The expression of PfGST1 in an Ara-bidopsis thaliana tt19mutant lacking the GST-like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in t
Trang 1forms of Perilla frutescens leading to comprehensive
identification of anthocyanin biosynthetic genes
Mami Yamazaki1,2, Masahisa Shibata1, Yasutaka Nishiyama1,3,*, Karin Springob1,,
Masahiko Kitayama3, Norimoto Shimada4, Toshio Aoki4, Shin-ichi Ayabe4and Kazuki Saito1,5
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 underlyunderstand-ing the production
of secondary products In particular, combinatorial
analysis of transcriptome and metabolic profiles pro-vides excellent clues for decoding the function of unidentified genes, if these analytical platforms are available, as in the case of a model plant, Arabi-dopsis thaliana [1–3] However, as no microarray chips
Keywords
anthocyanin; chalcone isomerase;
glutathione S-transferase; PCR-select
subtraction; Perilla frutescens
Correspondence
K Saito, Graduate School of Pharmaceutical
Sciences, Chiba University, Yayoi-cho 1-33,
Inage-ku, Chiba 263 8522, Japan
Fax: +81 43 290 2905
Tel: +81 43 290 2904
E-mail: ksaito@faculty.chiba-u.jp
Present address
*Ehime University, Matsuyama, Japan
Donald Danforth Plant Science Center,
St Louis, MO, USA
Database
The sequences reported in this article have
been deposited in the DDBJ under the
accession numbers AB362191 (PfGST1) and
AB362192 (PfCHI1)
(Received 16 February 2008, revised 30
March 2008, accepted 7 May 2008)
doi:10.1111/j.1742-4658.2008.06496.x
Differential screening by PCR-select subtraction was carried out for 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 tt19mutant 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.
Trang 2are 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]
Chemical analysis indicated that only red perilla, but
not green perilla, produces anthocyanins,
malonylsh-isonin [cyanidin
3-O-[6¢¢-O-(E)-p-coumaroyl]-b-d-gluco-pyranoside-5-O-(6¢¢¢-O-malonyl)-b-d-glucopyranoside]
being the main pigment [7] (supplementary Fig S1)
Health-beneficial properties of anthocyanins are widely
recognized, and they are mainly ascribed to the
antiox-idant activity of anthocyanins [8] In the Japanese
Pharmacopoeia, only red perilla is registered as a
tra-ditional Japanese⁄ Chinese crude drug Differential
gene expression analysis of the two forms by
cDNA-differential display led to the identification of several
genes involved in biosynthetic reactions (structural
genes) and regulation of the expression of biosynthetic
genes (regulatory genes) [4,9–11] However, the
cover-age of gene expression profile by cDNA-differential
display seems still incomplete, given the lack of a few
genes in the entire biosynthetic pathway of
anthocya-nins in the collected gene repertoire obtained to date
Therefore, a more complete analysis for differential
gene expression profiles is needed
The participation of glutathione S-transferase
(GST)-like protein in the vacuole transport of
anthocy-anin and proanthocyanidin is still ill-defined Three
genes, Bz2 from maize [12], AN9 from petunia [13],
and TT19 from A thaliana [14], encoding GST-like
proteins have been isolated by a forward genetic
approach for mutants with changed color of seeds and
flowers, and the involvement of these genes in
seques-tration of anthocyanin and proanthocyanidin into
vac-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 nonproducanthocyanin-produc-ing 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 were determined, and the sequence homologies were analyzed by blast-x (Fig 1 and supplementary Table S1) Of 120 red perilla-specific clones in Fig 1, nearly half of them (56 clones) were genes related to secondary metabolism, in particular for flavonoid bio-synthesis, indicating the preferential expression of genes involved in flavonoid metabolism in red perilla
Of 24 green perilla-specific clones, in contrast, no clones were related to secondary metabolism These results indicate a dominant contribution of secondary metabolism-related genes in the gene repertoire prefer-entially expressed in red perilla
