花卉園芸植物から単離したフラボノイド生産促進遺伝子EFP(Enhancer of flavonoid production)の解析

誌名誌名 名城大学農学部学術報告

ISSNISSN 09103376

著者著者

森田, 裕将棚瀬, 幸司大宮, あけみ久松, 完中山, 真義

巻/号巻/号 51号

掲載ページ掲載ページ p. 35-41

発行年月発行年月 2015年3月

農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

Scientific Reports of the Faculty of Agric叫turモ， Meijo University (名城大農学報)51 : 35-41 (2015)

Original

Isolation and Characterization of EnhαncerofFlαvonoid Production Genes from Floricultural Crops

*.** Yasumasa MORITA .，. ，Koji TANASE ，Akemi OHMIYA "，

TamotsuHIS刷 ATSU** and Masayoshi NAKAYAMA

Abstract Anthocyanins are major flavonoid pigments of seed plants， that are responsible for a wide range of flower

colours. In contrast， aurones are minor flavonoid pigments that confer a bright yellow colour on flowers， such as snapdragon.

τnose pigment concentrations have a directly effect on flower colour intensity. It has recently been shown that a chalcone

isomerase-like protein， called enhancer of flavonoid production (EFP)， has a role in the effective anthocyanin production

and flower colour intensity. Although EFP genes are widely distributed in terrestrial plants， studies of these genes have

been limited to only a few plants. Here we investigated the contribution of EFP gene to flower colouration of the popular

floricultural crops: carnation， chrysanthemum and snapdragon. EFP genes were isolated from carnation and chrysanthemum

and those mRNA expressions were abundantly detected in flower petals at the bud stage. In snapdragon， the mRNA

expression could be detected not only in anthocyanin accumulating flowers but also in aurone accumulating yellow flowers.

These results suggest that the EFP gene broadly contributes to flower colouration of floricultural crops by enhancing

flavonoid pigment production.

Key words: flower colour， anthocyanin， EFP gene， carnation， chrysanthemum， snapdragon

花井園芸植物から単離したフラボボ、ソノイド生産促進遺伝子EFP(Enhancer 01)βTαvon仰1ωoidproduction)の解析

(森田裕将山.棚瀬幸司

要約 アントシアニンは，植物二次代謝産物の一種，フラボノイドに分類される色素化合物である.アントシアニンが，

赤や紫，青色などの多彩な色調の花色発現に関わる一方で、，同じくフラボノイドの一種のオーロンは，黄色い花の花色素

として知られる.花の濃淡は，花弁細胞に蓄積するこれら花色素の濃度によって決まり，濃くなるほど色が濃く鮮やかに

なる.近年，アサガオの花色突然変異体を用いた解析から，アントシアニンのみならず，フラボノドイド全般の生合成へ

促進的に働く新規因子， Enhancer of Flavonoid Production (EFP:フラボノイド生産促進因子)が同定された EFP遺伝

子は，フラボノイドを生産する陸上植物のゲノム中に広く存在するため，植物のフラボノイド生合成に広範囲かつ重要

な役割を持つことが予想されている.しかしその解析例はアサガオを含め限られた植物種にとどまっている.そこで，

我々は，多彩な色調の花を咲かせる花井園芸植物とEFP遺伝子の関係について調査を行った.主要な切花品目であるカー

ネーション，キクからEFP遺伝子の単離を行い，アントシアニンの蓄積がみられる花弁の著から開花にかけてmRNAの発

現が増大することを確認した.加えて，オーロンを蓄積し，黄色の花を咲かせるキンギヨソウの花弁においてもEFP遺伝

子の発現を確認した.これらの結果は，EFP遺伝子が種々の花井園芸植物の花色発現，特にアントシニアンやオーロンな

どのフラボノイド生産を促進することで花色の濃淡の制御に関わることを示唆している.

