leaves (Table 2). Measurement of
the fragment peak at m/z 182, which corresponds to the
core tropane ring after ester cleavage, indicated that the
tropane ring itself is labeled (see Supplementary Material
Fig 2). These results suggested that the young stages of the
leaves are the active site of tropane alkaloid biosynthesis in
E. coca, a result very different from that reported for tro-
pane alkaloid production in Solanaceae species, where
roots are the major site of synthesis (Biastoff et al. 2009;
Hu and Du 2006; Palazon et al. 2008; Ziegler and Facchini
2008).
To determine if the ester side chains of cocaine and
cinnamoyl cocaine were biosynthesized in a different time
or place than the tropane ring, we compared the labeling
of the side chains in buds and young leaves with that of
the tropane ring by mass spectrometry and NMR. Label
from [U-13C]glucose was incorporated both into the core
tropane ring (Supplementary Material Fig. 2) and into the
cinnamic acid moiety of cinnamoyl cocaine (Supplemen-
tary Material Fig. 7). It was not possible to detect labeling
of the benzoic acid moiety of cocaine due to the low
Table 1 Amino acid content (nmol mg-1 Dw) in E. coca buds, leaves, stems and roots
AA Buds Leaves
stage #1
Leaves
stage #2
Leaves
stage #3
Stem Root
Ala 7.16 ± 1.65 7.36 ± 1.57 6.87 ± 0.42 5.79 ± 1.38 4.91 ± 0.72 4.39 ± 1.05
Arg 15.30 ± 4.40 23.41 ± 14.78 7.09 ± 2.34 4.08 ± 1.99 107.19 ± 76.39 9.02 ± 4.28
Asn 0.67 ± ±0.30 0.47 ± 0.07 0.38 ± 0.09 0.26 ± 0.09 1.42 ± 0.38 1.64 ± 1.43
Asp 11.03 ± 2.98 12.90 ± 2.18 14.05 ± 3.03 16.22 ± 1.41 12.76 ± 1.06 4.76 ± 0.92
Gln 50.84 ± 5.63 52.54 ± 9.77 48.86 ± 24.07 14.84 ± 4.47 330.00 ± 165.76 68.81 ± 28.09
Glu 46.32 ± 3.82 51.41 ± 3.54 48.41 ± 8.20 42.24 ± 4.89 47.65 ± 5.35 15.07 ± 2.61
Gly 3.49 ± 0.96 3.45 ± 0.44 3.17 ± 1.11 1.22 ± 0.22 1.59 ± 0.43 2.21 ± 0.82
His 0.50 ± 0.16 0.50 ± 0.24 0.42 ± 0.14 0.53 ± 0.17 1.58 ± 0.57 0.73 ± 0.26
Ile 0.34 ± 0.06 0.32 ± 0.02 0.37 ± 0.13 0.24 ± 0.02 0.20 ± 0.02 0.26 ± 0.05
Leu 0.21 ± 0.05 0.24 ± 0.03 0.25 ± 0.13 0.14 ± 0.05 0.13 ± 0.02 0.20 ± 0.03
Lys 0.38 ± 0.15 0.44 ± 0.08 0.38 ± 0.04 0.34 ± 0.07 0.57 ± 0.14 0.42 ± 0.16
Met N.D* N.D* N.D* N.D* N.D* N.D*
Orn 0.17 ± 0.07 0.15 ± 0.03 0.09 ± 0.02 0.10 ± 0.04 0.56 ± 0.20 0.34 ± 0.13
Phe 0.35 ± 0.09 0.34 ± 0.03 0.33 ± 0.05 0.20 ± 0.02 0.16 ± 0.03 0.20 ± 0.03
Pro 3.09 ± 0.98 7.65 ± 4.98 16.58 ± 5.86 2.57 ± 1.43 1.34 ± 0.41 0.62 ± 0.15
Ser 9.85 ± 2.20 10.13 ± 1.47 11.35 ± 2.40 13.97 ± 1.75 11.17 ± 1.11 5.54 ± 0.66
Thr 3.00 ± 0.34 2.72 ± 0.40 2.25 ± 0.20 1.70 ± 0.22 2.28 ± 0.33 1.57 ± 0.48
Trp 0.23 ± 0.02 0.15 ± 0.01 0.18 ± 0.04 0.10 ± 0.02 0.12 ± 0.03 0.78 ± 0.09
Tyr 0.09 ± 0.03 0.09 ± 0.01 0.07 ± 0.01 0.05 ± 0.01 0.07 ± 0.02 0.18 ± 0.05
Val 0.36 ± 0.05 0.36 ± 0.04 0.30 ± 0.04 0.28 ± 0.03 0.34 ± 0.03 0.36 ± 0.08
* ND indicates not detectable
0
1
2
3
4
5
6
Putrescine
n
m
o
l (
m
g
 D
w
)-1
Buds 1 2 Stem3 Roots
Leaf stage
Fig. 3 HPLC with fluorescence detection was employed to determine
the content of polyamine putrescine, in various E. coca leaf stages and
organs. Polyamines were extracted in MeOH and quantified as OPA–
FMOC derivatives. For details see ‘‘Materials and methods’’ section.
Concentrations were calculated by calibration with the relative
standard curve. Values presented are means ± SD (n = 3)
Plant Mol Biol (2012) 78:599–615 603
123
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604 Plant Mol Biol (2012) 78:599–615
123
amount present, but another benzoate derivative in the
same sample, methyl salicylate, was well-labeled (Sup-
plementary Material Fig. 8) indicating an active formation
of benzoic acid metabolites in young leaf stages. Thus
there is no evidence for any partitioning of tropane alka-
loid biosynthesis in E. coca, in which the tropane ring and
side chain moieties are formed at different sites and then
assembled.
