Sekiguchi lesion gene encodes a cytochrome P450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice

Article abstract of DOI:10.1074/jbc.M109.091371

Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N- acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the Gα subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

Full text of DOI:10.1074/jbc.M109.091371

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11308–11313, April 9, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Sekiguchi Lesion Gene Encodes a Cytochrome P450
Monooxygenase That Catalyzes Conversion of Tryptamine
□
S
to Serotonin in Rice*
Received for publication, December 4, 2009, and in revised form, February 2, 2010 Published, JBC Papers in Press, February 11, 2010, DOI 10.1074/jbc.M109.091371
Tadashi Fujiwara‡1, Sylvie Maisonneuve‡1, Masayuki Isshiki‡1,2, Masaharu Mizutani§, Letian Chen‡,
Hann Ling Wong‡, Tsutomu Kawasaki‡, and Ko Shimamoto‡3
From the ‡Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192
and the §Graduate School of Agricultural Science, Kobe University, Rokkodai, Nada, Kobe 657-8501, Japan
Serotonin is a well known neurotransmitter in mammals and
plays an important role in various mental functions in humans.
In plants, the serotonin biosynthesis pathway and its function
are not well understood. The rice sekiguchi lesion (sl) mutants
accumulate tryptamine, a candidate substrate for serotonin bio-
synthesis. We isolated the SL gene by map-based cloning and
found that it encodes CYP71P1 in a cytochrome P450 monooxy-
genase family. A recombinant SL protein exhibited tryptamine
5-hydroxylase enzyme activity and catalyzed the conversion of
tryptamine to serotonin. This pathway is novel and has not been
reported in mammals. Expression of SL was induced by the
N-acetylchitooligosaccharide (chitin) elicitor and by infection
with Magnaporthe grisea, a causal agent for rice blast disease.
Exogenously applied serotonin induced defense gene expres-
sion and cell death in rice suspension cultures and increased
resistance to rice blast infection in plants. We also found that
serotonin-induced defense gene expression is mediated by the
RacGTPase pathway and by the G␣ subunit of the heterotrimer-
ic G protein. These results suggest that serotonin plays an
important role in rice innate immunity.
tigated in many plant species, including rice, Arabidopsis, and
maize (3). Most of lesion mimic mutants are known to activate
immune responses in the absence of pathogens (2). A rice lesion
mimic mutant, Sekiguchi lesion (sl), exhibits unique orange
colored lesions that are induced by inoculation of pathogens,
including Magnaporthe grisea (4, 5). In the sl mutants, in-
creased activities of tryptophan decarboxylase and monoamine
oxidase are correlated with lesion formation (5). However, the
identity of the SL gene and its function in cell death and disease
resistance remain to be studied.
Plant Rac/Rop GTPases constitute a unique subfamily of the
Rho family of small GTPases and are highly conserved in plant
species. In rice, OsRac1 regulates a series of immune responses,
including cell death, reactive oxygen species production, acti-
vation of pathogenesis-related genes, liginification, and phyto-
alexin production (6). Recently, it was found that OsRac1 forms
a complex with RAR1, HSP90, HSP70, OsMAPK6, OsrbohB,
Sti1/Hop, and RACK1 (7–10), which play important roles in
rice innate immunity. Heterotrimeric G protein is also involved
in innate immunity through OsRac1. In fact, rice dwarf1 (d1)
mutants lacking a G␣ subunit of heterotrimeric G protein
reduces elicitor-induced defense gene expression and resis-
tance to M. grisea (11).
Plants have developed various immune responses to pro-
tect themselves from pathogen attack (1). One of them is the
hypersensitive response, which is often accompanied by
programmed cell death, and is effective to prevent the
growth of biotrophic pathogens (2). The hypersensitive
response is induced coordinately with production of reactive
oxygen species, lignification, production of phytoalexins,
and expression of defense-related genes.
