Isolation and characterization of differentially expressed genes in petals of chrysanthemum mutant cultivars developed by irradiation

Scientia Horticulturae 189 (2015) 132–138

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Isolation and characterization of differentially expressed genes in petals of chrysanthemum mutant cultivars developed by irradiation Sang Hoon Kim a,1 , Sang Yeop Sung a,1 , Ye-Sol Kim a , Yeong Deuk Jo a , Si-Yong Kang a , Jin-Baek Kim a , Joon-Woo Ahn a , Bo-Keun Ha b,∗ , Dong Sub Kim a,∗ a b

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea

a r t i c l e

i n f o

Article history: Received 13 February 2015 Received in revised form 1 April 2015 Accepted 4 April 2015 Keywords: Anthocyanin Chrysanthemum Mutation Radiation Suppression subtractive hybridization

a b s t r a c t Chrysanthemum is one of the most popular ornamental plants, whose petal colors are primarily determined by pigments including flavonoids/anthocyanins and carotenoids. To develop chrysanthemum cultivars with various petal colors, mutation breeding targeting alteration of pigmentation pattern has been performed. A radiation-induced mutant line, ‘ARTI-purple’, showed a flower color altered from the original bright pink to purple. In this study, we used suppression subtractive hybridization to analyze transcripts and characterize the differential gene expression of chrysanthemum petals between the mutant ‘ARTI-purple’ and its wild-type Chrysanthemum × morifolium cultivar ‘Argus’. One hundred and seventy-six genes were identified (e-value ≤ 1e − 5) and classified based on sequence homology to genes with known or putative functions. The genes were categorized functionally by gene ontology analysis and their tentative pathways were confirmed using the TAIR database. The analyses revealed that these genes were related to carbohydrate metabolism, biosynthesis of secondary metabolites, and lipid metabolism. Six genes in a Kyoto encyclopedia of genes and genomes (KEGG) pathway which included the largest number of differentially expressed genes were selected for validation by quantitative PCR, and most of them showed higher expression levels compared with the wild-type. In addition, we isolated two novel clones (PC06E06 and PC08C09) having glutathione S-transferase (GST) family conserved domains and one clone (PC02G08) having a Multidrug and toxic compound extrusion (MATE) family conserved domain based on analysis using conserved domain database (CDD). The expressions of PC08C09 and PC02G08 were upregulated in ‘ARTI-purple’, which implies that anthocyanin accumulation pattern might be altered in mutant. In this study, we identified several differentially expressed genes between ‘Argus’ and ‘ARTIpurple’. The analysis suggested that several metabolic genes as well as glutathione S-transferases and MATEs might be involved in the control of flower pigmentation in chrysanthemum. © 2015 Elsevier B.V. All rights reserved.

Abbreviations: 3GT: UDP-glucose, anthocyanidin 3-O-glucosyltransferase; 5 3GT, anthocyanidin 5 3-O-glycosyltransferase; ANS, anthocyanidin synthase; CDD, conserved domain database; CHS, chalcone synthase; Cy3G, cyanidin 3O-glucoside; DFR, dihydroflavonol 4-reductase; E3 G, epicatechin 3 -O-glucoside; EC number, enzyme commission number; F3H, flavanone 3-hydroxylase; GO, gene ontology; GSTs, glutathione S-transferases; KEGG, kyoto encyclopedia of genes and genomes; MAT, malonyl-CoA: anthocyanin 5-O-glucoside-6 -Omalonyltransferase; MATE, multidrug and toxic compound extrusion; SSH, suppression subtractive hybridization; TAIR, The Arabidopsis Information Resource. ∗ Corresponding authors at: Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Korea/Division of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Republic of Korea. Tel.: +82 62 530 205/+82 63 570 3311; fax: +82 63 570 3319. E-mail addresses: [email protected] (S.H. Kim), [email protected] (S.Y. Sung), [email protected] (Y.-S. Kim), [email protected] (Y.D. Jo), [email protected] (S.-Y. Kang), [email protected] (J.-B. Kim), [email protected] (J.-W. Ahn), [email protected] (B.-K. Ha), [email protected] (D.S. Kim). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2015.04.007 0304-4238/© 2015 Elsevier B.V. All rights reserved.