Isolation of ornithine decarboxylase (ODC)
and arginine decarboxylase (ADC) genes
Since the results of the metabolite analysis and the 13C
feeding studies pointed to young leaves as the organ con-
taining the highest biosynthetic activity, a kZAPII cDNA
library was constructed from rolled and expanding leaves
to search for biosynthetic genes and 8,000 clones were
sequenced. The ESTs were classified into different groups
according to the type of gene represented (see Supple-
mentary Material Fig. 3). Since the genes encoding the two
proposed first steps in TA biosynthesis, ornithine decar-
boxylase and arginine decarboxylase, are conserved in
many plant species, we searched our EST collection using
the BLAST algorithm (Altschul and Lipman 1990) for
sequences similar to ODC and ADC from the Solanaceae.
The search identified one cDNA corresponding to a puta-
tive ODC and one corresponding to a putative ADC.
The full length clone for ODC was isolated by using
N. tabacum ODC (AAK13622) as a probe. The complete
cDNA has an open reading frame that encodes a protein of
413 AA with a calculated molecular mass of 44.6 kD. The
protein sequence comparison with plant bacterial and yeast
ODC proteins indicated that E. coca ODC is most similar to
ODCs of Populus trichocarpa (79% AA identity) and The-
obroma cacao (77% AA identity) and also shares similarity
with ODCs of N. tabacum, Lycopersicum esculentum,
D. stramonium, Ricinus communis and Glycine max (68, 68,
68, 61 and 61 identity, respectively, while only sharing low
similarity with ODCs of E. coli and Saccharomyces cere-
visiae (18–25 and 43% AA identity) (Fig. 4a). The proteins
encoded by these genes all share the particular features of
other plant ODC decarboxylases, as revealed by Prosite
Scan (http://www.expasy.ch/tools/scanprosite/). E. coca
ODC contains the Orn/DAP/Arg decarboxylase family 2
pyridoxal-phosphate attachment site (PFYAVKCNPE-
PALLGS, positions 162–177) as well as the Orn/DAP/
Arg decarboxylase family 2 signature 2 sequence
(GMPRMTMLNIGGGFT, positions 337–352) (Poulin et al.
1992)(Fig. 4a).
The EcADC cDNA yielded an open reading frame
encoding a protein with 679 AA and calculated molecular
mass of 72.9 kD. Protein sequence comparisons were
similar to those found for EcODC, with the highest
similarity shown to an ADC of Populus trichocarpa (81%)
(Fig. 4c). The putative EcADC sequence shares similarity
on the amino acid level with ADCs from Nicotiana taba-
cum, Pisum sativum, L. esculentum, D. stramonium,
Brassica juncea and Arabidopsis thaliana and E. coli (75,
73, 73, 75, 71 and 72 and 36% identity, respectively).
EcADC also contains the Orn/DAP/Arg decarboxylase
family 2 pyridoxal-phosphate attachment site (YPVKCNQ
DRYVVEDIVKFG, positions 164–182) and the Orn/DAP/
Arg decarboxylase family 2 signature 2 sequence (VRL
GANMQVIDIGGGLG, positions 342–358)(Akiyama and
Jin 2007) (Fig. 4c).
To analyze the phylogenetic relationship between ODC
and ADC from E. coca and related decarboxylases from
other plants and organisms like E. coli and S. cerevisiae,
protein sequences were subjected to neighbor joining
analysis and phylogenetic trees were constructed based on
the results of the analyses. Figure 4b and d show the
relatedness of E. coca ODC and ADC with those from
other plants, E. coli and S. cerevisiae. The E. coca proteins
are most closely related to those of P. trichocarpa, and
most distantly related to the enzymes from either E.coli or
S. cerevisiae. Sequence analysis via iPSORT indicated that
EcADC has a putative chloroplast transit peptide at the
N-terminus, consistent with other known ADC enzymes
(Bannai et al. 2002; Borrell et al. 1995; Bortolotti et al.
2004).
Enzymatic activity of the heterologously expressed
ODC and ADC
To determine whether the isolated E. coca cDNAs encoded
proteins with genuine ODC and ADC activities, the com-
plete open reading frames of E. coca ODC and ADC were
heterologously expressed in BL21 E. coli cells using the
expression vector pET32. The recombinant ODC was
purified using Ni-chelating resin and the introduced thio-
redoxin encoding portion was cleaved using thrombin (see
‘‘Materials and methods’’). The resulting purified protein
was shown to exhibit ornithine decarboxylase activity by
converting ornithine to putrescine (Supplementary Fig 4).
The empty vector pET32 was used as control, and no
putrescine was formed in this assay. No decarboxylase
activity was detected for other substrates tested, including
arginine and lysine (Supplementary Material Fig 4). A
known suicide inhibitor of ornithine decarboxylase, DFMO
(Hao et al. 2005) completely blocked putrescine formation.
Maximum E. coca ODC activity was found at pH 8 and
a temperature of 28�C. None of the monovalent and
bivalent ions tested showed any stimulatory effects (Sup-
plementary Material Fig 5). The kinetic parameters of
E. coca ODC determined in the presence of 1 mM pyri-
doxal phosphate (PLP) as a cofactor showed a lower Km
Plant Mol Biol (2012) 78:599–615 605
123
http://www.expasy.ch/tools/scanprosite/
(395.3 lM) than that reported previously for Nicotiana
glutinosa ODC (Lee and Cho 2001a, b) and a higher kcat
indicating that the E. coca ODC is almost 20 times more
efficient (Table 3).
Heterologously expressed E. coca ADC had a low
affinity for the Ni-chelating resin used in purification most
likely due to the steric influence of the 15 kD thioredoxin
fusion protein on the His tag. Despite these difficulties in
Fig. 4 Alignments of predicted E. coca ODC and ADC sequences
with those from other species and organisms and their phylogenetic
relationships. a The predicted amino acid sequence of ODC from
E. coca was aligned with plant, bacterial and yeast ODC sequences
using the ClustalW and Bioedit sequence alignment programs.
