Plant Biotechnology Journal (2013), pp. 1–9 doi: 10.1111/pbi.

12105

Production of indole alkaloids by metabolic engineering

of the tryptophan pathway in rice
Joseph G. Dubouzet1,2,*, Fumio Matsuda1,3, Atsushi Ishihara1,4,5, Hisashi Miyagawa1,4,6 and Kyo Wakasa1,7,8,9
1
JST/CREST Plant Functions and Their Control, Japan Science and Technology Agency, Tokyo, Japan
2
Biotransformation Team, Scion Research, Rotorua, New Zealand
3
Department of Bioinformatic Engineering, Osaka University, Suita, Osaka, Japan
4
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Atsugi, Kyoto, Japan
5
Faculty of Agriculture, Tottori University, Tottori, Japan
6
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto, Japan
7
Biotechnology Laboratory, National Institute of Crop Science, Ibaraki, Japan
8
Faculty of Agriculture,Tokyo University of Agriculture, Atsugi, Japan
9
Department of Bio-Science, Tokyo University of Agriculture, Tokyo, Japan

Received 18 May 2013; Summary

revised 23 June 2013;
accepted 9 July 2013.
*Correspondence (Tel: +647- 343-5345;
fax +647- 343-5444;
e-mail joseph.dubouzet@scionresearch.com)
Keywords: tryptophan decarboxylase,
anthranilate synthase, serotonin,
tryptamine, b-carboline.
Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently
increasing the metabolic flow of tryptophan derivatives into the production of secondary
metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a
putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing
OASA1D, the feedback-resistant anthranilate synthase alpha-subunit mutant (OASA1D).
Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The
co-expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp
pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a
contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D.
Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of
serotonin and serotonin-derived indole compounds (potentially pharmacoactive b-carbolines)
that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel
system for the production of significant amounts of pharmacologically useful indole alkaloids in
rice.

OASA2) from rice (Oryza sativa L.) and introduced a point

Introduction
Indole alkaloids comprise a diverse class of plant metabolites
useful to man. Many of these compounds are produced in minute
quantities, and extraction and purification are often uneconom-
ical. Producing large amounts of indole alkaloids in common crop
plants may provide a better alternative. However, multiple
enzymes often control complex metabolic pathways, and some
routes may be regulated in a highly coordinated manner (Aharoni
and Galili, 2011).
Many indole alkaloids originate from tryptophan (Trp), and the
biosynthetic pathways of its various derivatives are shown in
Figure 1. Trp decarboxylase (TDC) catalyses the first committed
step of indole alkaloid synthesis by decarboxylating Trp to form
tryptamine (Glenn et al., 2011). The overexpression of TDC in
tobacco led to an accumulation of tryptamine and depletion of
the Trp pool (Guillet et al., 2000). Tryptamine and secologanin
are precursors for the biosynthesis of clinically useful indole
alkaloids (Gongora-Castillo et al., 2012).
Anthranilate synthase (AS) is sensitive to feedback inhibition by
Trp in the Trp biosynthetic pathway (Kreps et al., 1996). Feedback
inhibition of AS must be overcome to accumulate Trp. Tozawa
and coworkers cloned AS alpha-subunit genes (OASA1 and
mutation into OASA1 to obtain OASA1 (N323D), hereafter
referred to as OASA1D. The Trp levels in the vegetative tissues of
rice overexpressing this gene were 359 that of the control
(Tozawa et al., 2001). These transgenic plants produced rice
seeds with increased Trp content (Wakasa et al., 2006). Meta-
bolic profiling analysis of rice calli overexpressing OASA1D
indicated that trace amounts of other indole metabolites that
did not include immediate derivatives of tryptamine (Wakasa and
Ishihara, 2009). Targeted analysis of the OASA1D rice seedlings
resulted in the detection of tryptamine and serotonin, albeit at
much lower levels than Trp (Dubouzet et al., 2007). These results
indicated that TDC is relatively inactive in rice even in the
presence of a large amount of its substrate. The results also
suggested that the elevation of TDC activity in OASA1D-
transformed rice may convert excess Trp to tryptamine, which
could be used for the biosynthesis of other indole alkaloids.
Tryptophan decarboxylase is active in rice under some physi-
ological conditions. For example, tryptamine was detected in
lesions from a lesion-mimic mutant line of rice after infection with
the rice blast fungus Magnaporthe grisea (Ueno et al., 2003). The
causal gene was found to be a cytochrome P450 monooxygenase
that catalysed the conversion of tryptamine to serotonin (Fujiwara

Please cite this article as: Dubouzet, J.G., Matsuda, F., Ishihara, A., Miyagawa, H. and Wakasa, K. (2013) Production of indole alkaloids by metabolic engineering of
the tryptophan pathway in rice. Plant Biotechnol. J., doi: 10.1111/pbi.12105

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd 1
2 Joseph G. Dubouzet et al.