1. Introduction Chrysanthemum (Chrysanthemum × morifolium) is a popular ornamental plant whose flower colors are the most important factors affecting their market value. Flower color of chrysanthemum is determined by the compositions and concentrations of pigments including flavonoids and carotenoids. Among those pigments, anthocyanins are grouped as a class of flavonoids and represent various colors such as red, purple, orange, and blue following the expressions of species-specific enzymes of their biosynthetic pathway such as chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose: anthocyanidin 3O-glucosyltransferase (3GT) (Aharoni et al., 2001; Kobayashi et al., 2001; Suzuki et al., 2001; Shelagh et al., 2001; Kitamura et al., 2004; Tanaka et al., 2008; Davies, 2009). Flavonoid derivatives

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(e.g. Cy3G, and E3 G) are modified by glycosylation, acylation and/or methylation and accumulated in the vacuole by mediations of transporter proteins such as glutathione S-transferases (GSTs) or a multidrug and toxic compound extrusion (MATE) transporters (Kitamura et al., 2004; Conn et al., 2008; Zhao and Dixon, 2010). The biosynthetic pathway for anthocyanins and their regulation by a R2R3-MYB transcription factor have been well studied, yet the location and catabolism of the synthesized anthocyanins are still not clearly established (Zhao and Dixon, 2010). Mutation breeding is a useful technology for plant breeders and biological researchers (Broertjes, 1966; Dowrick and Bayoumi, 1966). Since the 1930s, plant breeders have used gamma-ray irradiation for mutation breeding in asexual and sexual ornamental plants (Ahloowalia et al., 2004). Irradiation at lower dose rate is useful especially for radiation breeding of vegetatively propagated crops because the obtained mutants would be directly used as new cultivars (Yamaguchi et al., 2008). Irradiated plants have been selected based on their visible characteristic, such as flower color, structure and size, or leaf shape and growth patterns. Among them, a flower color mutant can be characterized by accumulation of various pigments including flavonoids/anthocyanins, betalains, and carotenoids (Tanaka et al., 2008). In chrysanthemum, flavonoids/anthocyanins have been reported to be responsible for pink-purple coloration (Sung et al., 2013; Noda et al., 2013). A spray-type chrysanthemum cultivar ‘Argus’ and its derived mutant ‘ARTI-purple’ have been studied for their expressions of genes for anthocyanin biosynthetic enzymes, namely CmCHS, CmCHI, CmF3H, CmDFR, CmANS, Cm3GT, Cm5 3GT, and CmMAT and anthocyanin concentrations. Although no differences in the expressions of these genes were observed, the concentration of anthocyanin in ‘ARTIpurple’ was approximately 3.5-fold higher than that of ‘Argus’ (Sung et al., 2013). The results indicated that the expression of CmMYB1, a repressive regulator of anthocyanin, was reduced in ‘ARTI-purple’, which inhibited a metabolic process affecting anthocyanin content, rather than the direct transcriptional inhibition of anthocyanin biosynthetic genes (Sung et al., 2013). Suppression subtractive hybridization (SSH) is a useful method to identify and isolate genes differentially expressed between two individuals. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population, and the subtraction step excludes the common sequences between the target and driver populations. Based on this principle, only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of an abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNAs and simplifies the analysis of the subtracted library (Diatchenko et al., 1996, 1999). This technology has been successfully applied to the analysis of the transcriptomes of several plant species during development and in response to stress (Xu et al., 2007; Galla et al., 2009). The identified ESTs can be characterized by an analysis of the Kyoto encyclopedia of genes and genome (KEGG) pathways, gene ontology (GO), and the conserved domain database (CDD) family. Such analyses can expand our understanding by predicting differences in biological compounds and gene expression networks (Nabors, 1976; Xu et al., 2007; Galla et al., 2009; Sung et al., 2013). In this study, we aimed to analyze cDNA libraries by PCR-based SSH to characterize the differentially expressed genes in chrysanthemum petals between a mutant with purple-colored flowers and its wild-type cultivar with bright pink flowers. This approach successfully identified differentially expressed genes and enhanced our understanding of anthocyanin accumulation in chrysanthemums.