Identical amino acids are highlighted with black boxes. The motif
(PFYAVKCNPEPALLGS) binding the cofactor, pyridoxal 50-phos-
phate (PLP), in E. coca ODC is indicated as motif 1. The
decarboxylase motif (GMPRMTMLNIGGGFT) is marked as motif
2. These motifs are conserved in all ODC plant sequences. GenBank
accession numbers are as follows: Erythroxylum coca (JF909554)
Populus trichocarpa (EEE94752) Theobroma cacao (EF122792).
Lycopersicon esculentum (AAB82301), Datura stramonium
(CAA61121), Nicotiana tabacum (AAK13622), and Glycine max
(CAD91350), Ricinus communis (EEF52797), E. coli (BAE77028.1)
and Saccaromyces cerevisiae (DAA08982). b The phylogenetic tree
was constructed from ODC amino acid sequences by the neighbor-
joining method of ClustalW and TreeView. Numbers at branch points
indicate bootstrap values (1,000 replicates) c the predicted amino acid
sequence of ADC from E. coca was aligned with plant sequences and
the E. coli sequence using theClustalW and Bioedit sequence
alignment programs. Identical amino acids are highlighted with black
boxes. The motif (YPVKCNQDRYVVEDIVKFG) binding the
cofactor, pyridoxal 50-phosphate (PLP), in E. coca ADC is indicated
as motif 1. In addition, the decarboxylase motif (VRLGANMQVI-
DIGGGLG) is marked as motif 2. These motifs are conserved in all
ADC sequences. Gen Bank accession numbers are as follows: Erythr-
oxylum coca (JF909553), Populus trichocarpa (XP_002306141), Nico-
tiana tabacum (AAQ14851), Datura stramonium (CAB64599),
Lycopersicum esculentum (CAI39242), P. sativum (Q43075), Arabidop-
sis thaliana (AAB09723), B. juncea (AAF26434) and E. coli.
(BAE77001) d the phylogenetic tree of ADC sequences was constructed
as above for ODC sequences. Numbers at branch points indicate
bootstrap values (1,000 replicates)
606 Plant Mol Biol (2012) 78:599–615
123
purification, the fusion protein successfully displayed
arginine decarboxylase activity using arginine as substrate,
and the empty vector and boiled enzyme controls showed
no ADC activity (Supplementary Material Fig 6).
Complementation of a yeast ODC mutant by E. coca
ODC
To confirm the catalytic function of the E. coca ODC, we
tested whether it was able to complement a yeast strain
mutated in ODC. The Saccharomyces cerevisiae spe1D
mutant (a met15 ura3 leu2 his3 spe1D:: G418) is defective
for the ODC gene and thus not able to grow in the absence
of polyamines. We cloned EcODC into the pYES52 yeast
vector and used it to transform the spe1D ODC mutant.
Controls included spe1D transformed with empty pYES52
vector and a spe1D strain grown on a medium without
polyamines to deplete its intracellular stores (McNemar
et al. 1997). The mutant spe1D transformed with ODC
from E. coca completely recovered a wild-type phenotype
on polyamine-free medium. The empty vector transformant
also showed visible growth, while spe1D depleted of
internal polyamine stores was unable to grow. In liquid
culture (Fig. 5), the spe1D mutant complemented with
ODC grew exponentially much better than when trans-
formed with the empty vector, while the depleted,
untransformed mutant was not able to grow at all. Analysis
of the polyamine content in the three lines by HPLC with
fluorescence detection revealed that the ODC transformant
of spe1D had four times more spermidine (no other
Fig. 4 continued
Table 3 Kinetic properties of E. coca ornithine decarboxylase (ODC) compared to ODC of N. glutinosa
Enzyme Km (lM) Vmax (nmol/min/mg protein) kcat (s
-1) kcat/Km (M
-1 s-1) Optimal pH
E. coca ODC 395.3 ± 110.20 248.20 ± 30.25 0.18 ± 0.02 465.85 8.0
N. glutinosa ODC 562 ± 61 12.54 ± 1.0 0.93 9 10-2 ± 0.7 9 10-3 16.53 8.0
Plant Mol Biol (2012) 78:599–615 607
123
polyamine was detected) than the empty vector trans-
formant and 80 times more spermidine than the untrans-
formed spe1D (Table 4).
Variation in ODC and ADC transcript levels
among organs and developmental stages
To determine whether ODC and ADC are associated with
cocaine production, we examined their gene transcript
levels in different leaf development stages by qRT-PCR.
Since this was the first attempt to describe transcript
measurement in E. coca, it was first necessary to select the
best internal reference genes. A geNorm expression sta-
bility analysis (Vandesompele et al. 2002) was performed
for nine selected E. coca genes selected from our cDNA
library, and the 6409 (JN020149) and 10131 (JN020151)
genes were found to show the highest stability in the dif-
ferent tissues (Supplementary Material Table 1). Expres-
sion of both ODC and ADC genes was detectable in all the
tissues analyzed (Fig. 6a, b). The expression of EcODC
was highest in the youngest leaves, followed by buds and
older leaves. The lowest levels of EcODC transcripts were
observed in the stems and the roots with 15–38 times less
transcript than those found in the youngest leaves. The
expression pattern observed for EcADC was somewhat
different from that observed for EcODC. The highest
EcADC expression was found in the buds and youngest
leaves, with a level almost five times higher than in the
mature leaves, which contained the lowest EcADC tran-
script levels for all measured tissues. The expression level
of EcADC in stems and roots was 1.5 times higher than in
the mature leaves (Fig. 6a, b). Overall, the expression of
ADC was almost ten times higher that ODC in all the tis-
sues analized.