キーワード:花色，アントシアニン，フラボノイド生産促進遺伝子，カーネーション，キク，キンギョソウ

Introduction

35

* Experimental Farm， Faculty of Agriculture， Meijo Universi臥Kasugai，Aichi 486-0804， Japan (名城大学農学部附属農場フィールドサイエンス研究室 愛知県春日井市鷹来町菱ヶ池4311-2)** NARO Institute of Floricultural Science， Tsukuba， Ibaraki 305-8519， Japan (農研機構花き研究所茨城県つくば市藤本2-1)E-mail: ymorita@meijo-u.ac.jp ry Morita) Received 15 January 2015 Accept巴d23 February 2015

Flower colour at仕actsconsumer interest and is one of血e

most important廿aitsin f10ricultural crops.τ'here is a market

requirement for a variety of f10wer colours， thus

understanding of the mechanisms related to f10wer colour

expression is an important issue for plant breeding using

either traditional or biotechnical methods of genetic

36 Sci. Rep. Fac. Agr. Meijo Univ. (名城大農学報)51 (2015)

modification.

Flavonoids are a group of secondary plant products that

ubiquitously present in the terrestrial plant kingdom.

Anthocyanin and aurone pigments are members of the

flavonoid group that accumulate in the vacuole (Winkel-

Shirley， 2001; Tanaka et al.， 2008). Anthocyanin pigments

provide a wide range of colours from red-orange to blue-

violet in flower petals， whereas aurone pigments confer a

yellowish colour. Flower colour intensity is generally

dependent on the pigment concentrations; higher amounts of

the pigments result in deep-coloured flowers， and inversely，

lower amounts lead to pale colours (Morita et al.， 2014). In

addition， other factors， such as co-pigmentation， vacuolar pH

and cell shape， are known to affect flower hue (Grotewold，

2006).

百lemajor en可mes加 theanthocyanin and aurone

biosynthetic pathways are characterized in detail (Fig. 1). In

the flavonoid biosynthetic pathway， the first step is mediated

by the chalcone synthase (CHS)， and the resultant products，

chalcones， are transformed into flavanones via the

anthocyanin pathway and into chalcone glucosides via the

aurone pathway (羽Tinkel-Shirley，2001; Tanaka et al.， 2008).

The genes for flavonoid biosynthetic enzynles have been

cloned from model plants and also floricultural crops

(Grotewold， 2006; Tanaka et al.， 2008). Moreover， mutations

conferring white flowers were characterized in the genes for

production of anthocyanidin (Chopra et al.， 2006). In morning

p-Cournaroyl-CoA

αiS.J， n長}4'GT ，

Chal∞ne gJucosides _ Chal∞nes

錨↓削iAu~∞es Ravanones

間↓Dihydroflavonols

:t Anth∞yanidins

拘 Tt

3 x MaJonyJ心oA

FNS 四.... FJav，∞es

FLS 四 4・' FJavor羽Js

Anthα司yanidin3-0→gJuα>sides

! Diversean廿lOcyanins

Figure 1. Simplified flavonoid biosynthetic pathway. The enzymes in the pathway are: CHS， chalcone synthase; CHI， chalcone isomer宙 e; THC4'GT， UDP-glucose:te仕ahydroxychalcone4'-O-glucosyl仕組sferase;AS， aureusidin synthase; F3H， flavanone 3-s-hydro勾rlase; DFR， dihydroflavonol 4-reductase; ANS， anthocyanidin s戸lthase; 3GT， UDP-glucose:flavonoid 3-0-g1ucosyltransferase; FNS， flavone syn血aseand FLS， flavonol syn仕lase. 百le arrowheads indicate modification steps of 初出ocyanins 白at are mediated by glycosyltransferases， acyltransferases and methyltransferases (fanaka et al.， 2008).

glory (Ipomoea nil)， a仕aditionalornamental plant in ]apan，

mutations in the genes for CHS， chalcone isomerase (CHI)，

dihydroflavonol4-reductase (DF町andanthocyanin synthase

(即時，) conferred white flowers virtually containing no

anthocyanins (Chopra et al.， 2006). In a recent study， it was

shown that the deep colour of morning glory flower is

reduced by a mutation in the gene encoding a chalcone

isomerase-like protein仰oritaet al.， 201の.百legene for

chalcone isomerase-like protein was designated as an

enhancer 01 jlavonoidρroduction (EF:丹becausethe mutation

led to a reduction of the total flavonoid compounds， including

anthocyanins. Moreover， RNAi knockdown mutants of白e

EFP homologues in petunia (Petunia hybrida) and torenia

(Torenia hybrid，α) had pale colour flowers and low amounts of

anthocyanins and other flavonoids (Morita et al.， 2014).