Figure 2 Phylogenetic relationships of the protein translations of putative

aromatic amino acid decarboxylases in Oryza (AK103253, AK065830,
NM196176, NM196380, NM_001067504 and AK069031) relative to a
Figure 1 Metabolic pathways for the biosynthesis of aromatic amino
known tryptophan decarboxylase (Cr TDC, M25151) and tyrosine
acids and indole alkaloids. AS, anthranilate synthase; CM, chorismate
decarboxylase (Pc TYDC2, M96070). Cr and Pc stand for Catharanthus
mutase; Str, strictosidine synthase. TDC, tryptophan decarboxylase; TyDC,
roseus and Petroselinum crispum, respectively. TDC and TyDC correspond
tyrosine decarboxylase; feedback regulation of AS by Trp is indicated by a
to tryptophan and tyrosine decarboxylases, respectively. Branch lengths
curved arrow.
are drawn to scale while the numbers at the nodes correspond to
bootstrap support level.

et al., 2010). Indole acetic acid and its conjugates, which are
derived from Trp possibly via tryptamine, were increased in rice
calli and seeds overexpressing OASA1D (Wakasa et al., 2006).
Serotonin, a tryptophan derivative, accumulated at the infection
site of rice leaves inoculated with Bipolaris oryzae, probably as
reinforcement of the cell walls (Ishihara et al., 2008).
In this report, we examined the effect of simultaneous
expression of OsTDC together with OASA1D in rice calli and
analysed the metabolic changes in these calli. Activation of both
Trp synthesis and its rapid conversion to tryptamine led to the
production of new tryptamine derivatives in significant quantities.
Our results suggested that the tight regulation of metabolic flow
from Trp in wild-type rice plants may be to avoid the accumu-
lation of possibly harmful indole alkaloids. Further, we discuss the
utility of the OASA1D:OsTDC plasmid construct for metabolic
engineering of the Trp pathway in rice and other plants.
Figure 3 Response of OsTDC and OsTyDC expression to MJ in wild-type
(Nipponbare) and transgenic rice line overexpressing OASA1D (HW5).
Results methyl jasmonate (MJ) (1.25 mM) solution was sprayed at 24 h before
Phylogenetic analysis of putative aromatic amino acid tissue sampling. Expressions of AK069031 (OsTDC, white) and AK065830
decarboxylase genes in rice (OsTyDC, black) were determined with qRT-PCR methods.

The relationships among some aromatic amino acid decarboxy-

lases are shown in Figure 2. The TDC homologues in rice are
grouped into well-supported subclusters (numbers on the nodes
correspond to bootstrap support values):
AK103253 is clustered with PcTyDC2 (M96070) from Petro-
selinum crispum, whereas NM_001067504 and AK069031 form
another cluster with CrTDC (M25151) from Catharanthus roseus.
AK065830, NM196176 and NM196380 form the third cluster
that is more closely related to PcTyDC2 than to CrTDC. We refer
to AK065830 and AK069031 as OsTyDC and OsTDC in this
report.
Response of TDC homologues to elicitation by methyl
jasmonate (MJ)
Seedlings of Nipponbare and a high-Trp transgenic line overex-
pressing OASA1D (line HW5) were sprayed with 1.25 mM MJ and
incubated for 24 h. Figure 3 shows that MJ treatment increased
OsTDC expression in both genotypes, but the response in the
various replicates was quite variable, which led to high error rates.
In contrast, the OsTyDC transcript level was lowered by MJ
treatment.

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9

Generation of OASA1D/TDC and OASA1D/TyDC
transformants
Transformed calli containing OASA1D:TDC and OASA1D:TyDC
gene constructs (Figure 4a) were selected by growing Agrobac-
terium-inoculated rice calli in media containing 5-methyltrypto-
phan (5MT). Transformed calli containing OASA1D:TDC exhibited
a dark pigmentation throughout the callus mass (Figure 4b) that
persisted even after the calli were transferred to 2N6 mainte-
nance media. Callus growth was apparently unimpeded despite
this pigmentation.
The regenerated shoots were slow-growing and exhibited
bunch-type proliferation and dark-pigmented outgrowths (Fig-
ure 4b). OASA1D:TDC plants showed severe growth retardation,
excessive shoot proliferation and generally poor vigour, and only
two lines survived in the greenhouse. Adverse effects of unknown
metabolites may help explain the severe phenotypes of these
OASA1D:TDC regenerants.
In contrast, normal-looking and vigorously growing regener-
ants were easily produced from the pearly white calli overex-
pressing OASA1D:TyDC (data not shown). Leaf necrosis or
hypersensitive response symptoms were not observed in plants
regenerated after Agrobacterium-mediated transformation with
OASA1D:TDC or OASA1D:TyDC.
OASA1D, TDC and TyDC transcript levels in transformed
calli
Four callus lines expressing OASA1D:TDC and three lines express-
ing OASA1D:TyDC were obtained after PCR-based screening for
the presence of 35S-TDC and 35S-TyDC gene cassettes in 5MT-
resistant lines. The transcript levels of OASA1 and TDC genes in
both Nipponbare- and the OASA1D:TDC-transformed calli were
evaluated by real-time quantitative reverse-transcription PCR (qRT-
PCR). OASA1 (primarily OASA1D in the transformed lines)
transcript levels were 6–1629 more than those in Nipponbare
(Table 1). Similarly, TDC transcript levels were 8–399 more than
(a)
(b)
a b
Figure 4 Construction of the OASA1D:OsTDC
vector and phenotypes of transformants. (a) An
expression cassette containing CaMV 35S
promoter (35SP), OsTDC and Nos terminator
(NosT) was constructed in pBluescript (KS), excised
and then inserted upstream of the ubiquitin
promotor (UbiP) of vector pM159. (b) OASA1D:
OsTyDC (left panel) and OASA1D:OsTDC (middle
panel) phenotypes in callus and OASA1D:OsTDC
regenerated plant (right panel).
ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
Indole alkaloids in rice 3
those in Nipponbare. Much higher levels (>7009 that of the wild
type) of TyDC transcripts were obtained from OASA1D:TyDC
constructs. Expression of OASA1 under the control of the Ubi
promoter in the OASA1D:TyDC transformant is lower than or
almost the same as the OASA1D:TDC transformant. Both OsTyDC
and OsTDC genes were under the control of the 35S promoter,
but the up-regulation of OsTyDC is clearly much higher than the
latter.
Accumulation of Trp, tyrosine, phenylalanine,
tryptamine and tyramine in OASA1D:TDC, OASA1D:
TyDC lines and Nipponbare
The levels of the aromatic amino acids and the corresponding
decarboxylation products in the calli of OASA1D:TDC, OASA1D:
TyDC lines and Nipponbare are shown in Figure 5. The data were
obtained via ion monitoring with liquid chromatography–electro-
spray ionization-mass spectrometry (LC-ESI-MS). All of these
aromatic amino acids are derived from the shikimate pathway
(Figure 1) so differential activation of the respective branches by
overexpression of downstream genes may be the result of
competition.
Expression of feedback-insensitive OASA1D in transgenic rice
was expected to increase Trp. The levels of Trp are shown
in Figure 5a. Paired t-test of Trp levels indicated signifi-
cant (P < 0.05) differences among all treatment comparisons
(OASA1D:OsTDC vs. OASA1D:OsTyDC; control vs. OASA1D:
OsTDC or control vs. OASA1D:OsTyDC). The OASA1D:TDC lines
showed very low levels of Trp, whereas the OASA1D:OsTyDC
lines had 779 more Trp than the OASA1D:TDC lines.
The tryptamine content of the various lines is shown in Figure 5d.
Paired t-test of tryptamine levels revealed significant (P < 0.05)
differences between OASA1D:OsTDC vs. OASA1D:OsTyDC and
control vs. OASA1D:OsTDC. The high levels of tryptamine in the
OASA1D:OsTDC lines indicated that the excess Trp due to OASA1D
activity was decarboxylated by TDC into tryptamine. No tryptamine
was detected in Nipponbare and OASA1D:OsTyDC, whereas the
c
4 Joseph G. Dubouzet et al.