Among the genes specifically expressed in red perilla listed in supplementary Table S1, six genes, chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanidin syn-thase(ANS), flavonoid 3-glucosyltransferase (3GT), and anthocyanin acyltransferase (AAT), are the structural genes coding for the biosynthetic enzymes, which have been previously characterized [15–17] Two genes pre-sumably coding for CHI and GST have not been iso-lated and are thus new from perilla One of these two genes, PfGST1, was subjected to further analysis as described below With respect to the regulatory genes,
in addition to the previously isolated two regulatory genes, bHLH-F3G1 [18] and Myb-P1 [9], another basic helix-loop-helix (bHLH) protein gene was newly cloned The gene, 8R6, coding for an uncharacterized tonoplast membrane protein that has been obtained by mRNA differential display [6], was again isolated by 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
Trang 3activity 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
two forms Although the content of rosmarinic acid is
higher in green perilla than in red perilla [7], no genes
related to rosmarinic acid biosynthesis were specifically
expressed in green perilla This is presumably due to a
slight difference of gene expression that could not be
differentiated by the PCR-select method between two
chemovarietal forms, or a difference of accumulation
levels resulting simply from the dominant metabolic
flow of phenylpropanoid precursors to rosmarinic acid
formation rather than to anthocyanin production
Another possibility is that translational or post-trans-lational regulation operates for this pathway
Molecular characterization of PfGST1 encoding GST-like protein
Ten clones partially coding for GST were isolated as red-perilla-specific genes by PCR-select subtraction Of these 10 clones, four and six, respectively, encoded the N-terminal and the C-terminal parts of a GST-like protein homologous to AN9 [13] and TT19 [14], involved in transport of anthocyanin to the vacuoles
As the N-terminal clones contained a putative first ATG codon together with an in-frame stop codon in the upstream region, the full-length clones containing the entire coding region were amplified by PCR using cDNA obtained from red perilla leaves as a template The sequence analysis of 19 full-length clones indicated that they were divided into two groups, with a single nucleotide change that resulted in no amino acid sub-stitution This is presumably due to the microhetero-geniety of the genomic sequences The clone obtained 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
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)
Fig 1 Profiling of fragments with PCR-select cDNA subtraction in P frutescens.
Trang 4The expression pattern of the PfGST1 gene was
investigated by semiquantitative RT-PCR for the
RNA from leaves and stems of red and green perilla
(Fig 3) Predominant expression was observed in
leaves of red perilla, followed by stems of red perilla
Very weak but apparent expression was detected in
stems of green perilla; however, the transcript of
PfGST1was hardly detected in leaves of green perilla
These observed expression patterns were in good
agreement with the anthocyanin accumulation profiles
in those tissues of red and green perilla as reported
previously [7], indicating the involvement of PfGST1
in anthocyanin accumulation in perilla plants A
GST-like protein, presumably involved in anthocyanin
accumulation in orange fruit, was reported to be
pref-erentially expressed in pigmented orange fruit [19],
sug-gesting the general participation of GST-like proteins
in anthocyanin transport
Functional confirmation of the involvement of
PfGST1 in anthocyanin accumulation by using
Arabidopsis as a host plant
To investigate the function of PfGST1 in planta, the
PfGST1 cDNA was transferred by
Agrobacterium-based transformation into an A thaliana tt19 mutant [14] lacking the TT19 GST-like gene that is responsi-ble for uptake of anthocyanin into the vacuoles The expression of the PfGST1 cDNA was driven by the promoter from cauliflower mosaic virus 35S RNA (35S) in a constitutive manner (Fig 4A) Under 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
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
PfGST1 (×0.5)
Actin
PfGST1 (×1.0)
PfCHI1 (×0.1)
PfCHI1 (×0.3)
Fig 3 Expression of PfGST1 and PfCHI1 in Perilla frutescens Semiquantitative RT-PCR of PfGST1 using 0.5 lL or 1.0 lL of tem-plate cDNA and PfCHI1, with 0.1 lL or 0.3 lL of temtem-plate cDNA together with Actin as a standard The cDNAs from leaves and stems of red and green perilla were used as templates.