These observations suggest that EFP proteins generally play

a role in enhancing anthocyanin production resulting in

deeper flower colour. Moreover， the EFP gene is widely

distributed in terrestrial plants and its contribution to the

general flavonoid production including anthocyanin synthesis

is postulated (Morita et al.， 2014; Fujino et al.， 2014).

Carnation (Dianthus caryoPhyllus)， chrysanthemum

(Chrysanthemum mor扮lium)and snapdragon (Antirrhinum

majus) are popular cut flowers in the global floriculture

T

V lnEFP MG---TEMVMVDEIPFPPQVNLDNKLLSLMGHGITDVEIHFLQ工KYTA工GVYLDPEIVSH57 PhEFP MG---K羽田町DEIPFPSQFMMTT盟国r.MGHG工TDIEIHFLQIKFTAIGVYL世田V四日

DcEFP ー--MVNDVLVDDIAFAKETTLT-KPLS日.GYGCTDLEIHFLQIKHTAIGVYFEPTWDH55 CsEFP MG---SEMVMVDDISFPS官ITTS-KPLSLLGHGITDIEIHFLQIKFTA工GVYIDPEIVSH56 CmEFP MGー国-SEMVMVDDISFPSQITTS幽 KPLSLLGHG工TDIEIHFLQIKFTA工GVYIDPEIVSH56 ThEFP-A MAGGEVETVMVDEISFPRj官工QIT.“.RPLSLLGHGITDIEIHFLQI来FTAIGIYLDLQILDH59 ThEFP司 B MAGGEVETVTVDEISFPRQVQIT-KPLSLVGHGI官'DIEIHFLQIKFTAIG工玄LOLQ工VDH59 孟皿EFP MGSEVVDWMVDEVPFPSKITFT-KPLSLLGHGLTDIEIHFLQIKFTAIGVYLDPEIVAH 59

: * **::.*. : : *.1・:*:* **:*i・***i隆司烏合****:・・:: :: * Y N 可 守

主坦FP LQK問団世間'LAQDDDFFEAIIN.日四即日VWIKEIKGSQ四四日間VROLI沼田 117PhEFP LQQWKGKSGAELIENDEFFEAIVNAPVDKFLRVVVlKEIKGSQYGVQLESAVRDRLAEVD 117 DcEFP 叩 IKWKGKSGKEL招 DDEFFAALASAPVESVV四 WlKElKASQYGTQLEGAVRDRLAAVD115 CsEFP LQKllKGKSGT既.AEDDDFFDSVISAPVDKYLRIWlKEIKGSQYGVQLESSVRDRLAADD116 CIDEFP L官!KWKGKSGTELADDDDFFDSVISAPVDKYLRIVVlKEIKGSQYGVQLESSVRDRLAADD116 ThBFP-A L官!KWKGKSETELAQDDDFFEA工vs及.PVEKFIRVVVI笈EIKGSQYGVQLESAVRDRLAEED119 ThEFP・B LQKWKGKSETELAKDDDFFEAIVSAPVEKFFRVVVlKEIKGS官!YGVQLESAVRDRLAEED119 AmEFP L包KWKGKSATEVGEDDDFFDAIVSAPVDKIVRVVVlKEIKGSQYGVQLESAVRDRLADED119