Table 1 Transcript activity of transgenes in the calli of double transformants relative to control. The transformed lines overexpressed OASA1D
and OsTDC or OsTyDC. Quantitation was performed using SYBR Green-mediated qPCR

OASA1 + OASA1D OsTDC OsTyDC

Calli line Transcript level Fold change Transcript level Fold change Transcript level Fold change

Nipponbare 0.4  0.2 1.0 2.2  1.3 1.0 1.6  1.4 1.0
OASA1D:OsTDC.19 2.5  0.5 6.1 41.1  2.2 18.8 2.5  3.7 1.6
OASA1D:OsTDC.38 66.1  5.6 162.6 66.8  7.7 30.6 0.3  0.1 0.2
OASA1D:OsTDC.76 15.5  0.7 38.2 85.0  3.2 38.9 1.2  1.2 0.7
OASA1D:OsTDC.83 5.1  0.7 12.4 17.7  2.9 8.1 0.1  0.1 0.1
OASA1D:OsTyDC.27 9.7  3.0 23.7 0.7  0.1 0.3 41.3  19.4 25.9
OASA1D:OsTyDC 48 4.4  0.4 10.9 0.2  0.1 0.1 1256  276 787.0
OASA1D:OsTyDC 76 6.2  1.1 15.3 0.3  0.1 0.1 927  152 580.6

(a)
(d)

(b)
(e)

(c)

Figure 5 Aromatic amino acids and their primary

decarboxylated products in OASA1D:TDC,
OASA1D:TyDC and Nipponbare calli. Levels of Trp
(a), Tyr (b), Phe (c), tryptamine (d) and tyramine (e)
in each callus line are shown. Data are the
means  SD of three replicates.

OASA1D:TDC transformants contained tryptamine at an average
concentration of 140.7 nmol/g FW.
The Tyr content of the various lines is shown in Figure 5b.
Paired t-test of Tyr levels indicated significant (P < 0.05) differ-
ences between OASA1D:OsTDC vs. OASA1D:OsTyDC and control
vs. OASA1D:OsTDC. The OsTyDC lines had the highest Tyr
content.
The tyramine content of the various lines is shown in Figure 5e.
Paired t-test failed to detect significant differences in tyramine
levels. However, the introduction of OsTyDC increased levels of
tyramine in two lines. The levels of Tyr and tyramine in the
OASA1D:TDC were similar to those in Nipponbare.
The amounts of Phe in the various lines are shown in Figure 5c.
Paired t-test of Phe indicate significant (P < 0.05) differences
among all comparisons. On average, OASA1D:TDC lines showed
31.5% lower Phe than Nipponbare, whereas the OASA1D:TyDC
lines had 49.5% lower Phe than Nipponbare.
Metabolic profiles of OASA1D:TDC and OASA1D:TyDC
calli
The overproduction of tryptophan and tryptamine was expected to
affect the concentrations of other indole metabolites. Indole
metabolites are detected by ultraviolet at 280 nm, so high-
performance liquid chromatography equipped with photodiode