35S-PfGST1/tt19 35S-GUS/tt19
35S-PfGST1 (pGWB2)
RB
A
B
LB
Km r 35S promoter PfGST1 Nos-T Hyg r
Fig 4 Functional complementation of tt19 mutants with PfGST1 (A) T-DNA construct in the binary vector used in this study (B) Phe-notypes of T1seedling 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.
Trang 5ing 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 anthoantho-cyanin 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-lation and the interspecies functional compatibility of these proteins
To investigate whether PfGST1 can influence the accumulation of tannins (proanthocyanidins) in Arabidopsis seeds, the color of seed coats due to pro-anthocyanidin accumulation was examined for the transgenic plants (supplementary Fig S3) Apparently, PfGST1failed to rescue the function of the TT19 gene, which supports the accumulation of proanthocyanidins
in the Arabidopsis seed coat [14] Also, the AN9 gene from petunia was incapable of transporting proantho-cyanidin, although it could participate in the transport
of anthocyanin [14], as observed in the case of PfGST1 As the protein sequence of PfGST1 was clo-ser to that of AN9 than to that of TT19, this sequence difference may be responsible for functional discrimi-nation with respect to uptake of proanthocyanidin Further analysis of the peptide region or amino acid residues involved in this discrimination would be inter-esting
Molecular characterization of PfCHI1 encoding CHI
Two clones exhibiting high homology with CHI from grapevine were obtained by PCR-select subtraction in the list of red perilla-specific genes Sequence analysis
0
2
4
6
8
10
12
14
WT
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.
Trang 6revealed that one of them, designated PfCHI1,
con-tained the entire ORF coding for the CHI protein
The deduced 214 amino acid sequence exhibited 70%,
67% and 65% identities with those from Vitis vinifera,
Citrus sinensis and Lotus japonicus, respectively
(sup-plementary Fig S4) Phylogenetic analysis (Fig 7)
sug-gested that PfCHI1 belongs to the family of type I
CHIs, which comprises the CHI proteins utilizing only
6¢-hydroxychalcone (naringenin chalcone) as a
sub-strate, as opposed to the type II CHIs, which are
active on both 6¢-hydroxychalcones and
6¢-deoxychal-cones found in leguminous plants [21]
The mRNA accumulation pattern of PfCHI1 was
investigated by semiquantitative RT-PCR (Fig 3) The
most abundant accumulation was observed in red
leaves, followed by green leaves Low levels of
expres-sion were detected in both red and green stems These
expression patterns were slightly different from those
of PfGST1 and anthocyanin accumulation [7] This
difference is presumably due to the fact that
naringe-nin formed by CHI is the substrate for not only
antho-cyanins but also general flavonoids
To confirm the function of PfCHI1, the enzymatic
activity of the recombinant PfCHI1 protein was
deter-mined in vitro using naringenin chalcone as the
sub-strate As shown in Fig 8, the recombinant PfCHI1
protein could stereospecifically convert naringenin
chalcone to (2S)-naringenin, whereas the nonenzymatic
reaction gave racemic naringenin as product These
results provided evidence that PfCHI1 encodes the functional CHI protein
Conclusions
PCR-select subtraction provided comprehensive pic-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%
Fig 7 Phylogenetic tree of CHIs Neighbor-joining tree based on
deduced amino acid sequences of PfCHI1, Citrus sinensis CHI
(AB011794), Lotus japonicus CHI1(AB054801), CHI2 (AB054802),
and CHI3 (AB073787), Medicago sativa CHI (M91079),
Phaseo-lus vulgaris CHI (S54703), and Vitis vinifera CHI (X75963) The
deduced amino acid sequence of PfCHI1 exhibited 70% identity
with that from V vinifera.