Discussion
In keeping with our long-term goal to elucidate the bio-
synthesis of cocaine in coca (Erythroxylum coca), here we
investigated the site of biosynthesis in the plant and the
initial steps of the pathway. As part of this work, it was first
necessary to determine the distribution of cocaine and other
tropane alkaloids in different organs of E. coca. The
240 12 48
O
D
 6
0
0.0
0.2
0.4
0.6
0.8
1.0
 ODCspe1 
spe1 
Time (hours)
pYESDest52
Δ
Δ
Fig. 5 E. coca ODC complements a yeast ODC mutant. Growth of
the three strains in liquid polyamine-free culture on a rotary shaker at
30�C. Absorbance was monitored at 600 nm every 12 h
Table 4 Spermidine content (nmol g cell Fw-1) of S. cerevisiae
strains in ODC complementation experiments
Strains Spermidine
spe1D transformed with ODC 1.61 ± 0.28
Empty pYesDest52 vector 0.42 ± 0.27
Untransformed spe1D 0.02 ± 0.01
(B)
 
R
el
at
iv
e 
ex
pr
es
si
on
 
0
10
20
30
40
50
60
ODC 
R
el
at
iv
e 
ex
pr
es
si
on
0
100
200
300
400
500
600
700
ADC 
(A)
Buds 3 Stem
Buds
1 2 Roots
1 2 3 Stem Roots
 Leaf stage
 Leaf stage
Fig. 6 Relative transcript levels of ODC (a) and ADC (b) in various
E. coca organs and leaf development stages of 4-month-old plants.
The relative expression of transcripts in all tissues was normalized to
the internal reference genes 6409 and 10131. Displayed are
means ± SD of the three technical replicates from each of three
replicate samples from each of three plants
608 Plant Mol Biol (2012) 78:599–615
123
highest levels of cocaine were found to accumulate in buds
and mature leaves, while stems had less than half. These
values are in the same range as what has been reported
previously for E. coca (Plowman and Rivier 1983). How-
ever, such high concentrations of cocaine are characteristic
only for E. coca and another cultivated species, E. novo-
granatense, grown in Java (Plowman and Rivier 1983),
while wild Erythroxylum species accumulate much lower
amounts (Alyahya et al. 1979; Campos Neves and Campos
Neves 1967). Cocaine has been reported for at least 23 of
the approximately 250 wild Erythroxylum species (Bieri
et al. 2006).
In addition to cocaine, E. coca also contains the (E)- and
(Z)-isomers of cinnamoyl cocaine, in which the benzoyl
ester function is replaced by a cinnamoyl group. With the
(E)-isomer predominating, cinnamoyl cocaine is present in
up to fivefold greater amounts in buds (1.1% of dry weight)
than cocaine, a pattern also noted previously (Johnson and
Emche 1994; Rivier 1981). However, cinnamoyl cocaine
decreases in favor of cocaine during leaf development,
providing evidence for a dynamic regulation of tropane
alkaloid accumulation in E. coca (Johnson 1993; Johnson
and Elsohly 1991).
A detailed analysis of label incorporation from 13CO2
feeding provided information about where tropane alkaloid
biosynthesis is occurring in E. coca. Fed plants accumu-
lated labeled methylecgonine, cocaine, and cinnamoyl
cocaine in buds and young, expanding leaves. In contrast,
mature leaves did not incorporate significant amounts of
label supporting the view that de novo biosynthesis of
tropanes occurrs in young leaves with storage in the mature
leaves. Overall, methylecgonine was labeled up to three
times higher than both cocaine and cinnamoyl cocaine. A
significant incorporation of labeled methylecgonine deriv-
atives in the stem tissue showed that the stem might also
have biosynthetic capabilities. Thus, the production of
tropane alkaloidsin E. coca occurs in young shoot tissues
in stark contrast to the situation in the Solanaceae where
tropane alkaloids such as scopolamine, the calystegines and
nicotine are biosynthesized in the roots and translocated to
the leaves (Ziegler and Facchini 2008).
Until now there has been no information available with
regards to genes and enzymes involved in tropane alkaloid
biosynthesis in E. coca. In order to fill these gaps, we made
a cDNA library from young leaves which were shown by
13C incorporation studies to be sites of cocaine biosynthesis
and searched for potential candidates for the first step of
cocaine biosynthesis. We successfully identified genes
corresponding to two pyridoxal phosphate-binding decar-
boxylases, ornithine decarboxylase (ODC) and arginine
decarboxylase (ADC), thought to be involved in making
putrescine, the polyamine precursor of tropane alkaloids in
other species studied.
Phylogenetic trees constructed on the basis of amino
acid sequences showed that E. coca ODC and ADC were
most closely related to those from Populus trichocarpa, an
observation consistent with the fact that both Erythroxylum
and Populus belong to the order Malpighiales (Fig. 4b, d).
All of these sequences were distinct from those of the
Solanaceae where tropane alkaloid biosynthesis has been
previously studied. This separation illustrates the taxo-
nomic distance between the Erythroxylaceae and the
Solanaceae, whose most recent common ancestor existed at
the time of the basal split of the eudicots more than
116 million years ago (Brachet et al. 1997; Dräger 2004).
Thus tropane alkaloid biosynthesis likely arose more than
once in the evolution of higher plants.
Alignments of amino acid sequences between the ODC
and ADC of E. coca and those of other species showed that
the E. coca sequences also possessed motifs of the Orn/
DAP/Arg decarboxylase family 2 which are well-con-
served in prokaryotic and eukaryotic decarboxylases, as
well the pyridoxal phosphate binding site. Pyridoxal
phosphate is required for decarboxylase catalytic activity
(Moore and Boyle 1990). All of these features indicate that
the E. coca ODC and ADC decarboxylases are well con-
served in relation to other plant enzymes of this type
(Akiyama and Jin 2007). The less conserved regions of
these proteins are usually associated with the N- and
C-terminal regions, and might result in different regulatory
functions (Hummel et al. 2004a, b).