Serotonin is known as neurotransmitter in mammals, which
regulates mood, sleep, and anxiety in human (12). Although
serotonin was found in many plant species (13), the serotonin
biosynthesis and its function are unclear. Recently, serotonin
has been reported to be accumulated in rice during senescence
and defense response (14, 15). In addition, serotonin is incor-
porated into the cell wall at disease lesions infected with Bipo-
laris oryzae and M. grisea, suggesting that serotonin plays an
important role in control of the strength of the cell wall for the
mechanical barrier against pathogens (15).
To understand the roles of hypersensitive response cell death
in immune responses, a large number of lesion mimic mutants,
in which cell death is spontaneously induced, have been inves-
To understand how the SL gene regulates cell death and disease
resistance in rice, we have isolated the SL gene by a map-based
approach. We found that the SL gene encodes cytochrome P450
monooxygenase, which catalyzes 5-hydroxylation of tryptamine
to synthesize serotonin. In addition, our results suggest that sero-
tonin contributes to innate immunity by activating intracellular
signaling of immune responses.
* This work was supported by grants-in-aid from the Ministry of Agriculture,
Forestry, and Fisheries of Japan (Rice Genome Project IP4001), the Japan
Society for Promotion of Science Grant 13G0023 (to K. S.), and the Program
for Promotion of Basic Research Activities for Innovative Biosciences
(PROBRAIN) (to T. K.).
□S
The on-line version of this article (available at http://www.jbc.org)
contains supplemental “Experimental Procedures,” Tables S1 and S2,
and Figs. S1–S3.
1 These authors contributed equally to this work.
2 Presentaddress:KiharaInstituteforBiologicalResearch, YokohamaCityUni-
versity, Yokohama 244-0813, Japan.
EXPERIMENTAL PROCEDURES
3 To whom correspondence should be addressed. Tel.: 81-743-72-5500; Fax:
81-743-72-5502; E-mail: simamoto@bs.naist.jp.
Plant Materials and Genetic Mapping—The wild type japo-
nica rice cv. Kinmaze and four sl mutants (CM265, CM998,
11308 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 15•APRIL 9, 2010

Serotonin Biosynthesis in Plant
CM1019, and CM2229), induced by N-methyl-N-nitrosourea
in the Kinmaze background, were provided by the Japanese
National BioResource Project of Rice. The CM265 mutant
was used in a cross with a substitution line (SL239) carrying
a segment of chromosome 12 from an indica rice (cv. Kasal-
ath) in a japonica rice background (cv. Koshihikari). The F2
and F3 plants were analyzed using a sequence-tagged site and
cleaved amplified polymorphic sequence markers to con-
struct a physical map of the region containing the SL gene
(supplemental Fig. S1). Details of the mapping are provided
in the supplemental “Experimental Procedures”.
Plasmid Constructs and Rice Transformation—The SL
cDNA was cloned into the p2K1 vector containing the maize
Ubq1 promoter (16). For the RNA interference construct of
Sti1a/Hop, a 350-bp 3Ј-untranslated region of the Sti1a cDNA
was PCR-amplified and cloned in the reverse orientation
behind the Ubp1 promoter in the pANDA vector (16). The
RNA interference construct of OsRac1 was described previ-
ously (10). Details of plasmid construction are provided in
supplemental “Experimental Procedures”. Transgenic plants
were generated from Agrobacterium-transformed rice calli
(17).
Reverse Transcription-PCR and Real Time PCR—Total
RNAs were isolated from leaves and suspension cultures using
TRIzol reagent (Invitrogen) and treated with DNase I (Invitro-
gen). cDNA was synthesized using SuperScriptII reverse tran-
scriptase (Invitrogen) and used for reverse transcription-PCR
and real time PCR with gene-specific primers (see supple-
mental Table S1).
Rice Blast Infection—Rice seedlings were inoculated at the
four- to six-leaf stage by spraying with aqueous spore sus-
pensions containing 105 spores/ml of the compatible rice
blast races 003 and 007. Inoculated seedlings were kept in
a dark chamber with a moisture-saturated atmosphere at
24 °C for 20 h and then maintained in a greenhouse. Inocu-
lated leaves were harvested at the indicated time points and
used for real time PCR. To examine the effect of serotonin on
rice blast resistance, rice seedlings were watered with 400
␮g/ml serotonin for 22 h and then inoculated with race 007
as described above. Lesion development was scored on rice
leaves at 6 days after inoculation by measuring lesion length
with a ruler.