(2S)-Naringenin
Naringenin chalcone
(2R)-Naringenin
(2R)-Naringenin
A
B
No enzyme
Retention time (min)
20
Fig 8 Chiral HPLC profiles of the reaction products of naringenin chalcone with recombinant PfCHI1 expressed in Escherichia coli The recombinant E coli BL21AI protein extract carrying pD17– PfCHI1 was used for the assay as previously described [21] The 2R- and 2S-naringenins were separated by reverse-phase chiral chromatography with Chiralcel OD-RH (4.6 · 150 mm) (A) Recom-binant PfCHI1 (B) Control (incubation without protein).
Trang 7chamber 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¢) and an
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
TGTATGGTGCAAC-3¢) and GST-1-r (5¢-AGAAAGC
TGGGTTTATTTTGGGAGATCCATAACTTTTCTCC-3¢)
for the first round of PCR; and attB1 (5¢-GGGGACAAGT
TTGTACAAAAAAGCAGGCT-3¢) and attB2 (5¢-GGGG
ACCACTTTGTACAAGAAAGCTGGGT-3¢) for the
sec-ond round of PCR The GATEWAY-compatible entry
clone pD221–PfGST1 was obtained through recombination
of the PCR product with pDONR221 After the sequence
of the insert of pD221–PfGST1 was verified, the insert was
transferred into a binary vector pGWB2 downstream of the
CaMV35S promoter to give pGWB2–PfGST1 for plant
transformation The resulting binary vector pGWB2–
PfGST1 was introduced into A tumefaciens C58C1
(GV3101) by a freeze–thaw method [24]
The A thaliana tt19 mutant [14] was transformed with
pGWB2–PfGST1 and pGWB2–GUS, in which the
expres-sion of the Escherichia coli uidA gene, coding for GUS, was
controlled by the CaMV35S promoter, by the floral dip
method for in planta transformation [25] Selection of
trans-formants was carried out on GM agar medium [26] containing kanamycin (50 mgÆL)1) and hygromycin (20 mgÆL)1) The transformation state of A thaliana was confirmed by PCR using the primers GST-1-f and GST-1-r for the genomic DNA The expression of the transgene was studied by RT-PCR for the first-strand cDNA obtained from the transformed Arabidopsis with the primers GST-1-f 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 lL 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
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 induction by l-arabinose After centrifugation at 6000 g for
5 min, bacteria were suspended in the extraction buffer (50 mm potassium phosphate, pH 7.5, 50 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol) and disrupted by sonication The supernatant solution obtained by centrifugation was used as the soluble protein fraction for SDS⁄ PAGE and enzyme assay
Assay of CHI activity
CHI activity of the recombinant PfCHI1 protein was assayed in vitro as described previously [21] The enantio-meric naringenin was separated by reverse-phase chiral
Trang 8chromatography with Chiralcel OD-RH (4.6· 150 mm;
Daicel, Japan)
Semiquantitative RT-PCR
The first-strand cDNAs were obtained from RNA isolated
from leaves and stems of red and green perilla Using the
first-strand cDNAs as templates, semiquantitative PCR (20
cycles) was carried out to determine the expression levels of
PfGST1and PfCHI1 The expression of actin was used as
a control The sequences of primers were: 0 and
GST-R-1 (5¢-GATATGAGGGCATCTAAAAATTATT-3¢) for
PfGST1; PfCHI-2 (5¢-CAAAATGTCTGTGACTCAAGT
CC-3¢) and CHIseq-r-1 (5¢-GACATTCATTGGTCACTGA
TAAGCG-3¢) for PfCHI1; and Pf_actin-f (5¢-GATATG
GAGAAGATCTGGCACC-3¢) and Pf_actin-r (5¢-CTCC
TGCTCGAAGTCTAGTGC-3¢) for actin cDNA
General molecular technology
The DNA sequences were determined with the BigDye
Ter-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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Supplementary material
The following supplementary material is available online:
Fig S1 Biosynthetic pathway of anthocyanins in Perilla
Fig S2 Amino acid sequence alignment of GST Fig S3 Seed coat color of T2 seeds
Fig S4 Amino acid sequence alignment of chalcone isomerases
Table S1 cDNA fragments obtained by PCR-select subtraction from P frutescens
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