ーー.，・・4除脅脅: :: .:*:**:: ***:. .::・****・..合唱帳台・.合**.:*会合 .. .・

lnEFP KYEEEEEAA工.EKVVDFFQSKYFKKSSVITFSFPANTATAK工VF"宏EG司 -KEDS-SIEVEN 174 PhEFP KYEEEEEEALEKlVEFFQSKYFKRDSVVTYSFPATSGNVKISFATEG-帽 KEDS-EIEVQN174 DcEFP KYEDEEEAALEKVAEFFPIKYLKJ叩 SFFTFヌFP;及S-GTAE目班目EA--KEDS-KIEVGN171 CsEFP KYEEE盟国LEKVVEFFQTKYFKKDS'旺 .TFSFPAAS商工日IGFSSDE・-KEEPKTMKVEN 174 CmEFP KYEEEEEEALEKVVEFFQTKYFKKDSVLTFSFPAASNIAEIGFSSDE-同 KEEPKTMKVEN174 官坦FP-A KYEEEEESSLEKVVDFFQSKYFヌKDSVVTLHFPASSSIAEIVFASSDGDKEES-RIEVKN178 ThEFP-B KYEEEEEESLEKVV鷲FFQSKYFRKDSVVILHFPASSSIAEIVFASSDGDREES-RIEVKN176 AmEFP KYEDEEEEALEQIID世.QSKYFKKDSVLTYYFPATSATAEIVFATEG--KDDS周回EVKN176

合合脅:***:合合:::*: 骨骨::*.*.. ・* .:・ :::. :::. ::・・ 4・s マ

lnEFP ANVGGMIKKWYLGGSRAVSPST工SSLANILPD・・・・・ 206 PhEFP ANVAGEIKKWYLGGSRGLSPTT工SSLANTLSAELSK210 DcEFP ANVVEMIKKWYLGGSRAVSPSTIECLANNLSAELSK 207 CsEFP GNWD細工KKWYLGGTSAYSPSTISSLANTLSLELSK210 CmEFP GNVVDM工KKWYLGGTSAYSPST工SSLANTLSLELSK210 TbEFP-A ANVVEMIQKlIY工.GGTRAVSPTTVASLASGLSAELSK214 TbEFP司 .B ANVVEM工QKWYLDGTRSVSPTTVASLASGLYAELSK214 AmEFP ANVVDMLlO田YLGGTRGVSPTTIASLATGLSAELSK212

合合 ::i量..・4・.*: **;1・， .・.・

Figure 2. Multiple alignments of the deduced amino acid sequences of EFP proteins. Sequences were aligned wi仕1世leClustalW program v2.1. Identical arnino acids (*)， highly similar residues (:)， less similar residues (.)釦dgaps (-) are indicated. Arrowheads above白ealignment indicate血elocations of arnino acid residues出atare involved加仕leactive-site hydrogen-bond network in仕lecrystal structure of CHI complexed wi出仕lesubs仕a総 naringeninσezet al. 2000， Ngaki et al. 2012). Black arrowheads indicate the former two crystal residues of CHI that are conserved泊 EFPproteins. White arrowheads indicate the latter two crystal residues of CHI that are not conserved in EFP proteins.

MOR汀Aet al. -Isolation and Characterization of Enhancer 01 Flavonoid Production Genes from Floricultural Crops 37

market. Reddish and purplish flowers of these plants

accumulate anthocyanins， whereas yellowish snapdragon

flowers accumulate aurone pigmentsσanaka et al.， 2008).

To further elucidate the generality of EFP function in

enhancing flower colouration， we isolated the EFP

Figure 3. A phylogenetic tree for CHI superfamily. The amino acid sequences were aligned with the ClustalW program V2.1. Th巴 treewas constructed using the neighbour-joining method. Bootstrap values of 1000 replicates are shown next to the branches， and the scale bar indicates 0.1 amino acid substitutions per site. Abbreviations shown before each protein indicate the plant species: Am， Antirrhinum majzω; At， Arabidopsis thaliana; Cm， Chrysanthemu~月間orilolium; Dc， Dianthz俗

caryophyllus; Gm， Glycine max; In， IPomoea nil; Ms， Medicago sativa; Ph， Petunia hybrida and Th， Torenia hybrida. Crystal structures of AtCHI， AtFAP and MsCHI were previously reported σez et al.， 2000; Ngaki et al.，2012)

(a)

l ji lj| DcEFP(20)

(22)

Act(23)

Act(23)

2 3

(b)

CmEFP(20)

(22)

Act(23)

Act(23)

2 3

homologues from carnation and chrysanthemum， and

characterized their sequences and expressions in the flower

petals accumulating anthocyanins. In addition， we examined

the expression of EFP homologues in purple and yellow

flowers of snapdragon.

