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1 Korean Journal of Breeding Science Korean J. Breed. Sci. 50(4): ( ) Online ISSN: Print ISSN: 김다혜 박상규 박보라 이종렬 임선형 * 농촌진흥청국립농업과학원 Flavonoid Metabolic Engineering for Modification of Flower Color in Chrysanthemum Da-Hye Kim, Sangkyu Park, Bo-Ra Park, Jong-Yeol Lee, and Sun-Hyung Lim * National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, 54874, Republic of Korea Abstract In ornamental crops, the color and shape of flowers are one of the important traits. Generally, flower colors are determined by accumulating pigments such as carotenoids, flavonoids, and betalains. Among them, flavonoids are responsible for broad ranges of colors. Chrysanthemums are one of the most popular ornamental crops in the world, and there have been many efforts to change their flower color. In chrysanthemum flowers, cyanidin-based anthocyanin confers pink or red color, whereas terpenoid-based carotenoids are mainly responsible for yellow and green colors. However, blue colored chrysanthemums do not occur in nature. To date, there have been attempts to obtain blue or violet-colored chrysanthemum flowers through the introduction of a novel gene for accumulating delphinidin-based anthocyanins, while other studies have reported changing endogenous metabolites through the reconstruction of flavonoid biosynthesis. Since various transcription factors are involved in the regulation of flavonoid biosynthesis, it is important to understand not only the structural genes, but also the transcription factors required for the modification of flavonoid-based flower color. Therefore, in this paper, we describe the flavonoid biosynthetic pathway and its regulation, and review previous studies on the change in flower color through modification of flavonoid biosynthesis. This effort could be an important milestone in successfully achieving the modification of chrysanthemum flower color by means of plant biotechnology. Keywords Anthocyanin, Chrysanthemum, Flavonoid, Flower color, Metabolic engineering Received on August 20, Revised on August 27, Accepted on September 17, * Corresponding Author ( limsh2@korea.kr, Tel: , Fax: ) 서언국화 (Chrysanthemum morifolium) 는장미, 백합과함께세계 3대화훼작물중하나이며, 유럽과아시아에서주로생산된다. 국화는 6배체 (2n=6x=54) 작물로, 꽃색과꽃모양이다양하고화분용, 절화용, 정원용등다양한용도로사용되며, 세계적으로 200여종이보고되고있다 (Anderson 2006, Dowrick 1953). 2013년보고에의하면국화는전세계적으로인도 (18,000 ha), 중국 (8,475 ha), 일본 (5,230 ha), 멕시코 (2,467 ha), 태국 (2,199 ha) 순으로재배되고있으며, 유럽의시장거래규모액은 2억 5820만유로 ( 약 3357억원 ) 로보고되고있다 (Hanks 2015) 년도보고에의하면, 국내국화재배면적은 428 ha에이르며, 약 523억원의시장거래규모액을지니는경제적으로중요한작물이다 (MAFRA 2017). 화훼작물에서꽃모양과꽃색은소비자들에게심미적인만족감을부여하는중요한형질중의하나이다. 지금까지새로운 꽃모양과꽃색을개발하기위해전통적교배육종또는분자육종을바탕으로한노력들이지속적으로진행되고있다. 꽃색의경우, 식물의이차대사물질축적과밀접하게관련되어있고, 화훼작물별로물질생산과관련한생합성경로, 조절기작, 생리적기능등은분자생물학및대사공학분야에서많은연구가진행되고있다. 꽃색을결정하는물질은플라보노이드 (flavonoid), 카로티노이드 (carotenoid), 베타라인 (betalain) 등으로알려져있다 (Mol et al. 1998). 화훼작물은교배육종과돌연변이육종을통해흰색, 노란색, 분홍색, 주황색, 빨간색, 초록색등다양한꽃색의품종들이개발되어왔다. 일반적으로, 붉은색꽃잎의주요색소물질은플라보노이드계안토시아닌이며, 노란색또는초록색꽃잎의주요색소물질은카로티노이드계물질들이다 (Park et al. 2015). 