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2. Materials and methods 2.1. Plant materials The Chrysanthemum cultivar ‘Argus’ with bright pink flowers and its mutant ‘ARTI-purple’ with purple flowers which was derived by a gamma-ray mutagenesis program (40 Gy to plantlets which were regenerated in vitro) employed by us (Sung et al., 2013) were used as plant materials. ‘Argus’ and ‘ARTI-purple’ were developed by vegetative propagation of irradiated mutant lines for three generations under natural conditions in a plastic greenhouse at the Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute. Visual inspection indicated that the mutant’s purple color had been stably inherited for three years. The petals of each genotype were collected at 10 days after flowering from November to December. For sampling, flower development was divided into two stages after flowering. Stage 1 (ray floret stretched and pigmented) and 2 (ray florets fully developed) were defined as 13 and 25 days after the initiation of flowering, respectively (Supplementary Fig. S1). The stage 1 and 2 ray florets were mixed and frozen with liquid nitrogen to be preserved at −80 ◦ C before being used as a resource for SSH procedure and expression analysis. 2.2. mRNA isolation and construction of SSH libraries Total RNA was isolated from the ray florets of ‘Argus’ and ‘ARTIpurple’ using the TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s protocol. The chrysanthemum petals contained high levels of polysaccharides; therefore, the extracted total RNA in diethylpyrocarbonate was precipitated for 3 h using a constant concentration of 3 M LiCl2 at −20 ◦ C. Poly A+ mRNA was then isolated using a Poly Tract mRNA isolation kit (Promega, USA). A NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) was used to quantify the total RNA and mRNA at wavelengths of 230, 260, and 280 nm. The mRNA was adjusted to a final concentration of 1 ␮g/␮l. Electrophoresis on 1.2% agarose gels was used to verify the integrity of the total RNA and mRNA. The PCR-Select cDNA Subtraction Kit (Clontech, USA) was used to construct two subtraction cDNA libraries (forward and reverse) according to the manufacturer’s protocol. As the forward subtraction cDNA library, mRNA from both ‘Argus’, the tester, and ‘ARTI-purple’, the driver, was used for cDNA synthesis by a suppression PCR using subtractive hybridization. The reverse subtraction cDNA library was derived from ‘ARTI-purple’ as the tester and cDNA from ‘Argus’ as the driver. Two rounds of PCR using oligonucleotide primers complementary to the adapters amplified the subtractive hybridized products. A QIAquick PCR Purification Kit (Qiagen, China) was used to purify the products of the second nested PCR. The purified products were ligated into a vector pGEM-T (Invitrogen, USA) and transformed into Escherichia coli DH-5␣ cells. The SSH cDNA libraries were sequenced and edited to remove any vector and ambiguous sequences. 2.3. General molecular procedures and GO analysis To assign putative functions to the encoded proteins of the genes represented by the cDNAs, the sequences obtained by SSH were compared with sequenced deposited in the NCBI GenBank database using BLASTX in the Blast2GO tool. For the annotation, the default settings of filtration at an e-value of 1.0e − 3 (annotation cut-off of 33) were used. In addition, gene ontology, functional annotation, enzyme commission number (EC number), and PATHWAY were analyzed by Blast2GO. The tentatively identified KEGG pathways were confirmed by searching TAIR database. The sequences with homologous sequence shorter than 50 bp were classified as an

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‘unknown sequence’. Each sequence was classified by one or more GO terms into different GO categories at TAIR. The CDD family of the encoded proteins was identified according to their domain/motif similarity, which was imported from The CDD (http://www. ncbi.nlm.nih.gov/cdd). The CDD family analysis is useful for fast identification of conserved domains in protein sequences, which provides insights into the sequence, structure and function relationships.

2.4. Expression analysis by real-time quantitative RT-PCR (qPCR) For the qRT-PCR analysis, the total RNA (100 ng) was isolated from ‘Argus’ and ‘ARTI-purple’. The total RNA was treated with DNase (Promega, USA), and reverse transcription was performed according to the instructions of the transcript or high-fidelity cDNA synthesis Version 6.0 kit (Roche, Germany). The Primer 3 web interface (http://frodo.wi.mit.edu/primer3/) was used to design the primers of our cDNA sequence database from conserved sequence regions. The primers used in this study are listed in Table S1. The product sizes were approximately 80–160 bp. For the qPCR, 100 ng of total RNA was used for the reactions and the expression was estimated using a SYBR Green II Master Mix Kit (Takara, Japan) by the EcoTM Real-Time PCR System (Illumina, USA). Expression level of CmActin (Gene bank accession: AB205087) was normalized by Eco Software v.3.16.0 and applied as expression baseline control for each sample as previously reported (Morita et al., 2012). The reaction was performed in three replicates for each set of conditions, and the data were presented as means ± SD (n = 3).