Chitin, Serotonin, and Tryptamine Treatments—Rice sus-
pension cultures were treated with 2 ␮g/ml N-acetylchitooligo-
saccharide elicitor, 400 ␮g/ml (1.88 mM) serotonin (5-hy-
droxytryptamine hydrochloride (Mr 212.68)), or 400 ␮g/ml
(2.03 mM) tryptamine (tryptamine hydrochloride (Mr 196.68)),
and then incubated for various lengths of time. As reported
previously (15), less than 30% of applied serotonin is considered
to be incorporated by the feeding experiment. Total RNAs were
extracted from each sample and subjected to real time PCR. For
FIGURE 1. SL gene encodes a cytochrome P450 monooxygenase. A, struc-
ture of the SL gene. Arrows indicate the locations of mutations found in the sl
mutants. Arrowheads indicate locations of primers used in B. aa, amino acids.
B, expression of SL in the sl mutants. Reverse transcription-PCR was per-
formed using specific primers for SL and ubiquitin. WT, wild type. C, subcellu-
lar localization of SL-GFP. GFP (upper panel) and SL-GFP (lower panel) were
transiently expressed in rice protoplasts with ER-localized SECFP. Left panel,
GFP image; middle panel, SECFP image; right panel, merged images of GFP
andSECFP. Bars, 10␮m. D, expressionofSLafterelicitortreatment. TotalRNAs
were purified from rice suspension cultured cells either mock-treated or
treated with 2 ␮g/ml N-acetylchitooligosaccharide elicitor and used for real
time PCR. Ubiquitin (Ubq) was used as an internal control. Error bars indicate
standard deviations. E, expression of SL in seedlings after mock inoculation or
inoculation with compatible rice blast fungus (race 003 and 007). Real time
PCR was carried out using total RNAs prepared from the seedlings. Ubq was
used as an internal control. Error bars indicate S.D.
APRIL 9, 2010•VOLUME 285•NUMBER 15
JOURNAL OF BIOLOGICAL CHEMISTRY 11309

Serotonin Biosynthesis in Plant
the detection of cell death, suspension-cultured cells were
Immunoblotting—Microsomal fractions of the insect cells,
treated with serotonin or tryptamine for 24 h, stained for 10 expressing either SL or SL-G455D, were subjected to immuno-
blotting using a purified anti-SL antibody as described in
supplemental “Experimental Procedures.”
min with 0.05% (w/v) Evans Blue, and then washed four times
with distilled water (18). Dye bound to dead cells was dissolved
in a solution of 50% methanol and 1% SDS for 30 min at 50 °C
and quantified by absorbance at 595 nm.
RESULTS
Isolation of SL Gene by Map-based Cloning—To identify a
gene responsible for the sl phenotype, we used four sl mutants
(CM265, CM2229, CM998, and CM1019; supplemental Fig.
S1), which were generated by mutagenesis using N-methyl-N-
nitrosourea (21). The sl locus was mapped to a 350-kb genomic
region on chromosome 12, where 7 candidate genes are located
(supplemental Fig. S1). PCR experiments indicated that the
CM998 mutant has a 30-kb deletion containing genes for a
P450 and a zinc finger protein.5 Sequence analyses in the other
mutants revealed that SL encodes a P450 monooxygenase (Fig.
1A). SL gene expression levels were significantly reduced in
CM2229. CM998 showed no SL expression because of the dele-
tion of the entire P450 gene. CM1019 had two altered tran-
scripts as follows: an unspliced transcript resulting from a point
mutation at the 3Ј splice site of the single intron, and another
generated by cryptic splicing at a site near the 3Ј splice site (Fig.
1B). Overexpression of the P450 mRNA complemented the sl
phenotype (supplemental Fig. S2).
Measurement of SL Activities—The SL and SL-G455D
(CM265) cDNAs were cloned as SpeI-PstI fragments in the
pFastBac1 vector (Invitrogen) and used to transform E. coli
strain DH10Bac (Invitrogen). Preparation of the cDNA-
containing recombinant baculovirus DNAs and transfection
of Spodoptera frugiperda 9 (Sf9) cells were carried out
according to the manufacturer’s instructions (Invitrogen).
