Differential gene expression profiles of red and green
forms of Perilla frutescens leading to comprehensive
identification of anthocyanin biosynthetic genes
Mami Yamazaki
1,2
, Masahisa Shibata
1
, Yasutaka Nishiyama
1,3,
*, Karin Springob
1,
,
Masahiko Kitayama
3
, Norimoto Shimada
4
, Toshio Aoki
4
, Shin-ichi Ayabe
4
and Kazuki Saito
1,5
1 Graduate School of Pharmaceutical Sciences, Chiba University, Japan
2 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
3 Institute of Life Science, Ehime Women’s College, Uwajima, Japan
4 Department of Applied Biological Sciences, Nihon University, Fujisawa, Japan
5 RIKEN Plant Science Center, Yokohama, Japan
The plant chemovarietal forms, in which only the
chemical constituents of particular secondary products
differ, are interesting and useful for better understand-
ing of molecular regulation underlying the production

cDNAs from leaves of red and green perilla, two chemovarietal forms of
Perilla frutescens regarding anthocyanin accumulation. One hundred and
twenty cDNA fragments were selected as the clones preferentially expressed
in anthocyanin-accumulating red perilla over the nonaccumulating green
perilla. About half of them were the cDNAs encoding the proteins related
presumably to phenylpropanoid-derived metabolism. The cDNAs encoding
glutathione S-transferase (GST), PfGST1, and chalcone isomerase (CHI),
PfCHI1, were further characterized. The expression of PfGST1 in an Ara-
bidopsis thaliana tt19 mutant lacking the GST-like gene involved in vacuole
transport of anthocyanin rescued the lesion of anthocyanin accumulation
in tt19, indicating a function of PfGST1 in vacuole sequestration of antho-
cyanin in perilla. The recombinant PfCHI1 could stereospecifically convert
naringenin chalcone to (2S)-naringenin. PfGST1 and PfCHI1 were pre-
ferentially expressed in the leaves of red perilla, agreeing with the accumu-
lation of anthocyanin and expression of other previously identified genes
for anthocyanin biosynthesis. These results suggest that the genes of the
whole anthocyanin biosynthetic pathway are regulated in a coordinated
manner in perilla.
Abbreviations
CHI, chalcone isomerase; GST, glutathione S-transferase; GUS, b-glucuronidase.
3494 FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS
are available for most of the plants exhibiting interest-
ing chemovarieties, alternative technologies should be
applied to obtain comprehensive differential gene
expression profiles [4,5].
In Perilla frutescens (Labiatae), a medicinal plant
common in east Asian countries, there are two chem-
ovarietal forms, the red form (red perilla, ‘Aka-jiso’ in
Japanese) and the green form (green perilla, ‘Ao-jiso’),
differing in the accumulation of anthocyanins [6].

uoles has been experimentally proven. However, the
questions of whether this GST-like protein is com-
monly necessary for transport of anthocyanin in any
other plant species, and if so, how diverse the GST
proteins are in terms of their structures and functions,
remain to be solved by isolation and characterization
of functional orthologs from diverse plant species.
In the present study, we conducted differential gene
expression profiling between the anthocyanin-produc-
ing red form and the nonproducing green form by
PCR-select subtraction. This approach elucidated the
whole picture of differential gene expression behind
the differential anthocyanin production in the two
chemovarietal forms. The functions of two new differ-
entially expressed genes obtained by this method cod-
ing for GST and chalcone isomerase (CHI) have been
identified and characterized by in vivo and in vitro
studies.
Results and Discussion
PCR-select subtraction analysis gave the
comprehensive repertoire of genes differentially
expressed in red perilla
PCR-select subtraction analysis was conducted
between cDNAs from the leaves of red perilla and
green perilla. As a result of the first screening, 576
clones each were selected as specific candidates for red
perilla and green perilla. These clones were further
delimited to 120 clones specific for red perilla and 24
clones specific for green perilla by dot-blot hybridiza-
tion. The (partial) sequences of these delimited clones

