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 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 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]. 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 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 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 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 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. 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. 3496 FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS The 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 PfGST1 was 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 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 PfGST1 (×0.5) Actin PfGST1 (×1.0) PfCHI1 (×0.1) PfCHI1 (×0.3) Red stem Green leaf Green stem 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 template 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 Hyg r Nos-T 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 T 1 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- 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, PfGST1 failed 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 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%, 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. PfCHI1 (2S)-Naringenin (2S)-Naringenin Naringenin chalcone (2R)-Naringenin (2R)-Naringenin A B No enzyme Absorbance at 295 nm 010 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). M. Yamazaki et al. Anthocyanin biosynthetic genes from Perilla 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 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 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 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 Anthocyanin biosynthetic genes from Perilla M. Yamazaki et al. 3500 FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS chromatography 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 PfGST1 and PfCHI1. The expression of actin was used as a control. The sequences of primers were: GST-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. 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This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corre- sponding author for the article. Anthocyanin biosynthetic genes from Perilla M. Yamazaki et al. 3502 FEBS Journal 275 (2008) 3494–3502 ª 2008 The Authors Journal compilation ª 2008 FEBS . Differential gene expression profiles of red and green forms of Perilla frutescens leading to comprehensive identification of anthocyanin biosynthetic genes Mami. display led to the identification of several genes involved in biosynthetic reactions (structural genes) and regulation of the expression of biosynthetic genes