Heterologous expression in Sf9 cells and spectrophotomet-
ric analysis of P450s were carried out as described previously
(19).
Microsomal fractions of insect cells expressing either SL
or SL-G455D were obtained as follows. Infected suspension-
cultured cells (300 ml) were washed with phosphate-buff-
ered saline buffer and suspended in buffer A, consisting of 20
mM potassium phosphate (pH 7.25), 20% (v/v) glycerol, 1 mM
EDTA, and 1 mM dithiothreitol. The cells were sonicated,
and cell debris was removed by centrifugation at 10,000 ϫ g
for 15 min. The supernatant was further centrifuged at
100,000 ϫ g for 1 h, and the pellet was homogenized with
buffer A to provide the microsomal fractions. The microso-
mal fractions were stored at Ϫ80 °C before the enzyme
assays were performed.
SL Belongs to the CYP71 Family—The SL protein possesses
motifs conserved in P450 proteins, including the heme-bind-
ing motif, the EXXR motif, and the proline-rich motif
(supplemental Fig. S3). The G455D mutation in CM265 is in
the heme-binding motif and is likely to disturb the heme
coordination structure. The threonine residue at position
314, which is mutated in CM2229, is important for the
oxygen activation process in the P450 catalytic cycle
(supplemental Fig. S3). The SL protein shares 41% identity
with CYP71A1 from avocado fruit (GenBankTM accession
number AAA32913). In rice there are 90 genes and 28 pseu-
dogenes in the CYP71 family; however, the SL protein shows
less than 45% identity with any other members of the family.
Hence, the SL protein is a unique member of the CYP71
family and is designated CYP71P1.
SL activity was reconstituted by mixing the microsomes
with purified Arabidopsis NADPH-P450 reductase (20). The
reaction mixture consisted of 20 mM potassium phosphate
(pH 7.25), 50 pmol/ml recombinant P450 protein, 0.1
unit/ml NADPH-P450 reductase, 1 mM NADPH, and 100 ␮M
tryptamine. Reactions were initiated by the addition of
NADPH, and were carried out at 30 °C for 30 min. The sam-
ples were diluted with 20 mM phosphate (pH 2.5) and sub-
jected to HPLC4 as follows: column, COSMOSIL 5C18-PAQ
column (150 mm length ϫ 4.6 mm inner diameter; Nacalai
Tesque); solvent, 20 mM phosphate (pH 2.5); flow rate, 1.0
ml/min; detection, a fluorescence detector with an excita-
tion wavelength at 285 nm and an emission wavelength at
340 nm. Retention times of authentic samples were 6.5 min
for serotonin and 14.5 min for tryptamine. The identity of
the reaction product with tryptamine was examined using
HPLC equipped with an API-3000 triple stage quadrupole
mass spectrometer (Applied Biosystems) as described previ-
ously (15). For kinetic analysis, SL activity was assayed using
the substrate tryptamine at concentrations ranging from 1 to
40 ␮M. Reactions were initiated by the addition of NADPH
and carried out at 30 °C for 10 min. The kinetic constants
were calculated from triplicate data sets. Km and kcat values
were calculated from a double-reciprocal plot of the initial
velocity (v0) versus substrate concentration.
The intracellular location of the SL protein was investigated
using the transient expression of a GFP-fused protein in rice
protoplasts (Fig. 1C). The SL protein, with GFP fused to the C
terminus, co-localized with a marker protein that was designed
to localize to the endoplasmic reticulum (ER). The marker pro-
tein was constructed by adding the N-terminal signal peptide of
pro-2S albumin, which localizes to the ER (22), and the C-ter-
minal HDEL ER retention sequence, to SECFP (23). The results
indicate that SL localizes to the ER.