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
 Indole alkaloids in rice 5

array detector (HPLC-PDA) was used to characterize the profiles of

aromatic metabolites in wild-type and transgenic calli. Figure 6
shows the qualitative differences among phytochemicals detected
from the different genotypes included in this study. In the OASA1D
transformant, a large amount of Trp was accumulated along with
several minor metabolites, whereas the Trp peak is practically
absent in OASA1D:TDC lines. The metabolic profiles of OASA1D:
TyDC lines were almost identical to that of the OASA1D line.
Novel metabolites in OASA1D:TDC calli
High-performance liquid chromatography equipped with photo-
diode array detector (HPLC-PDA) analysis (Figure 6) revealed
several major peaks were detected in OASA1D:TDC transfor-
mants. This indicated that endogenous enzymes or enzyme
systems in these calli metabolized substantial fractions of the
tryptamine pool into other compounds. Four of these metabolites
(1–4) were purified from transformed calli by multi-step chroma-
tography and subsequently subjected to spectroscopic analyses.
Liquid chromatography–electrospray ionization-mass spectrom-
etry (LC-ESI-MS) analysis suggested that compound 1 (MW 176) is a
hydroxylated metabolite of tryptamine (MW 160). The 1H-NMR
spectrum of 1 was identical to that of authentic 5-hydroxytrypta-
mine (serotonin, Figure 7). Compound 2 was determined as
6-hydroxy-1-methyl-1,2,3,4-tetrahydro-ß-carboline (6-hydroxym-
ethyltryptoline, Figure 7) based on its 1H- and 13C-NMR data
(Salmoun et al., 2002). Compounds 3 and 4 had the same
molecular formula (C27H33N3O8) and UV spectra (kmax MeOH
282 nm). Based on a series of NMR spectroscopic data, including
1 Figure 7 Structures and elucidated biosynthetic pathways of novel indole
H-, 13C-NMR, H-H COSY, HMQC and HMBC, compounds 3 and 4
alkaloids in OASA1D:TDC transgenic rice calli. Enzymatically undefined
were deduced to have a similar structure, 2-hydroxy-3-(3′-amino-
routes are indicated by dotted arrows. The putative structures for
ethyl-5′-hydroxyindol-2′-yl)-3-indol-3′-yl-propyl ß-D-glucopyrano- compounds 1–4 are indicated by the corresponding numbers in bold font
side. Compounds 3 and 4 showed different optical rotations, (e.g. serotonin = 1).
suggesting that compound 4 is an epimer of compound 3. Their
absolute structures remain unknown. Compounds 3 and 4 are
indole–alkaloid glycosides, whose aglycone consists of serotonin effects of overexpressing TDC, the enzyme that funnels Trp into
and indole-3-glycerol moieties (Figure 7). To the best of our secondary metabolism (Figure 1).
knowledge, compounds 3 and 4 have not been described Methyl jasmonate activates the expression of genes that code
previously. The levels of 1, 2, 3 and 4 in OASA1D:TDC3 transformed for enzymes such as TDC that catalyse the formation of terpene
rice calli were 302, 25, 41 and 191 nmol/g FW, respectively. indole alkaloids (Zhou et al., 2010). Tryptophan decarboxylase is
responsive to MJ, and tryptamine derivatives produced by TDC
elicitation may function in ameliorating stress in rice. Accumula-
Discussion
tion of tryptamine derivatives like serotonin and its hydroxycin-
Metabolic flux in the Trp pathway is controlled by the feedback namic acid amides in leaves undergoing fungal attack indicated
inhibition of AS by Trp in plants. High levels of the Trp substrate in that these compounds may be an integral part of rice defences
calli overexpressing OASA1D facilitated the monitoring of the (Ishihara et al., 2011).

Figure 6 Profiles of active metabolites in

Nipponbare, OASA1D, OASA1D:TyDC and
OASA1D:TDC rice calli obtained by high-
performance liquid chromatography equipped
with photodiode array detector. The metabolic
profiles detected at UV 280 nm are presented in
the figure. The retention times of tryptophan,
tryptamine, tyramine and compounds 1–4 are
indicated by dotted lines.