PCR-select subtraction. The gene encoding caffeoyl-
CoA-3-O-methyltransferase, involved in phenylpro-
panoid metabolism leading to lignin formation, was
specifically expressed in red perilla, suggesting a higher
M. Yamazaki et al. Anthocyanin biosynthetic genes from Perilla
FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS 3495
activity of phenylpropanoid metabolism in red perilla
than in the green form. However, even by PCR-select
subtraction, not all genes that are differentially
expressed and involved in anthocyanin production
have been isolated. The gene encoding anthocyanin-5-
O-glucosyltransferase, predominantly expressed in red
perilla [10], failed to be cloned by this PCR-select
method. Thus, it would be desirable to apply several
different technologies to obtain the list of genes
expressing in a chemovariety-specific manner.
In addition to anthocyanin biosynthetic genes, red
perilla expressed a set of genes activated by light, such
as ATP synthase of photophosphorylation, one-helix
protein of photosystem II, Rieske [2Fe–2S] iron–sulfur
protein tic 55, RuBisCo activase, and T-protein of
glycine decarboxylase, involved in photorespiration.
This suggested that the gene expression regulated by
light signaling might be different between the red and
green forms of perilla, in addition to gene expression
for anthocyanin biosynthesis.
As a green perilla-specific gene, a gene encoding an
F-box protein was isolated. This might suggest the
possible involvement of F-box proteins in the degra-
dation of certain proteins related to the speciation of

from the majority of 16 clones was designated as
PfGST1. The deduced 214 amino acid sequences of
PfGST1 exhibited 61% and 50% identities, respec-
tively, with those of AN9 from petunia [13] and TT19
from A. thaliana [14] (supplementary Fig. S2). Phylo-
genetic analysis of deduced amino acid sequences of
GST-like proteins (Fig. 2) indicated that PfGST1
forms a subfamily together with AN9 and TT19, but
distinct from the maize Bz2 protein [12], which plays a
similar role in uptake of anthocyanin into vacuoles.
This presumably reflects the difference in the origin of
these proteins, either from eudicot or monocot plants.
Red specific (120) Green specific (24)
Others (13)
Cell wall protein (2)
Latex-like protein (2)
F-box protein (3)
Photo-response genes (1)
Signal transduction/Transcriptional factor (1)
Primary metabolism (2)
Others (34)
Transporter/membrane protein (7)
Photo-response genes (5)
Signal transduction/Transcriptional factor (14)
Secondary metabolism (56)
Primary metabolism (4)
AB
Fig. 1. Profiling of fragments with PCR-
select cDNA subtraction in P. frutescens.
Anthocyanin biosynthetic genes from Perilla M. Yamazaki et al.

sucrose stress, the accumulation of anthocyanin in
petioles was observed in the transgenic plants express-
AtGSTF10
AtGSTF12 (TT19)
AN9
PfGST1
AtGSTZ1
Bz2
AtGSTU19
AtGSTU5
AtGSTU7
AtGSTF8
AtGSTF2
AtGSTF6
AtGSTF7
61%
50%
Fig. 2. Phylogenetic tree of GSTs. The neighbor-joining tree was
constructed on the basis of deduced amino acid sequences of
PfGST1 (in this study), petunia AN9 (Y07721), maize Bz2 (X81971),
and Arabidopsis GSTs [AtGSTF2 (NM_116486), AtGSTF6
(NM_100174), AtGSTF7 (NM_100173), AtGSTF8 (NM_180148),
AtGSTF10 (NM_128639), AtGSTF12 (NM_121728), AtGSTU5
(NM_128499), AtGSTU7 (NM_128496), AtGSTU19 (NM_106485),
and AtGSTZ1 (NM_201671)]. The deduced amino acid sequence of
PfGST1 showed 61% and 50% identities, respectively, with those
of AN9 from petunia and AtGSTF12 (TT19) from A. thaliana. The
roles of PfGST1, AN9, AtGSTF12 (TT19) and Bz2 in anthocyanin
transport into vacuoles have been confirmed by experiments.
Red leaf