3. Results and discussion 3.1. cDNA library construction by SSH After constructing a cDNA library by SSH, we obtained 500 SSH clones, representing up- or down-regulated genes in the petals of ‘ARTI-purple’ compared with those of its wild-type ‘Argus’ (Table S2). The SSH clones were sequenced and sequences shorter than 100 bp were excluded. BLAST searching indicated that 34.8% (174/500) of the sequences were either unclassified or had no match in the NCBI database. Finally, 99 upregulated clones (99/250, 39.6%) and 77 down-regulated clones (77/250, 30.8%) were identified and annotated on the NCBI database using the Blast2go tool (Blast ExpectValue less than 1.0e − 3). The average valid readlengths of the unigenes were 583 bp (upregulated) and 519 bp (downregulated), respectively (Table S2). The e-value distribution of clustered unigenes was 1.0e − 4 to 1.0e − 180. These unigenes were then subjected to GO, KEGG, and CDD family member analyses. GO functional classifications were assigned for the 176 differentially expressed genes based on sequence similarities with known proteins using Blast2go. The percentage distributions of the GO terms between sequences from the two chrysanthemum petals were represented according to the cellular component, molecular function, and biological process (Supplementary Fig. S2). The most abundant groups among the upregulated genes in the mutant were a biological process (GO:0008150) and plasma membrane (GO:0005886), whereas the most abundant groups among the downregulated genes were plasma membrane (GO:0005886) and response to stress (GO:0006950) (Supplementary Fig. S2). Although accurate expression levels of the up- and downregulated genes were not measured in this study, these results indicated that in the mutant line, many genes had different expression levels compared with the wild type across the cellular component, molecular function, and biological process categories.

3.2. Pathway and conserved domain analysis Several metabolic pathway databases are available to analyze transcriptome and metabolome data. The KEGG classification system provides an alternative functional annotation of genes according to their associated biochemical pathways (Kanehisa and Goto, 2000). To further understand the molecular basis of the different flower color between the mutant and wild-type, the KEGG pathways were analyzed. Sixty-one genes encoded proteins with EC numbers, representing 41 different ECs. These EC numbers were mapped into 33 pathways. Among them, the representative pathways can be categorized into three groups: carbohydrate metabolism, biosynthesis of other secondary metabolites, and lipid metabolism (Table S3). The most abundant group was carbohydrate metabolism, which comprised eight KEGG pathways (Table S3). In the carbohydrate metabolism group, the starch and sucrose metabolism-related expressed sequence tags (ESTs) were the most abundant (Table S3). These included four genes encoding pectin methylesterase 2 (PME2; EC 3.1.1.11), pyrophosphorylase (UGP2; EC 2.7.7.9), cellulose synthase (CESA; EC 2.4.1.12), and beta-glucosidase (GBA3; EC 3.2.1.21) (Table 1). PME2 converts pectin to methanol and pectate, and the enzyme catalyzes the demethylesterification of cell wall polygalacturonans. In dicot plants, these ubiquitous cell wall enzymes are involved in important developmental processes, including cellular adhesion and stem elongation (Micheli, 2001; Johansson et al., 2002). UGP2 is primarily involved in the sucrose biosynthesis pathway and participate in sucrose breakdown according to the tissues or the extent of imported carbon (Leszek et al., 2004). CESA is involved in cellulose biosynthesis (Zhong and Ye, 2007; Kim et al., 2013). GBA3 plays an important role in diverse aspects of plant physiology, such as cell wall lignification and degradation, activation of phytohormones and plant defense systems (Halkier and Gershenzon, 2006; ˜ et al., 2006; Morant et al., 2008). Lee et al., 2006; Escamilla-Trevino In the group of biosynthesis of other secondary metabolites, phenylpropanoid biosynthesis-related ESTs were the most abundant (Table S3). These included three genes encoding betaglucosidase (GBA3; EC 3.2.1.21) and two O-methyltransferase (COMT1; EC 2.1.1.68, CCoAOMT; EC 2.1.1.104) (Table 1). COMT1 and CCoAOMT are involved in lignin and flavonoid biosynthesis, which are induced by stress conditions and are expressed during normal development (Chen et al., 2000; Anterola and Lewis, 2002). The expression levels of six genes involved in the starch and sucrose metabolism and phenylpropanoid biosynthesis were analyzed by qPCR in the two chrysanthemum petals (Fig. 1). Interestingly, most genes were highly expressed in the petals of ‘ARTI-purple’ compared with the wild-type ‘Argus’. Among them, the expression levels of PME2, GBA3, COMT1 and CCoAOMT increased by three-fold. Moreover, the expression of these genes, except PME2, was significantly up-regulated in Stage 2 compared to Stage 1. PME2 and GBA3, COMT1 and CCoAOMT play important roles in cell wall modification and lignin biosynthesis, respectively (Kanehisa and Goto, 2000; Halkier and Gershenzon, 2006; Pagadala et al., 2009; Moinuddin et al., 2010). However, there have been no reports on the function of these genes on flower coloration. Further researches are required for the clarification of the relationship between these genes and flower coloration. To further understand the functions of the differentially expressed genes in ‘ARTI-purple’, the unigenes were annotated based on their motif/domain similarities using NCBI’s Entrez database (Marchler-Bauer et al., 2003, 2007). The most common CDD families are shown in Table 2. The most frequently identified family was MIP with five members, followed by the Tubulin–FtsZ (four members).The GST-C and MATE members were identified in the ESTs database and these were considered as candidate proteins