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
6 Joseph G. Dubouzet et al.
We introduced two aromatic amino acid decarboxylase genes
into rice calli. One of the genes (AK065830) has been reported to
encode a TDC (Ueno et al., 2003). However, the same gene has
subsequently been identified as a TyDC (Kang et al., 2007). In our
experiments, rice calli expressing AK065830 accumulated tyra-
mine, not tryptamine. Thus, we confirm that this gene encodes a
TyDC, capable of converting tyrosine to tyramine. In contrast, the
callus simultaneously expressing OASA1D and AK069031 con-
tained tryptamine at an average concentration of 140.7 nmol/g
FW (Figure 5d). This indicates that AK069031 encodes a TDC,
consistent with its phylogenetic relationship to other aromatic
amino acid decarboxylases (Figure 2). Hence, OASA1D:OsTDC
can be used to modify the terpene indole alkaloid profile in rice.
Our results indicate that the excess Trp generated by OASA1D
was almost completely channelled into tryptamine by the addition
of 35S:OsTDC:NosT cassette. Tryptamine levels in OASA1D:TDC
calli were approximately 25% of the Trp levels in OASA1D:TyDC
calli. Analysis of OASA1D:TDC lines indicated that the callus lines
produced numerous novel substances that were not observed in
the OASA1D line (Figure 6). Similarly, ectopic expression of a
negative feedback-resistant anthranilate synthase gene from
Arabidopsis and the endogenous TDC gene driven by an inducible
promoter in C. roseus increased tryptamine levels by 14-fold and
ajmalcine by more than 40-fold (Hong et al., 2006).
Spectroscopic analysis of some of the new compounds
identified them to be serotonin and serotonin-derived indole
compounds (Figure 7). Serotonin, a mammalian neurotransmit-
ter, is accumulated in rice seedlings expressing OsTDC (Kang
et al., 2007). Tryptamine 5-hydroxylase, which catalyses the
conversion of tryptamine to serotonin, was recently detected in
the crude extract from rice seedlings (Kang et al., 2007). This
enzyme seemed to be quite active in rice calli as the average
serotonin contents of OASA1D:TDC callus lines are 302 nmol/g FW.
The level of tryptamine (140.7 nmol/gFW) in rice calli simulta-
neously expressing OASA1D and TDC was lower than the level in
tobacco leaves expressing TDC from C. roseus [4.13 lmol/gFW
(Di Fiore et al., 2002)]. The lack of metabolic pathways from
tryptamine in tobacco enables the accumulation of tryptamine at
an extremely high concentration (Di Fiore et al., 2002). The levels
of tryptamine and serotonin in the rice calli expressing OASA1D
and TDC in this study were much higher than those in rice
seedlings expressing TDC alone (Kang et al., 2007).
Overexpressing OASA1D enhanced Trp supply and increased
the production of downstream compounds. The OASA1D:TDC
callus lines exhibited dark pigmentation, slow growth rate and
bunch-type cell proliferation. The shoots regenerated from these
calli also exhibited dark pigmentation, growth retardation,
excessive shoot proliferation and poor vigour. These adverse
phenotypes are plausibly due to the synergistic effect of increas-
ing the conversion of anthranilate to Trp by overexpression of
OASA1D and the increased conversion of Trp to tryptamine and
other indole alkaloids by overexpression of OsTDC. Similar
phenotypes were reported when TDC genes were activated by
T-DNA tagging or overexpression in rice (Kanjanaphachoat et al.,
2012). High transgene-encoded TDC activity was also found to be
detrimental to the normal growth of C. roseus cultures (Canel
et al., 1998).
The other compounds identified in rice OASA1D:TDC calli were
1-methyl-6-hydroxy-1,2,3,4-tetrahydro-ß-carboline (2) and two
epimers of 2-hydroxy-3-(3′-aminoethyl-5′-hydroxyindol-2′-yl)-3-
indol-3′’-yl-propyl-ß-D-glucopyranosides (3 and 4) (Figure 7). 1-
Methyl-6-hydroxy-1,2,3,4-tetrahydro-ß-carboline was reported to
ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
be an endogenous constituent of the mammalian brain, and it
was suggested to be biosynthesized from serotonin with two
additional carbon atoms that originate from pyruvate or acetal-
dehyde derived from ethanol (Zhang et al., 1992). The enzyme
responsible for the formation of this compound has not been
identified. Upon injection of this compound into the mammalian
brain, the subsequent oxidation reaction generates dimers that
evoke behavioural responses similar to those caused by opioid
analgesics (Zhang et al., 1992). 1-Methyl-6-hydroxy-1,2,3,4-tet-
rahydro-ß-carboline is naturally found in cocoa-based food
products along with other pharmacologically active b-carbolines
that act as neuronal modulators (Herraiz, 2000). Some ß-
carbolines have antimicrobial properties (Tsuchiya et al., 1999).
Compounds 3 and 4 contain a serotonin unit in their structures
so they are probably serotonin derivatives as well. The remaining
part of these two compounds is the indole-3-glycerol structure
that is most likely derived from indole-3-glycerol phosphate. The
putative biosynthetic routes of newly identified compounds
are depicted in Figure 7. It is of interest to note that rice
plants expressing OASA1D accumulated 1,2,3,4-tetrahydroxy-
b-carboline-3-carboxylic acid and 2-[2-hydroxy-3-b-D-glucopyr-
anosyloxy-1-(1H-indole-3-yl) propyl] tryptophan. The former
compound has the same carboline skeleton with compound 2,
while the latter compound is analogous to compounds 3 and 4.
The compounds identified from OASA1D plants are biosynthe-
sized from Trp instead of serotonin for compounds 2–4.
In addition to the compounds identified in this study, there
remain several unidentified peaks that are specifically detected in
calli expressing TDC, as shown in Figure 6. This is a clear contrast
to the calli expressing TyDC, in which the accumulation of
tyramine was the only major metabolic change detected.
Rice plants expressing OASA1D gene did not show major
metabolic change except for high Trp accumulation (Tozawa
et al., 2001). This indicated that TDC is a key enzyme regulating
the flow of Trp into secondary metabolism. TDC is a critical
enzyme in the biosynthesis of ajmalicine, an indole alkaloid in
Rauvolfia verticillata (Liu et al., 2012). RNAi-mediated suppression
of tryptophan decarboxylase in Catharanthus roseus hairy root
culture eliminated all production of monoterpene indole alkaloids
(Runguphan et al., 2009). Our results indicate that one of the
reasons for the tight regulation of TDC activity in rice is to
regulate the formation of potentially harmful metabolites. For
example, the gene responsible for the hypersensitive response of
Sekiguchi-lesion mutants is a p450 monooxygenase that catalyses
the conversion of tryptamine to serotonin in rice (Fujiwara et al.,
2010). HR-induced lesions are only useful in the context of
resistance to pathogen attack.
The total concentration of serotonin and its derivatives was
much higher than the concentration of tryptamine, indicating the
rapid conversion of tryptamine to serotonin. We think that a
serotonin pathway may be functional and important in rice under
some physiological conditions such as biotic stress. The rice
suspension culture treated with chitosan elicitor enhanced the
expression of the OASA2 gene that encodes AS alpha-subunit 2
(Tozawa et al., 2001), and the expression of TDC was
up-regulated in rice seedlings treated with MJ (Figure 3). Indeed,
serotonin is accumulated in rice leaves infected with Bipolaris
oryzae (Ishihara et al., 2011).
The OsTDC transcript level in OASA1D calli is higher than that
in Nipponbare (Figure 3). This may be because OsTDC is
responsive to the higher levels of its substrate, tryptophan, in
the OASA1D calli. However, the tryptophan levels generated in