SL Gene Is Expressed during Innate Immune Responses—Ex-
pression of the SL gene in rice suspension cultures was analyzed
after treatment with a chitin elicitor that is known to induce a
series of defense responses in rice (Fig. 1D) (24). A clear
increase in SL mRNA levels was observed at 1 h after chitin
treatment (Fig. 1D). We also analyzed SL expression after infec-
tion with M. grisea. Infection by two races of compatible blast
fungus caused the activation of SL gene expression in rice plants
4 The abbreviations used are: HPLC, high pressure liquid chromatography;
TDC, tryptophan decarboxylase; ER, endoplasmic reticulum; GFP, green
fluorescent protein; SECFP, super enhanced cyan fluorescent protein.
5 M. Isshiki, unpublished results.
11310 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 15•APRIL 9, 2010

Serotonin Biosynthesis in Plant
at 48 h after infection, and expression was further increased at These results indicate that SL protein is indeed a cytochrome
72 h (Fig. 1E). These results suggest that SL has a role in rice P450 enzyme. The microsomal fractions from insect cells ex-
innate immunity.
pressing the recombinant SL proteins were used in enzyme
SL Protein Possesses Tryptamine 5-Hydroxylase Activity—It assays with tryptamine as the potential substrate. HPLC analy-
was shown previously that tryptamine levels were higher in an sl sis of the reaction products showed a specific peak at the same
mutant than in wild type plants and that serotonin levels were retention time as that of serotonin when fractions containing
very low (4, 5, 15). These results suggest that SL converts the wild type SL protein, but not the SL-G455D mutant protein,
tryptamine to serotonin. To test this possibility, we generated were used (Fig. 2C). The SL protein catalyzed the 5-hydroxyla-
recombinant SL protein with or without the G455D mutation tion of tryptamine with a high affinity and efficiency (Km
ϭ
found in the CM265 allele, using a baculovirus expression sys- 7.3 Ϯ 0.8 ␮M and kcat ϭ 45 Ϯ 1.5 minϪ1), although the SL
tem in insect cells. The presence of the recombinant SL and protein did not metabolize tryptophan, indole 3-acetic acid, or
SL-G455D proteins in the microsomal fractions was confirmed tyramine. Thus, it is likely that the SL protein is tryptamine
by immunoblot analysis (Fig. 2A). Fractions containing the wild 5-hydroxylase that catalyzes the conversion of tryptamine to
type SL protein had a cytochrome P450-specific reduced CO serotonin.
difference spectrum with a clear absorption peak at 450 nm
Serotonin Plays a Role in Innate Immune Responses—Sero-
(Fig. 2B). In contrast, the fractions containing the SL-G455D tonin has been reported to be accumulated at a high level (1.9
protein exhibited no reduced CO difference spectrum (Fig. 2B). mM) in disease lesions infected with B. oryzae and M. grisea (15),
whereas the high accumulation of
serotonin is abolished in the sl
mutant. In addition, the sl mutant is
more susceptible to infection of B.
oryzae as compared with wild type,
and application of serotonin sup-
presses the fungal growth in the
leaves of the sl mutant (15). Thus, it
is possible that serotonin is involved
in innate immunity. To elucidate
how serotonin regulates immunity
responses, we analyzed the expres-
sion of four defense genes, probena-
zol 1 (PBZ1), phenylalanine ammo-
FIGURE 2. SL is a tryptamine 5-hydroxylase. A, production of recombinant SL proteins in insect cells using a
baculovirus expression system. Microsomal fractions from the insect cells expressing the recombinant SL
proteins were separated by SDS-PAGE and analyzed on an immunoblot using the anti-SL antibody. CBB, Coo-
massie Brilliant Blue. B, CO difference spectrum of the microsomal fractions expressing SL proteins. C, HPLC
nia-lyase 1(PAL1), chitinase 1 (Cht1),
and chitinase 3 (Cht3) in rice cell
suspension cultures. These four
analysis of reaction products after the microsomal SL proteins were assayed for enzyme activity using
genes are induced by chitin elicitor
tryptamine as a substrate.