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Table 1 Up-regulated genes of the ‘ARTI-purple’ mutant in two metabolic pathways. Related pathway (pathway ID)

Enzyme (enzyme ID)

Gene abbreviation

Clone ID

Starch and sucrose metabolism (map00500)

Pectin methylesterase 2 (3.1.1.11) Pyrophosphorylase (2.7.7.9) Cellulose synthase (2.4.1.12) Beta-glucosidase (3.2.1.21) Beta-glucosidase (3.2.1.21) O-methyltransferase (2.1.1.68) O-methyltransferase (2.1.1.104)

PME2 UGP2 CESA GBA3 GBA3 COMT1 CCoAOMT

PC28E10 PP05E10 PP16G02 PP06F09 PP06F09 PP06G10 PC12F04

Phenylpropanoid biosynthesis (map00940)

Fig. 1. Expressions of PME2, UGP2, CESA, GBA3, COMT1 and CCoAOMT transcripts in the petals of ‘Argus’ and ‘ARTI-purple’. The values are the mean ± SD (standard deviations) of at least three independent experiments, relative to the fold change of the transcripts. S1, Stage 1; S2, Stage 2.

which may related with anthocyanin metabolism or accumulation referring previous researches. 3.3. Characterization of GSTs and MATE in chrysanthemum Involvements of GSTs and MATEs in flavonoid/anthocyanin synthesis or transport have been reported in previous researches. In our study, two GSTs and one MATE were upregulated in the mutant (Table 3). To further investigate them as candidates affecting anthocyanin accumulation, the amino acid sequences coded by the genes from the two families were aligned with known protein

sequences from Arabidopsis and the other plant species using conserved regions and then subjected to phylogenetic analysis. The deduced amino acid sequences of the GSTs were classified as phiclass GSTs. GST PC08C09 was grouped with PM24 and ERD11 from Arabidopsis and a GST protein from bladder campion (Silene vulgaris) while GST PC06E06 was grouped with AN9 from petunia, GST4 from grapevine, and TT19 from Arabidopsis (Fig. 2A). MATE PC02G08 was placed in one clade with proteins such as FFT from Arabidopsis and MATE2 from Medicago (Fig. 2B). We further evaluated the relationship between anthocyanin concentration and the expressions of the isolated CmGSTs and CmMATE using a real-time PCR analysis with designed gene-specific primers (Table S1). In real-time PCR analysis, GST PC08C09 and MATE PC02G08 were shown to be expressed strongly in the ‘ARTIpurple’ petals. However, the expression level of PC06E06 was similar between ‘Argus’ and ‘ARTI-purple’ (Fig. 3). These suggested that GST PC08C09 and MATE PC02G08 can be candidates which might be related with flower color alteration detected in ‘ARTIpurple’. In plants, GSTs have been studied for their ability to detoxify chemically diverse herbicides and other toxic organic compounds (Alfenito et al., 1998). However, a group of plant GSTs were reported to combine with anthocyanidin in the last step of the anthocyanin biosynthetic pathway (Kitamura et al., 2004; Marinova et al., 2007; Conn et al., 2008). These anthocyanin-transporting GSTs have been reported in various species, such as Petunia (AN9; Mueller et al., 2000), Arabidopsis (Kitamura et al., 2004; Marinova et al., 2007), and grapevine (Conn et al., 2008) implying possible association of PC08C09 with the pigmentation process in chrysanthemum petals. However, GST PC08C09 was grouped with other GSTs such as PM24 and ERD11 in phylogenetic analysis although GSTs known to be related with anthocyanin transport (AN9, TT19, GST4) and GST PC06E06 were grouped together in a different sub-clade. Arabidopsis PM24 and ERD11, which have high similarity with PC08C09,