OASA1D calli are so high [1779 wild type (Tozawa et al., 2001)]
that it must be concluded that normal OsTDC activity in OASA1D
calli is insufficient to convert a large portion of the tryptophan
into tryptamine.
Serotonin and its derivatives are oxidized by nonenzymatic
reactions with oxyhemoglobin (Blum and Ling, 1959), ferricyto-
chrome (Alivistotos and Williams-Ashman, 1964) and diverse
inorganic oxidants (Eriksen et al., 1960). In addition, serotonin
serves as a substrate for peroxidase in the presence of hydrogen
peroxide (Siraki and O’Brien, 2002). The oxidized products of
serotonin show a dark brown colour indicating that the dark
pigmentation of the OASA1D:TDC transgenic lines may be due to
oxidized serotonin derivatives.
We converted excess Trp (produced by the negative feedback-
insensitive OASA1D enzyme) into tryptamine by co-expressing
OsTDC in rice calli. Tryptamine was subsequently converted into
its various indole alkaloid derivatives, such as serotonin and
b-carbolines, via enzyme systems already present in the rice
cytoplasm. Further manipulation of the existing pathways in rice
may lead to designer drugs that may find use in pharmacology.
Concurrent up-regulation of the melatonin pathway and/or
overexpression of certain methyl transferases can lead to the
biosynthesis of potent psychotropic drugs such as methylated
tryptamines. Cotransformation of the OASA1D:TDC vector with
vectors containing secologanin and/or strictosidine synthases may
be able to direct metabolic flux towards the production of terpene
indole alkaloids, some of which are prized for their anticancer
properties. Lastly, the OASA1D:TDC vector may be most useful in
transforming plant species that are known to produce desirable
indole alkaloids such as C. roseus and C. acuminata.
Experimental procedures
Jasmonate treatment of rice seedlings
Ten seeds of rice Nipponbare (Oryza sativa L.) and 15 seeds of the
transgenic line, HW5 (Wakasa et al., 2006), were sown in
separate jars. Surface-sterilized seeds were placed on metal mesh
screens over 10 mL water and cultured for 7 days. After
decanting the excess water, 2 mL of an aqueous solution
containing 1.25 mM methyl jasmonate (MJ) and 0.1% Tween
20 (0.6 lL of 95% MJ/2 mL) was sprayed onto the plants.
RNA extraction, reverse transcription and RT-PCR
Total RNA from 100 mg calli or leaves was extracted with
RNeasy (QIAGEN, Valencia, CA) followed by DNase treatment
according to the manufacturer’s instructions. Reverse transcrip-
tion of 5 lg total RNA from each sample was performed with
Superscript III (Invitrogen, Carlsbad, CA) at 42 °C following the
manufacturer’s protocol.
Forward and reverse primers for PCR were designed with the
help of Primer Express (ABI, Perkin Elmer Co., Ltd., Foster City,
CA) as follows: OsTyDC (5′-CCACCCTCGCACTGCCAC-3′ and
5′-GAGCCAGTCCACGACCACC) and OsTDC (5′-CAACCCCA-
CGGCCTTCTC and 5′-CGCCCAGTGCGTCATCC-3′). PCR was
performed with ExTaq with the following thermal parameters:
initial denaturation at 98 °C for 3 min, followed by 25–50 cycles
of 98 °C for 10 s, 55 °C for 15 s, 72 °C for 30–60 s and a final
extension step at 72 °C for 4 min.
Phylogenetic analysis
The putative protein translations were aligned via ClustalW set to
bootstrap 1000 NJ trees as implemented in BioEdit v.7.0.5 (http://
ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
Indole alkaloids in rice 7
www.mbio.ncsu.edu/BioEdit/page2.html). The alignment was
uploaded to BioNJ (http://www.phylogeny.fr/version2_cgi/one_-
task.cgi?task_type=bionj) to generate a treefile. The resulting
phylogeny was illustrated using NJplot (http://pbil.univ-lyon1.fr/
software/njplot.html).
OASA1D:TDC3 vector construction
We added a gene cassette consisting of 35SP-OsTDC-NosT
PM159 into a binary vector containing OASA1D under the
control of an ubiquitin promoter and Nos terminator. The
promoter, 35SP, was amplified from pIG121-Hm with primer
pair 1 (5′-ATAAGAATGCGGCCGCGTTTAAACGTCCCCAGATTA
GCC and 5′-CGCTCTAGACTGCAGCCCGGGGTCCCCCGTGTTC