FIGURE 3. Serotonin induces defense gene expression and cell death in rice suspension cell cultures. Total RNAs were prepared from rice suspension cells
either mock-treated or treated with 400 ␮g/ml serotonin or tryptamine. The samples were subjected to real time PCR to analyze expression of PBZ1 (A), PAL1
(B), Cht1 (C), and Cht3 (D). Ubiquitin (Ubq) was used as an internal control. Error bars indicate S.D. E, rice suspension cultured cells were treated with 400 ␮g/ml
serotonin or tryptamine. The levels of cell death were measured using Evans Blue staining.
APRIL 9, 2010•VOLUME 285•NUMBER 15
JOURNAL OF BIOLOGICAL CHEMISTRY 11311

Serotonin Biosynthesis in Plant
Because serotonin was able to
induce immune responses in rice
cell cultures, we examined the pos-
sibility that known components of
rice innate immunity are involved in
serotonin-induced defense gene
induction. It has been shown that
the small GTPase OsRac1 and its
signaling partners, including Sti1/
Hop and a heterotrimeric G protein,
are involved in rice innate immunity
(8, 10, 26). For these experiments,
we used rice cell cultures expressing
RNA interference knockdown con-
structs for OsRac1 and Sti1/Hop,
and a cell culture of the dwarf1 (d1)
mutant, which lacks a functional G␣
subunit of the heterotrimeric G pro-
tein (11). The results clearly showed
that these proteins are involved in
serotonin-induced defense gene
induction (Fig. 4, A–D).
Because our results suggest that
serotonin plays a role in rice innate
immunity, we examined whether
serotonin treatment of rice seed-
lings (ϳ1 month old) had any effect
FIGURE 4. Involvement of OsRac1, Sti1/Hop, and the G␣ subunit in serotonin-induced defense gene
expression. Expression of PBZ1 (A), PAL1 (B), Cht1 (C), and Cht3 (D) in wild type (WT), OsRac1, and Sti1a RNA
interference and d1 mutant cells treated with serotonin (S) or water (mock; M). Total RNA samples were ana- on infection with a compatible race
lyzed by real time PCR. Ubiquitin (Ubq) was used as an internal control. Error bars indicate S.D.
of M. grisea. The results indicate
that seedlings treated with sero-
tonin are significantly more resistant to fungal infection than
mock-treated seedlings (Fig. 5), suggesting that serotonin has
the ability to induce resistance against rice blast infection.
DISCUSSION
Serotonin is a well known neurotransmitter in mammals and
plays an important role in various mental functions in humans
(27). In mammals, serotonin is synthesized from tryptophan in
two steps involving L-tryptophan 5-hydroxylase and aromatic
L-amino acid decarboxylase (Fig. 6) (28). Tryptophan hydrox-
ylase belongs to a family of aromatic amino acid hydroxylases
and requires iron and the coenzyme tetrahydropterin for cata-
lytic activity (29). It is interesting that the SL gene encodes
CYP71P1 in the P450 superfamily, which is different from the
tetrahydropterin-dependent hydroxylase family in mammals
FIGURE 5. Induction of rice blast resistance by serotonin treatment. Rice
(Fig. 6). Serotonin has been found in more than 42 plant species
seedlingswereinoculatedwiththecompatibleblastfungusrace007. Disease
(13), suggesting that enzymes for serotonin biosynthesis are
lesion development was scored at 6 days after inoculation by measuring
lesion length. Error bars indicate standard error (n ϭ 78). An asterisk indicates
a significant difference by the t test at p Ͻ 0.01.
conserved in plants. However, the SL orthologs have not been
found in other plants, including Arabidopsis. Among the Ara-
bidopsis P450s, CYP83B1/SUR2 shows the highest identity
from fungal pathogens (25).6 Expression of these defense genes
(39%) with the SL protein. CYP83B1/SUR2 catalyzes the N-hy-
was clearly induced at 12 h after serotonin treatment, but no
droxylation of indole-3-acetaldoxime to synthesize indole glu-
induction was found with tryptamine (Fig. 3, A–D). Serotonin
cosinolate (30).
also induced cell death in cultured rice cells, as shown by Evans
It has been reported that some serotonin derivatives such as
Blue staining (Fig. 3E), although reduced levels of cell death
coumaroylserotonin and feruloylserotonin, which are hydroxy-
occurred after tryptamine treatment.