Erythroxylum coca ODC is predicted to be present in the
cytoplasm based on the absence of any identifiable tar-
geting signals. This localization is consistent with the bulk
of literature reports (Martin-Tanguy 2001; Descenzo and
Minocha 1993; Schwartz et al. 1986) except for some ODC
that accumulate in the nucleus (Schipper et al. 2004).
Unlike ODC, the E. coca ADC contains a hypothetical
chloroplast transit peptide at the N-terminus which is
consistent with reports for ADC enzymes isolated from rice
and tobacco (Akiyama and Jin 2007; Bortolotti et al. 2004).
Other properties of E. coca ODC and ADC are also gen-
erally similar to those previously described for these
enzymes from different plant species. The purification and
thrombin cleavage of ODC yielded to a single band of
44.6 kD very similar to the mass of 46.5 kD given for the
N. glutinosa ODC (Lee and Cho 2001a, b). However, the
purified recombinant ODC from E. coca was not able to
decarboxylate lysine as the N. glutinosa enzyme does. In
addition, the E. coca ODC has a Km that is substantially
lower than that of the N. glutinosa ODC, although the
differences in the assay conditions complicate this com-
parison. The recombinant E. coca ADC protein resolved on
a gel to a single band of 93.1 kD which was subsequently
reduced to 79.2 kD following thrombin cleavage of the
thioredoxin tag compared to 77 kD for ADC isolated from
Plant Mol Biol (2012) 78:599–615 609
123
tobacco. The substrate specificity of the E. coca enzyme
towards arginine was similar to that for the tobacco ADC.
To determine whether ADC, ODC or both are involved
in cocaine biosynthesis, we first compared the levels of
arginine and ornithine in the presumed sites of synthesis,
buds and young leaves. Both amino acids were present in
both tissues, although arginine is over 100-fold more
abundant than ornithine. Next, we compared the transcript
levels of ODC and ADC in E. coca organs, and the pattern
for both genes was found to be similar with the most
abundant transcripts found in buds and young leaves,
decreased transcript levels in mature leaves, and very low
transcript levels in the roots. From these data it is not
possible to argue that one of these decarboxylases is more
involved in cocaine biosynthesis than the other. Among
other tropane alkaloid-producing plants studied, D. stra-
monium and L. esculentum show nearly the opposite pat-
tern to what we have found in E. coca for ODC, with high
transcripts in roots and lower levels in shoots (Kwak and
Lee 2001; Michael et al. 1996). In general, ODC and ADC
proteins have been proposed to have different roles corre-
sponding to their differential cellular localization. ODC has
been considered responsible for putrescine production in
tissues undergoing active cell division, while ADC is
thought to be principally involved in producing putrescine
in non dividing tissues or tissues subjected to environ-
mental stresses (Bortolotti et al. 2004). In summary, our
data are consistent with both ODC and ADC contributing
to cocaine biosynthesis in E. coca, a situation that might be
different from that in other tropane alkaloid-producing
species.
Another difference between tropane alkaloid biosyn-
thesis in E. coca and that in other species may involve the
role of putrescine, considered to be the first intermediate of
the pathway (Sato et al. 2007; Biastoff et al. 2009). In most
plant species, putrescine levels are high in young leaves
and decrease with age (Evans and Malmberg 1989).
However, putrescine in E. coca shows the opposite pattern
with the highest amounts found in mature leaves. Perhaps
much of the putrescine in the buds and rolled leaves of
E. coca is diverted to tropane alkaloid formation. The high
amounts of putrescine in mature leaves might function to
retard leaf senescence or serve some other role in stress
protection (Galston 1983; Kusano et al. 2008).
Based on the biochemical and molecular differences we
observed between E. coca and other tropane alkaloid-pro-
ducing plants, we suggest that the tropane alkaloid pathway
is differently regulated in E. coca and may have arisen in
an independent fashion. Further studies to compare bio-
synthetic steps in the tropane alkaloid pathway in E. coca
with those from other known tropane alkaloid-containing
species will provide more insights into the evolution of
tropane alkaloid production in plants and its regulation.
Materials and methods
Plant material
Seeds of E. coca var coca were obtained from the Bonn
(Germany) Botanical Garden. Plants were grown at 22�C
under a photoperiod of 12 h light/12 h dark, humidity of 65
and 70%, respectively. New plants were propagated by
cuttings in pots with Perlite and fertilized once a week with
Ferty 3 (15-10-15) and Wuxal Top N (Planta, Regenstauf,
Germany). In order to standardize samples taken for dif-
ferent developmental stages of the leaf, three stages were
chosen. The youngest expanding leaves that remained in a
rolled state were designated as stage (1). Developmental
stage (2) refers to the young leaves which have unrolled
and begin to expand to their full mature (stage 3) state
(Supplementary Material Fig. 1).
Alkaloid, polyamine and amino acid analysis
Cocaine and cinnamoyl cocaine extracted from E. coca
plants were analyzed and quantified by HPLC with UV
detection. Frozen 100 mg samples of E. coca buds, leaves
(several different developmental stages), stems and roots
were ground in N2 and then extractedin 50% MeOH/H2O
(v:v). Separation of cocaine and cinnamoyl cocaine was
achieved on a Hewlett Packard HP 1100 Series HPLC
system with autosampler and diode-array detector (Agilent
Technologies, Böblingen, Germany). The procedure
employed a C-18 reversed phase column (Nucleodur
Sphinx RP, 250 9 4.6 mm, 5 lm, Macherey–Nagel,
Düren, Germany) operated at 1 ml min-1 and 25�C.
Injection volume was 5 ll. Elution was accomplished with
a gradient (solvent A: 0.05% trifluoroacetic acid (v:v),
solvent B: acetonitrile) of 10–31% B (21 min), 31–71% B
(10 min) followed by a cleaning cycle (71–100% B in
0.10 min, 2.5 min hold, 100–10% B in 0.1 min, 10% B
(4.9 min). Eluting compounds were monitored at 235 and
280 nm for quantification of cocaine and cinnamoyl
cocaine, respectively. Cocaine and cinnamoyl cocaine were
identified by comparison of retention times and UV spectra
with those of commercial standards (Sigma Chemical, St.