TCC-3′), whereas the Nos terminator was amplified from pM159
with primer pair 2 (5′-CCATCGATGAATTTCCCCGATCGTT and
5′-GGGGTACCTTAATTAACCGATCTAGTAACA-3′). These two
fragments were then serially introduced into pBluescript KS via
their respective primer adaptor sites to form a shuttle vector
known as pBSK35SP-NosT. OsTDC was amplified from genomic
rice DNA with primer pair 3 (5′-CCTTAATTAAGGATGGGCAGCT
TGGACACC and 5′-TTGGCGCGCCAATTAGTTCATCATCTCGGT-
3′) and ligated into PCR2.1. The OsTDC sequence was amplified
from the PCR2.1: OsTDC plasmid with KOD polymerase and
primer pair 4 (5′-CGGGATCCCGATGGGCAGCTTGGACACC and
CGGAATTCCGTTAGTTCATCATCTCGGT-3′) and then ligated into
the EcoRI/BamHI site in pBSK35SP-NosT. The pBSK35S-OsTDC-
NosT segment was excised from the plasmid vector by digestion
with PacI and PmeI and inserted into the corresponding site in
pM159. This plasmid, containing the gene cassettes for OASA1D
and OsTDC, was used to transform DH5a. A similar procedure
was followed to produce the OASA1D:TyDC vector. Plates
containing the OASA1D:TDC plasmid in DH5a, Agrobacterium
strain C58 C1 and E. coli HB101 harbouring the helper plasmid
pRK2013 were cultured separately, and single colonies were
selected for proliferation by liquid culture. Aliquots of 100 lL
from each culture were mixed, incubated for 6 h at 30 °C,
streaked over LB-Rif/Kan and incubated for 36 h at 30 °C. Single
colonies were obtained, and the presence of the inserts was
verified by restriction analysis.
Agrobacterium-mediated transformation of rice callus
Protocols previously established in our laboratory were used for the
generation of transgenic rice via selection with 5MT. Expression of
OASA1D gene confers resistance to 5MT (Tozawa et al., 2001).
qRT-PCR
Forward and reverse PCR primers [5′-GAGCTGATTGACCAGATG
GA-3′ and 5′-CATGTCTCCACGGAAAGAAA-3′ for OASA1D; 5′-
CGACGTCACCATGAAGGA-3′ and 5′-TATTCGTGGAGGGGAAA
AAC-3′ for OsTDC; and, 5′-CAAGGGCCGTGTTCCCTAGT-3′ and
5′-CTTCTGCCCCATTCCTACCAT-3′ for Actin (AK072796)] were
designed with Primer 3 (http://frodo.wi.mit.edu/cgi-bin/primer3/
primer3_www.cgi). The last 5 nucleotides of each primer were
limited to no more than two C or G residues to minimize the
frequency of primer–dimers and false priming products. Real-time
qRT-PCR was performed in an ABI Prism 7000 Sequence
Detection System (Perkin-Elmer Applied Biosystems, Foster City,
CA) using a SYBR Green RT-PCR Reagent kit in 10 lL reaction
volumes containing 2.5 ng cRNA template and 2.5 pmol primer.
The standard 2-step thermal cycling protocol was repeated 50
times. The melting point of the amplified products was deter-
mined with the prescribed dissociation protocol to verify that the
8 Joseph G. Dubouzet et al.
signal came from a single amplicon. The actin gene was used to
normalize expression data of the other genes.
Metabolic profiling analysis
Rice calli (approximately 0.9 g fresh weight) were homogenized
with a Polytron disrupter (Kinematica AG, Luzern, Switzerland) in
five volumes of ice-cold methanol–water (80 : 20, v/v). The
homogenate was centrifuged at 2,900 g for 10 min, and the
resulting supernatant was applied to a Sep-Pak Plus C18 cartridge
(Waters, Milford, MA). The 1.0 mL of eluate was evaporated in a
vacuum, and the residue was dissolved in 200 lL of water, and
then subjected to reversed-phase HPLC (LC-10Avp system;
Shimadzu, Kyoto, Japan) with a Cadenza CD-C18 column (250
by 4.6 mm (inner diameter); particle size, 3 lm, Imtakt, Kyoto,
Japan). Elution was performed with a mixture of 0.02% triflu-
oroacetic acid in water and acetonitrile (100 : 0, v/v, at 0 min;
98.2 : 1.8 at 10 min; 95.5 : 4.5 at 27.5 min; 70 : 30 at 60 min;
5 : 95 at 75 min; 100 : 0 at 75.5 min; 100 : 0 at 90 min) at a
flow rate of 0.85 mL/min and a temperature of 38 °C and was
monitored with a photodiode array detector (Shimadzu SPD-
M10Avp) over a wavelength range of 200 to 400 nm.
The levels of Phe, Tyr, Trp, tyramine and tryptamine in the
extracts were determined by using LC-MS, as previously described