Materials and Methods

Plant materials and cultivation

Carnation cultivar ‘Francesco' displaying standard red

flowers has been describedσ'anase et al.， 2012; Yagi et al.，

2013).百lechrysanthemum line ‘94-765' showing red-purple

flowers was provided by Seikoen Co. Ltd. (Fuchu， J apan) and

has been used in previous studies (Yoshioka et al.， 2012;

Noda et al.， 2013). Snapdragon cultivars‘Athlete Purple' and

‘Athlete Yellow' were from Sakata Seed Co. (Yokohama，

Japan). The plants were grown under natural daylight

conditions in a green house at the National Agriculture and

Food Research Organization， Institute of Floricultural

Scienceσ'sukuba， Japan). Flower petals were harvested at

three different stages of development， and immediately

frozen in liquid nitrogen and stored at -800

C until used for

total RNA preparation. Petal development was divided into

stages 1-3: stage 1， early bud stage; stage 2， late bud stage;

stage 3， opened or opening flower stage (Fig. 4).

Total RNA preparation

τne total RNA from flower petals was extracted using the

Get pureRNA kit (Dojindo Molecular Technologies，

Gaithersburg， MD， USA). RNA samples were subsequently

(c)

2 3 2 3

RT+

Ubi

RT-

Figure 4. Flower phenotypes and expression of the EFP homologues. Petal development stages for total RNA preparation (upper panel) and RT-PCR analysis of the temporal expression of the EFP homologues Oower panel) of carnation (a)，

chrysanthemum (b) and purple and yellow snapdragons (c). The numbers in parentheses for RT-PCR results indicate the number of cycles of PCR amplification.ηle constitutively expressed genes for the actin (Act) of carnation and chrysanthemum and the ubiquitin (Ubi) of snapdragon were used as internal controls.ηle lowest lanes represent negative controls without reverse transcriptase (RTー).

38 Sci. Rep. Fac. Agr. Meijo Univ. (名城大農学報)51 (2015)

purified using the RNeasy Plant Mini Kit (Qiagen， Hilden，

Germany).

Cloning of DcEFP， CsEFP and CmEFP cDNAs

To obtain DcEFP cDNA sequence， we first screened an

expressed sequence tag (ES1) library constructed from the

carnation cultivar‘Francesco'σanase et al.， 2012). Four

partial cDNA fragments for carnation EFP (DcEF丹 were

obtained.百leωtalRNA (5μg) prepared from白estage 2

petals was used to obtain the 5' and 3' ends of DcEFP by

using an Invi仕ogenGeneRacer Kit (Invi廿ogen，Carlsbad，

CA).百leGeneRacer RNA Oligo from the kit was ligated to

the 5' end of the 5' cap-removed mRNAs using T4 RNA ligase

followed by first-strand cDNA synthesis using Invitrogen

SuperScript III reverse transcriptase wi白 aGeneRacer Oligo

dTprimer.τne 5' and 3' ends ofDcEFPcDNA were amplified

using PrimeSTAR GXL polymeraseぐTaKaRaBio， Shiga，

]apan) with the gene-specific primer DcCHI4-R1 (5'-CITCAT

CCTCGTACITGTCGACTG-3')， GeneRacer 5' Primer that

was included in仕lekit， gene-specific primer DcCHI4-F1

(5'-AGGGACCGTCITGCAGCAGTC司3')and GeneRacer 3'

Primer.百leresultant polymerase chain reaction σCR)

products were cloned into the pCR II Blunt TOPO vector

(Invi仕ogen)and sequenced using仕leABI3130XL sequencer

(Applied Biosystems， Foster City， CA).百leassembled

DcEFP cDNA sequence contained the full-length open

reading frame.