Louis, MO, USA), and by comparison of mass spectra by
liquid-chromatography-mass spectrometry on a Bruker
Esquire 6000 ion trap instrument (Bruker Daltonics, Bre-
men, Germany). Cocaine was quantified by a calibration
curve obtained from a standard, while for cinnamoyl
cocaine calibration employed a 1:1 mixture of cocaine and
cinnamic acid standards with the molar response factor
assumed to be similar to that of cinnamoyl cocaine.
Amino acids extracted from E. coca plants were ana-
lyzed and quantified by LC–MS/MS. Samples were ground
in N2 and then extracted in MeOH–water–formic acid
610 Plant Mol Biol (2012) 78:599–615
123
(50:49.9:0.1, v:v:v) and diluted extracts were directly
analysed by LC–MS/MS. The analysis method was modi-
fied from a protocol described by Jander et al. (2004).
Chromatography was performed on an Agilent 1200 HPLC
system (Agilent Technologies, Boeblingen, Germany).
Separation was achieved on a Zorbax Eclipse XDB-C18
column (50 9 4.6 mm, 1.8 lm, Agilent Technologies,
Böblingen, Germany). Formic acid (0.05%) in water and
acetonitrile were employed as mobile phases A and B
respectively. The elution profile was: 0–1 min, 100% A;
1–3 min, 0–100% B in A; 3–4 min 100% B and 4.1–7 min
100% A. The mobile phase flow rate was 0.8 ml/min. The
column temperature was maintained at 25�C. The liquid
chromatography was coupled to an API 3200 tandem mass
spectrometer (Applied Biosystems, Darmstadt, Germany)
equipped with a Turbospray ion source operated in positive
ionization mode. The instrument parameters were opti-
mized by infusion experiments with pure standards (amino
acid standard mix, Fluka, St. Louis, USA). The ion spray
voltage was maintained at 5500 eV. The turbo gas tem-
perature was 700�C, nebulizing gas 70psi, curtain gas
35psi, heating gas 70psi and collision gas 2psi. Multiple
reaction monitoring (MRM) was used to monitor analyte
parent ion ? product ion conversion with MRMs chosen
as in Jander et al. (2004) except for Arg (m/z 175 ? 70),
Lys (m/z 147 ? 84), Orn (m/z 133 ? 70), and Gly that
was detected nonfragmented (m/z 76 ? 76). Both Q1 and
Q3 quadrupoles were maintained at unit resolution. Analyst
1.5 software (Applied Biosystems, Darmstadt, Germany)
was used for data acquisition and processing. Linearity in
ionization efficiencies were verified by analyzing dilution
series of standard mixtures (amino acid standard mix,
Fluka plus Gln, Asn and Trp, also Fluka). All samples were
spiked with 13C, 15N labeled amino acids (algal amino
acids, Isotec, Miamisburg, USA) at a concentration of
10 ug of the mixture per ml. The concentration of the
individual labeled amino acids in the mix was first deter-
mined by HPLC-fluorescence detection analysis after
pre-column derivatization with ortho-phthaldialdehyde-
mercaptoethanol using external standard curves made from
standard mixtures (amino acid standard mix, Fluka plus
Gln, Asn and Trp, also Fluka). Individual amino acids in
the sample were quantified by the respective 13C, 15N
labeled amino acid internal standard, except for tryptophan,
asparagine, and ornithine. Tryptophan was quantified using
[13C, 15N]Phe applying a response factor of 0.42, aspara-
gine was quantified using [13C, 15N]Asp applying a
response factor of 1.0, ornithine was quantified using [13C,
15N]Lys applying a response factor of 1.0.
Polyamines were analyzed and quantified by HPLC with
fluorescence detection. Samples were prepared as reported
above for the amino acid analysis and aliquots of the
supernatant were analyzed as ortho-phthaldialdehyde/
ethanethiol/fluorenylmethoxycarbonyl (OPA/Et/FMOC)
derivatives, according to a previous protocol (Hanczko
et al. 2005). The double derivatized samples were analyzed
on HPLC with fluorescence detection with the following
gradient (solvents A and B as above): 100% B (10 min),
followed by 3 min hold 100% B, 100–50% B in 0.1 min,
50% B (1.5 min). Polyamines were quantified by calibra-
tion curves constructed with commercial standards of
putrescine, spermidine and spermine (Sigma).
Yeast polyamines were extracted as described by Car-
ruthers et al. 2007, then analyzed and quantified according
to the method reported above.
13CO2 feeding
Ten E. coca plants of about 20 cm height were placed in a
closed plastic bag. Prior to the labeling period, the bag was
flushed with synthetic air containing only oxygen and
nitrogen. Then, the plants were exposed to synthetic air
containing 700 ppm 13CO2 (Ba
13CO3, 99%
13C abundance,
Euriso-Top GmbH, Saarbrücken, Germany) as the only
carbon source at 22�C on two successive days for 12 h on
each day. Subsequently, the plants were kept for 12 h in the
dark and then allowed to grow under standard growth
chamber conditions for 3 days. As controls, ten plants were
treated under the same conditions to synthetic air con-
taining 700 ppm 12CO2 (BaCO3, Sigma). Samples of
stems, buds and leaves of different stages were separately
harvested and frozen.