cinnamic acid amides, are involved in plant defense responses
against pathogen infection (31). The accumulation of hydroxy-
6 S. H. Kim, unpublished results.
cinnamic acid amides in the cell wall could provide an unde-
11312 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 15•APRIL 9, 2010

Serotonin Biosynthesis in Plant
REFERENCES
1. Hammond-Kosack, K. E., and Parker, J. E. (2003) Curr. Opin. Biotechnol.
14, 177–193
2. Hofius, D., Tsitsigiannis, D. I., Jones, J. D., and Mundy, J. (2007) Semin.
Cancer Biol. 17, 166–187
3. Lorrain, S., Vailleau, F., Balague´, C., and Roby, D. (2003) Trends Plant Sci.
8, 263–271
4. Arase, S., Ueno, M., Toko, M., Honda, Y., Itho, K., and Ozoe, Y. (2001) J.
Phytopathol. 149, 409–413
5. Ueno, M., Shibata, H., Kihara, J., Honda, Y., and Arase, S. (2003) Plant J.
36, 215–228
6. Kawasaki, T., Henmi, K., Ono, E., Hatakeyama, S., Iwano, M., Satoh, H.,
and Shimamoto, K. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10922–10926
7. Lieberherr, D., Thao, N. P., Nakashima, A., Umemura, K., Kawasaki, T.,
and Shimamoto, K. (2005) Plant Physiol. 138, 1644–1652
8. Thao, N. P., Chen, L., Nakashima, A., Hara, S., Umemura, K., Takahashi,
A., Shirasu, K., Kawasaki, T., and Shimamoto, K. (2007) Plant Cell 19,
4035–4045
9. Wong, H. L., Pinontoan, R., Hayashi, K., Tabata, R., Yaeno, T., Hasegawa,
K., Kojima, C., Yoshioka, H., Iba, K., Kawasaki, T., and Shimamoto, K.
(2007) Plant Cell 19, 4022–4034
10. Nakashima, A., Chen, L., Thao, N. P., Fujiwara, M., Wong, H. L., Kuwano,
M., Umemura, K., Shirasu, K., Kawasaki, T., and Shimamoto, K. (2008)
Plant Cell 20, 2265–2279
11. Suharsono, U., Fujisawa, Y., Kawasaki, T., Iwasaki, Y., Satoh, H., and Shi-
mamoto, K. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 13307–13312
12. Gingrich, J. A., and Hen, R. (2001) Psychopharmacology 155, 1–10
13. Roshchina, V. V. (2001) Neurotransmitters in Plant Life (Roshchina, V. V.,
ed) pp. 4–81, Science Publisher, Enfield, NH
FIGURE 6. Model for serotonin biosynthetic pathways in plants and
mammals.
gradable mechanical barrier for pathogens (31, 32). In fact, it was
shown that serotonin itself is polymerized and incorporated into
cell walls in rice, leading to the suppression of growth of the fungus
14. Kang, K., Kim, Y. S., Park, S., and Back, K. (2009) Plant Physiol. 150,
1380–1393
B. oryzae (15). It was also shown that hydroxycinnamic acid 15. Ishihara, A., Hashimoto, Y., Tanaka, C., Dubouzet, J. G., Nakao, T., Mat-
suda, F., Nishioka, T., Miyagawa, H., and Wakasa, K. (2008) Plant J. 54,
481–495
amides possess anti-fungal activity (33). These studies suggest that
serotonin may act as a phytoalexin, an antimicrobial compound
that is produced by plant cells in response to pathogen infection.
However, wefoundnoeffectofserotoninonthegerminationof M.
grisea spores (supplemental Table S2). In this study, we found that
serotonin induces immune responses including defense gene
expression and cell death through the pathway involved in G␣,
OsRac1, and Sti1. Taken together, it is suggested that serotonin
may have dual functions that regulate the strength of the cell wall
for the physical barrier against pathogens as well as activate intra-
cellular signaling for immune responses.