To obtain the chrysanthemum EFP cDNA information，

we first screened an EST library constructed from a wild type

chrysanthemum (c. seticuspe) (Higuchi et al.， 2013) and

obtained血efull-length CsEFP cDNA sequence. The total

RNA (2μg) prepared from stage 2 petals of 出e

chrysanthemum line ‘94-765' was used for first-strand cDNA

synthesis using SuperScript III reverse transcriptase

(Invi仕ogen).τnefull-length CmEFP cDNA was amplified by

PCR using the PrimeSTAR GXL polymerase (faKaRa Bio)

with CsCHI4-F1 (5'・AAACATCTATCTACAI口寸CCITC-3')

and CsCHI4-R1 (5'-TICACGGAAATITτA.GTACAGAC-3')

designed on白e5' and 3' untranslated regions of CsEFP

cDNA， respectively. The resultant PCR product was cloned

into pCR II Blunt TOPO vector (Invitrogen) and sequenced.

RT-PCR

百letotal RNA (2μg) prepared from stage 1-3 petals of

carnation， chrysanthemum and snapdragon was used for

first-strand cDNA synthesis using SuperScript III reverse

仕anscriptase(Invitrogen). PCR amplification was performed

using Ex Taq polymeraseσaKaRa Bio). We used血eprimer

sets DcCHI4-LF1 (5'-TCAGAAGTICAATITCITCATCC

AC♂) and DcCHI4-LR1 (5'-GAAGACACGAAAATACGCAGA

TCG-3')， CmCHI4-LF1 (5'-CITC.AτTITGTATCTACTGτTI

TCTC-3') and CmCHI4-LR1 (5'-AAGCGGACGGTICAAACT

CATITG司3')， and AmCHI4-F1

(5'-AAGTGAAGAATGCGAACGTGGTG-3') and AmCHI4-

LR1 (5 '-CCATCCAAAGCACACCTCATAGC-3') to amplify

DcEFP， CmEFP and AmEFP cDNA fragments， respectively.

Actin cDNAs for carnation (Accession number， AY007315)，

chrysanthemum (AB205087) and Ubiquitin cDNA for

snapdragon (X67957) were amplified using the primer sets

DcACT1-F1 (5'-AGCACGGTATCGTCACCAACTι3') and

DcACT1-R1 (5'-TCATCGATGGCTGGAAGAGGAC-3')，

CmACT1・F1 (5'-GAGCACCCAGτTCITCTCACTG・3') and

CmACT1-Rl (5'-GTCCITCCTAATATCCACGTCAC-3')， and

AmUBIG3540 (5'-ATI寸GGTGCTGAGGTIGAGAァ3') 如 d

AmUBIG3543 (5'・ACAACTGACTCCAGCAAACι3')，

respectively.

Results

Isolation of the full-Iength cDNAs for EFP homologue of

carnation and chrysanthemum

We screened the expressed sequenced tag (ES1)

databases for carnationぐTanaseet al.， 2012) and a wild type

chrysanthemum (Higuchi et al.， 2013). Partial cDNA

fragments of白ecarnation EFP homologue (DcEF丹 were

isolated from仕lecarnation EST database， and full-length

DcEFP cDNA sequence (Accession number: LC022793) was

determined by rapid amplification of cDNA end (RACE)

methods.τne DcEFP cDNA has a 135 bp 5' un仕anslated

region (UTR)， a 624 bp open reading flame (ORF) of 207

amino acid residues， and a 200 bp 3' UTR. We obtained cDNA

sequence for the EFP homolog of C. seticuspe (CsEF丹from

the EST contigs. The CsEFP cDNA sequence (LC022794)

contained a 91 bp 5' UTR， a 633 bp ORF of 210 amino acid

residues， and a 108 bp 3' UTR. Full-leng白 cDNAsequence

for the EFPhomolog of C. mor扮lium(CmEFlうwasobtained

byR下PCRmethod using the primers set for amplification of

the entire coding sequence of CsEFP.τne CmEFP cDNA

(LC022794) has a 633 bp ORF that has 98% sequence identity

wi白血e CsEFP ORF sequence. EFP homologue of

snapdragon was isolated from a pu叩leflower line as

AmCIPF4 (chalcone isomerase弗ldprotein 4)σujino et al.，

2014)， although in the current study we refer to AmCIPF4 as

AmEFP.