(Matsuda et al., 2005) The mass numbers of protonated mole-
cules ([M + H]+) of the compounds were chosen for selected ion
monitoring. Paired t-test for the levels of the metabolites in
Figure 5 were performed in Excel.
Isolation of compounds 1–4
About 74 g of transgenic rice callus (OASA1D:TDC38) was
homogenized in three volumes of methanol–water (80 : 20)
and then maintained for 1 day in the dark at 4 °C. The
homogenate was filtered through filter paper, and then the
filtrate was evaporated in a vacuum. The dried residue (3.1 g) was
dissolved in 200 mL of water. The solution was washed with
200 mL of hexane and then evaporated under reduced pressure.
The residue (2.5 g) was dissolved in 10 mL of a mixture of
methanol and 0.1% acetic acid in water (5 : 95, v/v) and then
applied to an ODS column (Cosmosil 75C18-OPN; Nacalai
Tesque, Kyoto, Japan) that had been equilibrated
with the same solvent. The column was washed with 500 mL
of the solvent, and the target metabolites were eluted
with 500 mL of methanol–0.1% acetic acid (60 : 40). The eluate
was evaporated to dryness (130 mg), and the residue was
dissolved in methanol–0.1% acetic acid (20 : 80) and then
subjected to HPLC on a Cadenza CD-C18 column [150 by
10 mm (inner diameter); particle size, 3 lm, Imtackt, Kyoto,
Japan]. Elution was performed with 0.1% trifluoroacetic
acid–acetonitrile (90 : 10, v/v, at 0 min; 75 : 25 at 30 min;
5 : 95 at 35 min; 90 : 10 at 35.1 min; 90 : 10 at 50 min) at a
flow rate of 3.0 mL/min and was monitored at a wavelength of
280 nm. The fractions containing compounds 1–4 were collected
and evaporated to yield purified metabolites. The respective
amounts of compounds 1–4 were 8.5, 4.2, 6.5 and 1.0 mg.
Spectroscopic data for compound 3
Yellow amorphous solid; UV kmax MeOH nm: 221, 282; [a]D26 41 °
(MeOH, c 0.11); ESI-MS (positive mode) m/z: 528 [M + H]+; HI-FAB-
MS (positive mode) m/z: 528.2360 [M + H]+ (calcd for
C27H33N3O8,528.2346); 1H-NMR (500 MHz, MeOH-d4) d (ppm):
7.41 (1H, s), 7.38 (1H, d, J = 8.1 Hz), 7.33 (1H, d, J = 8.2 Hz), 7.16
(1H, d, J = 8.7 Hz), 7.05 (1H, t, J = 7.8 Hz), 6.91 (1H, t,
ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–9
J = 7.5 Hz), 6.84 (1H, d, J = 2.2 Hz), 6.63 (1H, dd, J = 8.5 Hz,
2.3 Hz), 4.75 (1H, d, J = 6.4 Hz), 4.61 (1H, m), 4.23 (1H, d,
J = 7.9 Hz), 4.08 (1H, dd, J = 7.1 Hz, 3.0 Hz), 3.84 (1H, dd,
J = 11.7 Hz, 2.1 Hz), 3.64 (1H, dd, J = 11.8 Hz, 5.7 Hz), 3.47 (1H,
dd, J = 7.0 Hz, 3.2 Hz), 3.34 (1H, m), 3.30 (1H, m), 3.29 (1H, m),
3.28 (1H, m), 3.27 (1H, m), 2.95 (1H, m); 13C-NMR (125 MHz,
MeOH-d4) d (ppm): 151.5, 141.2, 142.3, 147.7, 150.8, 128.8,
124.2, 122.5, 119.8, 119.5, 114.3, 112.7, 112.3, 112.1, 105.9,
104.9, 102.9, 78.1, 78.0, 75.2, 74.2, 74.1, 71.5, 62.6, 40.9, 38.6,
23.8.
Spectroscopic data for compound 4
Yellow amorphous solid; UV kmax MeOH nm: 223, 282; [a]
D
26
+ 58 ° (MeOH, c 0.48); ESI-MS (positive mode) m/z: 528;
[M + H]+ HI-FAB-MS (positive mode) m/z: 528.2360 [M + H]+
(calcd for C27H33N3O8, 528.2346); 1H-NMR (500 MHz, MeOH-
d4) d (ppm): 7.58 (1H, d, J = 8.0 Hz), 7.34 (1H, s), 7.32 (1H, d,
J = 8.3 Hz), 7.21 (1H, m), 7.17 (1H, d, J = 8.7 Hz), 7.08 (1H, t,
J = 7.3 Hz), 6.99 (1H, t, J = 7.1 Hz), 6.84 (1H, d, J = 2.2 Hz),
6.63 (1H, dd, J = 8.6 Hz, 2.3 Hz), 4.75 (1H, d, J = 7.6 Hz),
4.66 (1H, m), 4.27 (1H, d, J = 7.6 Hz), 3.97 (1H, dd,
J = 8.4 Hz, 3.2 Hz), 3.78 (1H, dd, J = 8.2 Hz, 2.1 Hz), 3.63
(1H, dd, J = 8.1 Hz, 5.5 Hz), 3.61 (1H, m), 3.32 (1H, t, J =
8.9 Hz), 3.30 (1H, t, J = 8.5 Hz), 3.27 (1H, t, J = 9.2 Hz), 3.17
(1H, m).3.12 (1H, m), 3.11 (1H, m); 13C-NMR (125 MHz,
MeOH-d4) d (ppm): 151.4, 139.1, 137.8, 132.5, 129.5, 128.0,
123.6, 122.7, 119.9, 119.2, 115.3, 112.5, 112.4, 111.9, 106.7,
105.1, 102.8, 78.0, 77.9, 75.3, 73.9, 73.6, 71.4, 62.3, 41.4,
37.1, 23.4.
Acknowledgements
The authors would like to thank Dr. Kawagishi (NICS) for
providing plasmid pM159. This work was supported by the
CREST program of the Japan Science and Technology Corpora-
tion. The authors have no conflict of interest.
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