Table 2 The most common conserved domain database (CDD) families of differentially expressed genes in the ‘ARTI-purple’ mutant. Domain name

CDD family

MIP Tubulin–FtsZ

cl00200 cl10017

Mito-carr nsLTP1

cl02813 cl07890

Auxin-repressed

cl05238

GST-C p450 MATE-like

cl02776 cl12078 cl09326

Family description

Major intrinsic protein superfamily Tubulin/FtsZ: family includes tubulin alpha-, beta-, gamma-, delta-, and epsilon-tubulins as well as FtsZ, all of which are involved in polymer formation. Mitochondrial carrier protein Non-specific lipid-transfer protein type 1 (nsLTP1) subfamily; Plant nsLTPs are small, soluble proteins that facilitate the transfer of fatty acids, phospholipids, glycolipids, and steroids between membranes. This family contains several plant dormancy-associated and auxin-repressed proteins the functions of which are poorly understood. alpha helical domain of the Glutathione S-transferase family Cytochrome P450 Multidrug and toxic compound extrusion family and similar proteins

CDD family: A link to the record for the CDD superfamily to which this domain belongs. Up, upregulated; down, downregulate.

Occurrence Down

Up

3 2

2 2

0 1

3 2

0

2

0 0 0

2 2 1

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Table 3 Identification of GST-C and MATE-like family genes upregulated in the ‘ARTI-purple’ mutant. Clone ID GST-C family PC06E06 PC08C09 MATE-like family PC02G08

Sequence description [Taxon]

ACC

EC number

Positives (%)

e-Value

Glutathione s-transferase [Dahlia pinnata] Glutathione s-transferase [Nicotiana tabacum]

BAM72333.1 P30109

EC:2.5.1.18 EC:2.5.1.18

89.00 79.60

8e − 49 8e − 55

Protein transparent testa-like [Medicago truncatula]

XP 003628949.1

EC:2.7.6.5

84.00

1e − 66

ACC, accession number of best matching sequence.

Fig. 2. Phylogenetic analysis of GSTs and MATEs of Arabidopsis and other plants. (A) GSTs, (B) MATEs. Panels of GSTs and MATEs were adopted from Conn et al. (2008) and Zhao et al. (2011), respectively. In phylogenetic tree, species names and GenBank accession numbers were written in the parenthesis located on the right side of protein name. The name of proteins isolated in this research were emphasized by rectangles. The phylogenetic tree was generated by neighbor-Joining method in the MEGA 6 software. Numbers at branch points refers to bootstrap support.

do not seem to be associated with anthocyanin accumulation and their expressions were non-specifically increased by treatment with hormones and by abiotic stress, according to The ATTED-II database. A group of MATEs have been suggested to transport the flavonoid and anthocyanin derivatives in plants and be requisite for the accumulation of those metabolites (Marinova et al., 2007). In phylogenetic analysis, MATE PC02G08 was grouped with MATEs such as MATE2 (Medicago) and FFT (Arabidopsis) which are reported to be related with flavonoids/anthocyanin transports (Thompson et al., 2010; Zhao et al., 2011). Especially, MATE2 has been shown to transport anthocyanins and malonylated flavonoid glycosides efficiently in leaves and flowers. Loss of MATE2 function caused drastic decrease of anthocyanins and flavonoids which resulted in pale flower color (Zhao et al., 2011). On the other hand, the clade which contained MATEs that are not related with flavonoid transport (HvAACT1, FRD3, etc.) was separated from the clade in which MATE PC02G08 was included. Therefore, MATE PC02G08 might be related to accumulation of anthocyanins or flavonoids in chrysanthemum and participate in alteration of flower color in ‘ARTI-purple’. The regulators which act in the upstream of differentially expressed genes which includes MATEs and GSTs as well as metabolic proteins found in this study were remained unknown. In our previous study, we isolated CmMYB1 whose expression