Characterization of EFP homologues

CHI is the second committed enzyme of the flavonoid

pathway σig. 1). Recent phylogenetic and structural studies

MORITA et al. -Isolation and Characterization of Enhancer 01 Flavonoid Production Genes from Floricultural Crops 39

Table 1. Protein sequence similarity between the EFP homologues of floricultural crops and 出esequences of CHI and CHI-related proteins

cluster 1 cluster II cluster III cluster IV

AtCHI (246) MsCHI (222) AtFAP (287) InEFP (206) PhEFP (210)τbEFP -A (214)

DcEFP (20η le-14 7e-17 3e-13 6e-83 7e-89 3e-87

CsEFP (210) 7e-17 4e・19 3e-22 le-98 5e-l03 5e-l05

CmEFP (210) 2e-16 ge-19 6e・22 2e-98 le-l02 ge-l05

AmEFP (212) le-14 2e-17 2e-17 le-96 2e-l04 4e・104

Protein sequence similarity was analyzed using NCBI BlASf and represented by e-value. Numerals in parentbeses are tbe number of amino acid residues. Arabidopsis thaliana CHI (AtCHD and FAP (AtFAP) were used on behalf oftbe cluster 1 and III protein， respectively. Medicago sativa CHI (MsCHD was used on behalf oftbe cluster II protein.

in CHIs and CHI-related proteins have revealed that the

protein families are phylogenetically divided into four clusters

(Ralston et al.， 2005; Ngaki et al.， 2012). The proteins

belonging to cluster 1 and II have CHI enzymatic activity

侭alstonet al.， 200町， and those from cluster III and IV are

shown to work as a fa均Tacid protein (FAP) and EFp， respectively (Ngaki et al.， 2012; Morita et al.， 2014).百le

deduced amino acid sequences of EFP homologues for

carnation， chrysanthemum and snapdragon had a similar

size of about 200引 oaa with cluster IV proteins and showed a high homology to the previously reported EFPs from

morning glory (InEFP) ， petunia (PhEFP) and torenia

何百EFP-Aand τbEFP-B) σable 1).百lecluster-IV EFP

proteins show a close structural similarity with the cluster 1

and cluster II CHIs; however， they contain amino acid

substitutions in several catalytic residues of CHI enzyme

(Ralston et al.， 2005; Ngaki et al.， 2012). DcEFp， CmEFP and

AmEFP also contained the substitutions (Fig. 2). Moreover，

phylogenetic analysis clearly indicated that EFP homologues

belong to the cluster-IV EFP clade (Fig. 3).

Temporal expression of EFP genes during the development

of flower petals

To elucidate whether EFP homologues from carnation，

chrysanthemum and snapdragon participate in flower

colouration with accumulation of flavonoid pigments， we

detected the expression of出egenes in flower petals by RT-

PCR. We used a major standard-type carnation ‘Francesco'

(Fig. 4a) that has previously been used for EST cons仕uction

and whole genome sequencing analysesぐTanaseet al.， 2012;

Yagi et al.， 2013) and the chrysanthemum line・94-765'(Fig.

4b)出athas been used for the studying flower colouration

(Yoshioka et al.， 2012; Noda et al.， 2013). These flowers

display reddish and purplish colour wi白 anthocyanin

pigments. For仕leanalysis of snapdragon， we used ‘Athlete

Purple' (left panel of Fig. 4c) and ・AthleteYellow' (right panel ofFig.4c) as anthocyanin and aurone accumulating cultivars，

respectively. Likewise， carnation， chrysanthemum and

snapdragon， which accumulate anthocyanins in their flower

petals， were found to abundantly express EFP mRNA at bud

stage σig. 4). In addition，仕leabundant EFP expression was

also observed in a yellow snapdragon (Fig.4c).