16. Miki, D., and Shimamoto, K. (2004) Plant Cell Physiol. 45, 490–495
17. Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994) Plant J. 6, 271–282
18. Hayashi, M., Takahashi, H., Tamura, K., Huang, J., Yu, L. H., Kawai-
Yamada, M., Tezuka, T., and Uchimiya, H. (2005) Proc. Natl. Acad. Sci.
U.S.A. 102, 7020–7025
19. Saito, S., Hirai, N., Matsumoto, C., Ohigashi, H., Ohta, D., Sakata, K., and
Mizutani, M. (2004) Plant Physiol. 134, 1439–1449
20. Mizutani, M., and Ohta, D. (1998) Plant Physiol. 116, 357–367
21. Satoh, H., and Omura, T. (1979) J. Fac. Agr. Kyushu Univ. 24, 165–174
22. Hara-Hishimura, I., Takeuchi, Y., Inoue, K., and Nishimura, M. (1993)
Plant J. 4, 793–800
We have identified SL as tryptamine 5-hydroxylase enzyme 23. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., and Miyawaki,
A. (2002) Nat. Biotechnol. 20, 87–90
for serotonin biosynthesis, and we showed that in rice, sero-
tonin is synthesized in different pathways found in animals.
Recently, it was reported that serotonin is accumulated during
senescence in rice (14). However, whether SL is involved in
senescence response remains to be analyzed. Two genes, tryp-
24. Yamaguchi, T., Yamada, A., Hong, N., Ogawa, T., Ishii, T., and Shibuya, N.
(2000) Plant Cell 12, 817–826
25. Midoh, N., and Iwata, M. (1996) Plant Cell Physiol. 37, 9–18
26. Ono, E., Wong, H. L., Kawasaki, T., Hasegawa, M., Kodama, O., and Shi-
mamoto, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 759–764
tophan decarboxylase and SL, required for serotonin synthesis 27. Geyer, M. A., and Vollenweider, F. X. (2008) Trends Pharmacol. Sci. 29,
445–453
from tryptophan have now been identified in plants. It is there-
28. Mockus, S. M., and Vrana, K. E. (1998) J. Mol. Neurosci. 10, 163–179
fore possible to control the serotonin levels in plants by manip-
29. Fitzpatrick, P. F. (2003) Biochemistry 42, 14083–14091
ulating the expression of these genes.
30. Bak, S., Tax, F. E., Feldmann, K. A., Galbraith, D. W., and Feyereisen, R.
(2001) Plant Cell 13, 101–111
Acknowledgments—We thank Yuko Tamaki for technical assistance
and Nagao Hayashi for help with the rice blast infections. We thank
the Rice Genome Resource Center for providing a substitution line
and a full-length cDNA clone, and the National BioResource Project
of Rice for providing the sl mutant lines.
31. Hahlbroch, K., and Scheel, D. (1989) Annu. Rev. Plant Physiol. Plant Mol.
Biol. 40, 347–369
32. Keller, H., Hohlfeld, H., Wray, V., Hahlbrock, K., Scheel, D., and Strack, D.
(1996) Phytochemistry 42, 389–396
33. Miyagawa, H., Ishihara, A., Nishimoto, T., Ueno, T., and Mayama, S.
(1995) Biosci. Biotechnol. Biochem. 59, 2305–2306
APRIL 9, 2010•VOLUME 285•NUMBER 15
JOURNAL OF BIOLOGICAL CHEMISTRY 11313

Plant Biology:
Sekiguchi Lesion Gene Encodes a
Cytochrome P450 Monooxygenase That
Catalyzes Conversion of Tryptamine to
Serotonin in Rice
Tadashi Fujiwara, Sylvie Maisonneuve,
Masayuki Isshiki, Masaharu Mizutani, Letian
Chen, Hann Ling Wong, Tsutomu Kawasaki
and Ko Shimamoto
J. Biol. Chem. 2010, 285:11308-11313.
doi: 10.1074/jbc.M109.091371 originally published online February 11, 2010
Access the most updated version of this article at doi: 10.1074/jbc.M109.091371
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2010/02/11/M109.091371.DC1.html
This article cites 32 references, 15 of which can be accessed free at
http://www.jbc.org/content/285/15/11308.full.html#ref-list-1