For analysis of 13C incorporation the major tropane
alkaloids (methylecgonine, cocaine and cinnamoyl
cocaine) were ground under N2, and extracted in MeOH–
water-formic acid (50:49.9:0.1, v:v:v) then analyzed by
liquid chromatography-orbitrap mass spectrometry. LC–
MS analysis was performed using the Ultimate 3000 series
RSLC (Dionex, Sunnyvale, CA, USA) system and the
Orbitrap XL mass spectrometer (Thermo Fisher Scientific,
Bremen, Germany) equipped with an ESI source. HPLC
was accomplished using an Acclaim C18 Column
(150 9 2.1 mm, 2.2 lm; Dionex) at a constant flow rate of
300 ll min-1 with a gradient of 0.1% (v/v) formic acid in
water (solvent A) and 0.1% formic acid in acetonitrile
(solvent B) as follows: 0.5–10% (v/v) B (10 min), 10–80%
B (4 min), 80% B (5 min), 80–0.5% (v/v) B (0.1 min),
0.5% B (6 min). Five ll were injected into the HPLC
gradient system. ESI source parameters were set to 35 V
for capillary voltage and 275�C for capillary temperature.
All the samples were measured in positive mode using the
Orbitrap analyzer. Full scan mass spectra were generated
using 30,000 resolving power. Analysis of data was
accomplished using XCALIBUR (Thermo Fisher Scien-
tific, Waltham, MA, USA) software. Controls for com-
parison of natural abundance were run on the same day.
Plant Mol Biol (2012) 78:599–615 611
123
The incorporation of 13C into methylecgonine and its
derivatives was calculated as follows: Proportion of
M + n½ � in % = signal M + n½ �= sum of signals M½ � þ
M + 1½ �þ M + 2½ �þ M + 3½ �þ M + 4½ � � 100 where [M]
represents the unlabeled mass spectrum peak, [M ? n] is the
peak expected from the isotopically labeled analyte. Results
are reported for the enrichment of cocaine (m/z of unlabeled
ion = 304), cinnamoylcocaine (m/z of unlabeled ion = 330),
and methylecgonine (m/z of unlabeled ion = 200).
To compare the proportion of 13C between control and
treated plants the proportions of [M ? n] were arcsin
square root transformed to stabilize the variance of the
proportions. The transformed datawere compared using
analysis of variance (ANOVA). If the variances were still
unequal and errors not normally distributed, the non-
parametric Kruskal–Wallis rank sum test was applied.
Statistical analyses were done in R 2.11.1 (R Development
Core Team 2010).
cDNA library and cloning
Young E. coca leaves were used to construct an enriched
cDNA library. Total RNA was isolated using the RNeasy
Plant Mini Kit (Qiagen, Hilden, Germany). A cDNA
library was constructed from poly(A) ? RNA following
the protocol described by the manufacturers (cDNA Syn-
thesis Kit, ZAP-cDNAm Synthesis Kit and ZAP-cDNAm
Gigapackm III Gold Cloning Kit). The library obtained was
amplified once. Plaque lifts on replicate filters were pre-
pared on nylon membranes (Hybond N1, Amersham, GE
Healthcare, Munich, Germany). N. tabacum ODC
(AAB65826) RT-PCR product was used as a probe for
E. coca ODC, after labeling by random priming with
[a-32P] dCTP using the Ready to Go DNA labeling kit
(Pharmacia, GE Healthcare) and purification by Sephadex
G50 Quick Spin columns (Boehringher, Ingelheim, Ger-
many). Filters were hybridized essentially as described by
Church and Gilbert (1984), and high-stringency washes
were made according to the method of Sambrook et al.
(1989). Filters were exposed to Hyperfilm MP (Amersham)
with an intensifying screen at 270�C. Positively hybridiz-
ing phages were plaque-purified once or twice. In vivo
excision of pBluescript SK (2) phagemids was carried out
according to the manufacturer’s instructions (Stratagene,
Agilent Technologies, Santa Clara, CA, USA). The full
length ODC of 1.2 Kb was sequenced in pBluescript by
using M13 universal primers. For E. coca ADC, ESTs
initially annotated as PLP-binding proteins, were aligned
by sequence comparison with non-redundant proteins from
GenBank using the BLASTX search algorithm (Altschul
and Lipman 1990). Four selected sequences were aligned
to the ADC, and were re-sequenced using the Amplitaq
DNA polymerase and fluorescence cycle sequencing using
an ABI Prism (Applied Biosystems, Carlsbad, CA, USA).
The EcADC full length clone of 2.1 Kb was used for fur-
ther work. The accession numbers for the newly obtained
EcODC and EcADC are JF909553 and JF909552,
respectively.
Sequence alignment and phylogenetic analysis
Full length E. coca ODC and ADC cDNA sequences were
used to search homologous sequences via BLASTX in
NCBI (National Center for Biotechnology Information).
Multiple alignments of amino acid sequences were per-
formed between E. coca and other plant, bacterial and yeast
sequences using CLUSTALW (http://align.genome.jp/). A
phylogenetic tree was then constructed by the neighbor–
joining method with TreeView program (http://taxonomy.
zoology.gla.ac.uk/rod/treeview/). Typical motifs of ODCs
and ADCs were analyzed via ScanProsite (http://www.
expasy.ch/tools/scanprosite/). Signal sequence predictions
were performed using the iPSORT program at the website
http://ipsort.hgc.jp (Bannai et al. 2002).
Protein production and enzyme assays
The ODC and ADC open reading frames were subcloned into
the ‘‘pET-32attR expression vector’’, which was a gateway
compatible vector based on pET32 vector from Qiagen
GmbH (Hilden, Germany) (Zimmermann et al. 2004)
Expression and harvesting of recombinant proteins in BL21
(DE3) E. coli was accomplished as previously described
(D’Auria et al. 2002). Desalted enzymes were analyzed by
SDS-PAGE gel electrophoresis followed by Coomassie
Brilliant Blue staining of the gel. His-tagged ODC protein
was His-Tag cleaved by using the thrombin cleavage kit
(Novagen, Merck, Darmstadt, Germany) and the cleavage
was checked by western blotting and the absence of the His
tag verified by using the mouse anti-His antibody. ODC
protein concentration was determined as previously descri-
bed (Bradford 1976). EcODC purified protein was tested for
ornithine decarboxylase activity in 1 mM Hepes buffer, pH
8, containing 1 mM DTT and 1 mM PLP at RT for 30 min
using 1 mM L-ornithine as substrate.