Discussion

In a previous study， it was reported that EFP gene has a

role in flower colour intensity by controlling anthocyanin

concentration. For example， mutations and RNAi knockdown

of the gene for morning glory， petunia and torenia resulted in

pale flowers， which were caused by the reduction of

anthocyanins (Morita et al.， 2014). EFP gene encodes a CHI-

like protein belonging to the cluster IV clade of the CHl司fold

proteins侭alstonet al.， 2005; Ngaki et al.， 2012; Morita et al.，

2014).τbe cluster-IV EFP genes are widely distributed in

terrestrial plants (Ngaki et al.， 2012) and are assumed to act

as EFP to ensure that sufficient amounts of flavonoids are

produced αtlorita et al.， 2014). Here we isolated EFP gene

from two floricultural crops， carna廿on (DcEFP) and

chrysanthemum (CsEFP and CmEFP). In addition to these

EFP genes of two floricultural crops， expression of EFP gene

for snapdragon was tested to elucidate participation of EFP in

flower colouration.

Despite the s仕ucturalsimilarity between EFP and CHI， it

is unlikely that EFP has a role in anthocyanin biosynthesis as

bona fide CHI because the cluster-IV EFP proteins lack

catalytic residues for CHI enzymatic activity (Ralston et al.，

2005; Ngaki et al.， 2012; Morita et al.， 2014). The deduced

amino acid sequence comparison showed that EFPs from

carnation， chrysanthemums and snapdragon also lacks the

catalytic residues σ'ig. 2). Expression of EFP mRNA was

observed in血eflower petals of each examined floriculture

crop that accumulated anthocyaninsσig. 4). It was clearly

that the expression levels were higher at也ebud stages由加

出eopened stage in every plant. Expression of EFP gene in

40 Sci. Rep. Fac. Agr. Meijo Univ. (名城大農学報)51 (2015)

flower petals at developmental bud s匂geswas previously

reported in morning glory (Morita et a1.， 2014). Moreover，

PCR products for EFP mRNA were detected in ear1y cyc1es，

suggesting that EFP genes were abundantly expressed in the

flower peta1s. Bud stage expression of EFP mRNA in the

flower petals of carnation， chrysanthemum and snapdragon

is coupled with those of anthocyanin biosynthesis genes that

were reported in previous studies (Schwinn et a1.， 2006;

Chen et a1.， 2012; Sasaki et al.， 2012). In morning glory， the

expressions of both EFP and anthocyanin biosynthesis genes

were coordinately promoted by genes encoding仕leMYB

and WD40 family transcriptiona1 regulators αtIorita et a1.， 2014). Our results confirmed仕lecontribution of EFP to

anthocyanin production and flower colouration. A11elic

differences in the expression level of EFP gene are

considerable candidates for plant breeding to expand flower

colour range.

The yellow colour of snapdragon flowers is mainly

provided by 6-0-g1ucosides of aurones (Sato et a1.， 2001).

Aurones are synthesized through enzymatic steps wi血CHS，

UDP-glucose:te仕ahydro勾Tchalcone4'-0・glucosyltransferase

σHC4'G司 andaureusidin syn世lase(AS) (Fig. 1). Our

results showed that EFP mRNA expression was observed for

yellow snapdragon as well as the purple ones accumulating

anthocyaninsσig. 4c). It is tempting to speculate白紙 EFP

may also enhance aurone synthesis. If knockdown of EFP in

yellow snapdragon resulted in reduction of aurones and pale

yellowish flowers， then EFP has a role for efficient production

of aurones. Structural sirnilarity of the ligand-binding pocket

between EFP and CHI suggests出atEFP could capture

chalcone and/or related products (Ngaki et a1.， 2012; Morita

et a1.， 2014). We speculate that EFP physically interacts wi出

chalcone and/or related products to perform its roll with up-

regulating CHS activity， resulting in efficient production of

anthocyanins and aurones. Further research could test this

hypothesis.

Acknowledgments

明Te白ankKazuo Ichimura and Naonobu Noda for

providing snapdragon and chrysanthemum plants. This work

was funded by the Development of Innovative Crops through

the Molecular Analysis of Useful Genes Program (No. 5213)

of the N ational Agriculture and Food Research Organization.

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