Fig. 3. Relative expression levels of clones PC08C09, PC06E06, and PC02G08, as determined by real-time PCR. The values are the mean ± SD (standard deviations) of at least three independent experiments, relative to the fold change of the transcripts.

was decreased specifically in ‘ARTI-purple’. The CmMYB1 protein contained a motif sequence (PDLNLELRIG) which was similar to FaMYB1 C2-motif (pdLNLD /E LxiG /S ) indicating that CmMYB1 may act as a transcriptional repressor. Further study to link the function

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of transcriptional regulators including CmMYB1 to the metabolism or anthocyanin transport-related proteins found in this study is required to understand flavonoid regulatory mechanism and engineer metabolic process of pigmentation (Tanaka et al., 2008). 4. Conclusions In this study, we identified differentially expressed genes that may be associated with different petal pigmentation pattern found in chrysanthemum irradiated mutant, ‘ARTI-purple’, when compared to wild type cultivar, ‘Argus’, by SSH analysis. We hypothesized that the several metabolic proteins, GSTs, and MATEs might be involved in the control of flower pigmentation of chrysanthemum based on difference in their transcription patterns between ‘Argus’ and ‘ARTI-purple’. Further biochemical and cell biological investigations of the candidate genes, including GSTs and MATE, should be performed to verify this hypothesis and reveal the underlying molecular basis of pigmentation in chrysanthemum petals by anthocyanin accumulation. Acknowledgements This work was supported by the R&D program of the Korea Atomic Energy Research Institute and the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIP) (No. 2012M2A2A6010572). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2015. 04.007 References Aharoni, A., De Vos, C.H., Wein, M., Sun, Z., Greco, R., Kroon, A., Mol, J.N., O’Connell, A.P., 2001. The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319–332. Ahloowalia, B.S., Maluszynski, M., Nichterlein, K., 2004. Global impact of mutationderived varieties. Euphytica 135, 187–204. Alfenito, M.R., Souer, E., Goodman, C.D., Buell, R., Mol, J., Koes, R., Walbot, V., 1998. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell Online 10, 1135–1150. Anterola, A.M., Lewis, N.G., 2002. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61, 221–294. Broertjes, C., 1966. Mutation breeding of chrysanthemums. Euphytica 15, 156–162. Conn, S., Curtin, C., Bézier, A., Franco, C., Zhang, W., 2008. Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins. J. Exp. Bot. 59, 3621–3634. Chen, C., Meyermans, H., Burggraeve, B., De Ryclce, R.M., Inoue, K., De Vleesschauwer, V., Steenackers, M., Vans Montagu, M.C., Engler, G.J., Boerjan, W.A., 2000. Cell-specific and conditional expression of caffeoyl-coenzyme A-3-Omethyltransferase in poplar. Plant Physiol. 123, 853–868. Davies, K.M., 2009. Modifying anthocyanin production in flowers. In: Gould, K., Davies, K., Winefield, C. (Eds.), Anthocyanins. Springer, New York, NY, pp. 49–80. Diatchenko, L., Lau, Y., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. 93, 6025–6030. Diatchenko, L., Lukyanov, S., Lau, Y.F., Siebert, P.D., 1999. [20] Suppression subtractive hybridization: a versatile method for identifying differentially expressed genes. Methods Enzymol. 303, 349–380. Dowrick, G.J., Bayoumi, A., 1966. The induction of mutations in chrysanthemum using X- and gamma radiation. Euphytica 15, 204–210. ˜ L.L., Chen, W., Card, M.L., Shih, M.C., Cheng, C.L., Poulton, J.E., 2006. Escamilla-Trevino, Arabidopsis thaliana ␤-glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides. Phytochemistry 67, 1651–1660. Galla, G., Barcaccia, G., Ramina, A., Collani, S., Alagna, F., Baldoni, L., Cultrera, N., Martinelli, F., Sebastiani, L., Tonutti, P., 2009. Computational annotation of

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