Identity of products in ODC assays was verified by
HPLC with fluorescence detection using the method
described above for the polyamine analysis. The EcADC
partially purified protein was tested for arginine decar-
boxylase activity in 1 mM Hepes buffer, pH 8, containing
1 mM DTT and 1 mM PLP at RT for 60 min using
L-arginine as substrate. The detection of ADC assays
products was achieved by liquid chromatography-triple
quadrupole mass spectrometry with the method reported
above for amino acids and polyamine analysis.
612 Plant Mol Biol (2012) 78:599–615
123
http://align.genome.jp/
http://taxonomy.zoology.gla.ac.uk/rod/treeview/
http://taxonomy.zoology.gla.ac.uk/rod/treeview/
http://www.expasy.ch/tools/scanprosite/
http://www.expasy.ch/tools/scanprosite/
http://ipsort.hgc.jp
Biochemical characterization
In all the kinetics studies, appropriate enzyme concentra-
tion and incubation were chosen so that the reaction
velocity was linear during the incubation time period. To
determine the Km value, the substrate concentration was
fixed at a saturated level. The kinetics parameters and the
biochemical properties for the purified ODC enzyme
including pH range, temperature stability, effectors were
performed as previously described (D’Auria et al. 2002).
ODC complementation of spe1D-yeast mutant
YPD medium was used for stock cultures of Saccaromyces
cerevisiae ODC-negative mutant (spe1D) (Balasundaram
et al. 1994). The strain S. cerevisiae spe1D provided by
Professor Herbert Tabor was transformed with E. coca
ODC, previously sub cloned into the pYESDEST52
Gateway expression vector for the yeast URA- mutant
strain (Invitrogen). Polyamine-free medium (SC) minus
uracil (0.05%) was used for selection of transformants.
Liquid cultures were grown on a rotator shaker at 30�.
Solid growth of mutant and transformant was visually
monitored, then liquid growth was monitored by absor-
bance readings at 600 nm every 12 h.
RNA extraction and real-time PCR analysis
Organs from E. coca rooted cuttings (4 months old) were
used for RNA extraction. Three biological replicates were
performed for all samples. Total RNA was extracted from
100 mg of fresh plant tissue, using the total RNA extrac-
tion kit from Invitek (Berlin, Germany). Genomic DNA
was removed by treatment with RNAse-free DNAse I
(Qiagen, Hilden, Germany). RNA quality was assessed on
an Agilent Bioanalyzer 2100 using a RNA 6000 Nano Kit
(Agilent, Böblingen, Germany). RNA quantification was
carried out using a NanoDrop 2000c (NanoDrop Tech-
nologies, Wilmington, USA). For cDNA synthesis, super-
script III reverse transcriptase (Invitrogen, Karlsruhe,
Germany) was employed to transcribe 5 lg total RNA with
random hexamer primers according to the manufacturer’s
instructions. Each PCR reaction contained 12.5 ll Brilliant
Sybr Green (Agilent/Böblingen, Germany), 0.375 ll Rox,
0.4 lM primers and 1 ll cDNA (diluted 1:20). All reac-
tions were performed in three technical replicates in a
Stratagene Mx3000P (La Jolla, USA).
Potential reference genes were chosen based on homol-
ogy to known reference genes in A. thaliana (Czechowski
et al. 2005). Sequences of the reference genes were obtained
from an in-house 454 cDNA sequencing database. Primers
for ODC and ADC and 9 potential reference genes (6409,
10131, 11242, Actin, APT2, EF1a, PTPB1, PEX4 and
TIP41) were designed using Beacon Designer (Premier
Biosoft International, Palo Alto, USA) using the default
parameters. PCR efficiency of primer pairs was optimized to
be between 79 and 97% with R2 values [ 0.985. PCR
product melting curves were analyzed for the presence of a
single peak, showing that only one PCR product is formed.
PCR products were cloned and sequenced to verify that all
primer pairs target the desired RNA. No template control
reactions were included on each plate, and each sample was
tested for gDNA contamination using a minus RT control. Alist of the analyzed genes, accession numbers, primer
sequences can be found in Supplementary Material Table 1.
Relative quantity values of all potential reference genes
were exported from Stratagene MXPro software and
imported into GeNORM v3.5 (Vandesompele 2002) to
determine the most stable reference genes thorough the M
value (Supplementary Material Table 2) and the number of
reference genes needed for normalization. ODC and ADC
expression was normalized to 6409 and 10131 expression
using qBASE v1.3.5 (Hellemans et al. 2007).
Nuclear magnetic resonance spectroscopy
13C NMR spectra were recorded on a Bruker AV500 NMR
spectrometer (Bruker Biospin, Karlsruhe, Germany) oper-
ating at resonance frequencies of 500.13 MHz for 1H and
125.75 MHz for 13C. The sample was measured in MeCN-
d3 at 297� K using a 5 mm TCI Cryoprobe. 13C NMR
spectra were recorded into 64 K data points with spectral
width 29985 Hz.
Acknowledgments We thank Dr. Michael A. Phillips, for his help
with the E. coca cDNA library, Katrin Luck for technical assistance,
Dr Christian Paetz for help with the 13CO2 experiment, Gregor
Schmidt for his help with qPCR normalization and Jan Jirschitzka for
helping with the editing of the manuscript. In addition, we thank Prof
Herbert Tabor for kindly providing the S. cerevisie D mutant. Lastly,
we thank Dr. Tamara Krügel, Andreas Weber and the rest of the
gardening staff of the MPI-ICE for their help in plant maintenance.
This work was supported by the Max Planck Society and an Alex-
ander von Humboldt Foundation postdoctoral fellowship to J.D.
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