seedling of transgenic Arabidopsis plants transformed
with 35S-PfGST1 or 35S-GUS as control. Left panel: 35S-
PfGST1 ⁄ tt19. All of 10 resistant plants accumulated anthocyanin in
the petiole, as indicated by arrowheads. Right panel: 35S-GUS ⁄ tt19.
All of five resistant plants did not accumulate anthocyanin.
M. Yamazaki et al. Anthocyanin biosynthetic genes from Perilla
FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS 3497
ing the PfGST1 cDNA, whereas the nontransformed
tt19 plants and the negative control plants expressing
the bacterial b-glucuronidase (GUS) gene did not
accumulate anthocyanins (Fig. 4B). All 10 indepen-
dent transgenic plants expressing the PfGST1 cDNA
checked by RT-PCR contained more anthocyanin
than tt19 plants, and three of them accumulated
higher amounts of anthocyanins than the wild-type
plants (Fig. 5). There was a rough correlation between
the accumulation of anthocyanin and the expression
of PfGST1 (data not shown). The patterns of antho-
cyanin molecules that accumulated in the transgenic
plants were analyzed by HPLC-MS (Fig. 6). The pat-
tern of the transformant was almost identical to that
of the wild-type plants, showing a cyanidin-derived
anthocyanin [7] as the main compound. All these
results indicated that PfGST1 can functionally com-
plement the mutation of the TT19 gene encoding
GST-like protein that participates in uptake of antho-
cyanin into vacuoles. A carnation anthocyanin mutant
was complemented by the expression of maize Bz2
and petunia AN9 [20], indicating again the universal
necessity of GST-like proteins in anthocyanin accumu-

10
12
14
WT
Total peak area
(Absorbance at 520 nm)
35S-PfGST1/tt19
16
tt19
X 10
6
Fig. 5. Anthocyanin contents of T
2
plants. Anthocyanin contents in
the leaf extracts are represented as total peak area of the chroma-
tograms at 520 nm. Extracts were prepared from rosette leaves of
10 independent transgenic plants transformed with 35S-PfGST1.
Fig. 6. HPLC chromatograms of anthocya-
nins at 520 nm in the extracts of rosette
leaves of transgenic Arabidopsis. (A) tt19.
(B) 35S-GUS ⁄ tt19. (C) 35S-PfGST1 ⁄ tt19.
(D) Wild-type plant. (E) Structures of three
major anthocyanins accumulated in Arabid-
opsis [1]. Glu, glucose; Xyl, xylose; p-Cou,
p-coumaroyl; Sin, sinapoyl; Mal, malonyl.
Anthocyanin biosynthetic genes from Perilla M. Yamazaki et al.
3498 FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS
revealed that one of them, designated PfCHI1, con-
tained the entire ORF coding for the CHI protein.
The deduced 214 amino acid sequence exhibited 70%,

tures of differential gene expression profiling between
the anthocyanin-producing red form and the nonpro-
ducing green form of P. frutescens. Among the differ-
entially expressed genes, two new genes have been
identified as coding for a GST-like protein involved in
anthocyanin transport in vacuoles and a type I CHI,
and their roles have been confirmed by in vivo and
in vitro studies. The expression levels of all the genes
involved in anthocyanin accumulation, including
PfGST1 and PfCHI1, was higher in red perilla. These
results indicate the tightly coregulated transcription of
all genes of the anthocyanin pathway in perilla.
Experimental procedures
Plant materials
The red and green forms of P. frutescens var. crispa were
grown on rock wool with a nutrient solution of Hyponex
(5-10-5) in a plant growth room for 16 weeks with a photo-
period of 18 h light (4500 lux) ⁄ 6 h dark at 25 °C. A. thali-
ana (ecotype Columbia) plants were grown in a growth
Vitis vinifera CHI
Citrus sinensis CHI
Medicago sativa CHI
Lotus japonicus CHI1
PfCHI1
Phaseolus vulgaris CHI
Lotus japonicus CHI2
Lotus japonicus CHI3
Type I
Type II
70%

FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS 3499
chamber and used for transformation as described previ-
ously [1].
PCR-select subtraction
Total RNA was isolated from young leaves of red and
green P. frutescens around 4–5 h after exposure to light by
RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan). PCR-select
subtraction was carried out between cDNAs from leaves of
red and green perilla as described previously [22,23].
cDNA cloning of PfGST1
To obtain a cDNA coding for the entire PfGST1 protein,
PCR amplification was carried out using a primer (GST-0,
5¢-ATGGTGGTTAAAGTGTATGGTGCAACC-3¢)andan
oligo-dT primer with the first-strand cDNA reverse-tran-
scribed from RNA of red perilla with Pyrobest DNA poly-
merase (Takara, Japan). The sequence of the GST-0 primer
containing the first Met codon was designed by alignment
analysis of four fragments obtained by PCR-select sub-
traction with the known GST genes. The protruding dA
residues were attached to the amplified fragment by Ex Taq
polymerase (Takara, Japan), and then the resulting frag-
ment was cloned into pGEM-T Easy (Promega, KK,
Tokyo, Japan) to give pGTE-PfGST1.
Construction of Agrobacterium-Ti plasmid
vector and plant transformation for PfGST1 by
GATEWAY technology
To attach attB sequences on both sides of PfGST1 cDNA,
two rounds of PCR reactions were performed with pGTE–
PfGST1 as the template. The sequences of primers were:
GST-1-f (5¢-AAAAAGCAGGCTACATGGTGGTTAAAG

and GST-1-r.
Anthocyanin determination
Aseptic Arabidopsis plants were grown on GM agar plates
for 2 weeks, and then transferred to GM agar plates supple-
mented with 10% sucrose for 1 week to induce the produc-
tion of anthocyanins by sucrose stress. Anthocyanins were
extracted with 5 l L of extraction solution (5% acetic acid,
45% methanol, and 50% water) per 1 mg of leaves with a
Mixer Mill MM300 (Qiagen). After centrifugation at 12 000 g
for 10 min, the supernatant solution was subjected to anal-
ysis of anthocyanins by LC-photodiode array-MS [Agilent,
ThermoQuest ⁄ Finnigan (San Jose, CA, USA) LCQ DECA]
as described previously [1].
Construction of the expression vector for
PfCHI1 by GATEWAY technology and expression
in E. coli
As one of two cDNA fragments isolated by PCR-select
subtraction designated as PfCHI1 was suggested to encode
the entire protein of CHI, by sequence comparison with
known CHI, the GATEWAY-compatible entry clone
pD221–PfCHI1 was constructed by attaching attB sites
using the primers CHI-1-f (5¢-AAAAAGCAGGCTA
CATGTCTGTGACTCAAGTCCAAGTGG-3¢) and CHI-
1-r (5¢-AGAAAGCTGGGTGCTAATTCTGGTTGAAC
AAGTGGGACAATCT-3¢). The PfCHI1 gene in pD221–
PfCHI1 was introduced into pDEST17, an expression vec-
tor in E. coli, to afford pD17–PfCHI1. E. coli BL21 AI
(Invitrogen, Carlsbad, CA, USA) was transformed with
pD17–PfCHI1, and the recombinant protein of PfCHI1
with a 6His tag at the N-terminus was expressed upon

minator sequencing Kit (ABI) and a PRISM 3100 genetic
analyzer (ABI) in the CREST-Akita Satellite Laboratory
for Plant Molecular Sciences. Sequence analysis was carried
out by blast and blast x programs against the GenBank
database at National Center for Biotechnology Infor-
mation. The molecular phylogenetic tree was constructed
with clustalw and visualized using tree view software.
Standard molecular techniques for recombinant DNA and
protein were according to published protocols [27].
Acknowledgements
We thank Dr S. Kitamura for providing Arabidopsis
seeds of the tt19 mutant and 35S-TT19 ⁄ tt19 transgenic
plants and CREST-Akita Satellite Laboratory for
Plant Molecular Sciences for DNA sequencing. This
work was supported, in part, by the Grants-in-Aid for
Scientific Research from the Japan Society for the Pro-
motion of Science (JSPS), and by CREST of Japan
Science and Technology. K. Springob was a recipient
of a postdoctoral fellowship from the JSPS.
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