베타라인은석죽목에속하는작물에서주로축적되며밝은노란색에서보라색에이르는색을나타낸다. 최근베타라인생합성경로의주요유전자의기능이밝혀지고베타라인이축적된담배꽃이최초로보고되었다 (Polturak et al. 2017). Copyright c 2018 by the Korean Society of Breeding Science This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 韓育誌 (Korean J. Breed. Sci.) 50(4), 2018 베타라인은방향족아미노산인타이로신 (tyrosine) 을기질로하여노란색을띄는베타잔틴과붉은색을띄는베타시아닌이생산된다. 베타잔틴생성에필수유전자인 cytochrome ( 시토크롬 ) P450 계열의 CYP76AD6와 l 3,4 dihydroxyphenylalanine (l DOPA) 4,5-dioxygenase 를도입하여노란색의담배꽃을개발하였고, 베타시아닌생성에필수유전자인시토크롬 P450 계열의 CYP76AD1과 l DOPA 4,5-dioxygenase, 그리고 betanidin-5-oglucosyltransferase를도입하여붉은색의담배꽃을개발하였다 (Polturak et al. 2017). 색소물질베타라인의생합성기작규명은앞으로화훼작물에서의다양한꽃색개발에응용되리라예상된다. 기존의교배육종을통해서개발되지못하던새로운꽃색개발은최근발전한생명공학기술을기반으로다양한화훼작물에서이루어지고있다 (Azadi et al. 2016). 한예로, 전통적교배육종으로는개발할수없었던파란색장미의경우, 장미의내재 DFR 유전자의발현을억제시키고파란색소물질인델피니딘 (delphinidin) 생합성유전자인제비꽃의 flavonoid 3',5'-hydroxylase (F3 5 H) 와붓꽃의 dihydroflavonol 4-reductase (DFR) 를도입함으로파란장미가개발되었다 (Katsumoto et al. 2007). 식물의플라보노이드와카로티노이드와같은이차대사물질들의경우서로다른독립적생합성경로를지니지만대사물질들의생합성은상호영향을주고받는다 (Bedon et al. 2010, Ben Zvi et al. 2012). 최종형질인꽃색은주요색소물질과조색소 (copigment) 의축적및상호작용에의해생성된다. 따라서물질각각의대사경로들을명확히이해하고생명공학적기술을활용하여꽃색관련대사물질의생성을정교하게조절한다면화훼작물의새로운꽃색개발이보다효율적으로달성될수있으리라기대한다. 본논문에서는화훼작물의플라보노이드계물질생합성에초점을맞추어생합성경로와조절기작에대해전반적으로서술하고, 지금까지시도된플라보노이드물질대사조절을통한다양한꽃색생성에대한선행연구들을검토함으로써경제적으로중요한화훼작물인국화의다양한꽃색개발을위한생명공학적전략들을제시하고자한다. 플라보노이드생합성경로플라보노이드생합성은페닐프로파노이드 (phenylpropanoid) 생합성경로로부터유래한한분자의 p-coumaroyl-coa와세분자의 malonyl CoA 들이 chalcone synthase (CHS) 활성에의해축합되어서찰콘 (chalcone) 이생성된다 (Fig. 1). 찰콘을기질로하여 chalcone isomerase (CHI) 에의해플라바논 (flavanones) 이 생성되거나, chalcone 4'-O-glucosyltransferase (C4 GT) 와 aurone synthase (AS) 의순차적활성에의해노란색소물질인오론 (aurone) 이생성된다. 플라바논은 flavanone 3-hydroxylase (F3H) 에의하여다이하이드로켐페롤 (dihydrokaempferol, DHK) 로변환되며, 다이하이드로켐페롤은 flavonol synthase (FLS) 와 DFR에의해경쟁적으로촉매되어각각의조색소인플라보놀 (flavonol) 과안토시아니딘의전구체인루코안토시아니딘 (luecoanthocyanidin) 이생성된다. DHK는 flavonoid 3 -hydroxylase (F3 H) 또는 F3 5 H의활성에의해플라보노이드 B-ring 에서이중또는삼중수산화 (di- or tri-hydroxylation) 를촉매하여플라보노이드물질군의다양성을증가시킨다. 루코안토시아니딘은 anthocyanidin synthase (ANS) 효소에의해안토시아니딘 (anthocyanidin) 으로변환된후, 당화 (UDP glycosyltransferase, UGT), 메틸화 (O-methyltransferase, OMT), 아실화 (acyltransferase, AT) 효소에의해수식화 (modification) 됨으로써최종적으로안토시아닌이생성되어액포내에저장된다. 안토시아닌계물질들은안토시아니딘의기본구조 (Fig. 2A) 에당화, 메틸화, 아실화등의수식화과정등을통해서구조적다양성이증가되며, 분자적특성이변경된다 (Grotewold 2006, Lepiniec et al. 2006). 안토시아닌의세포내안정적축적을위해서는당화가필수적이다. 안토시아닌의당화는글루코스 (glucose), 람노스 (rhamnose), 갈락토스 (galactose), 아라비노스 (arabinose), 자일로스 (xylose) 순으로의다중적결합양상을보인다 (Fig. 2B, Ünligil & Rini 2000, Zhang et al. 2014). 안토시아닌의당화는일반적으로 C3 위치와 C5 위치에서일어난다. C3 위치의당화는안토시아닌의안정성을향상시키는반면, C5 위치의당화는안정성을감소시킬수있다 (Mazza & Brouillard 1987). 또한, C7 위치와 B-ring의 C3 위치및 C5 위치에서도당화가될수있으며, 아실기가당에추가로결합됨으로써색소의안정성을증가시킨다 (Zhang et al. 2014). 플라보노이드계물질들은당공여체로써 UDP-당을사용하는 UDP glycosyltransferases (UGT) 에의해당화된다 (Gachon et al. 2005, Jadhav et al. 2012). UGT에는당공여체의 UDP 잔기에기질을결합시키는 44개의아미노산으로구성된 plant secondary product glycosyltransferase (PSPG) 모티프가관여하며, PSPG 모티프의마지막잔기에의해당선택성이결정된다 (Offen et al. 2006, Wang 2009). 식물의 UGT들은거대유전자군을형성하며, 작용하는기질에따라두가지유형으로분류된다 (Liu et al. 2018). 첫번째유형은 flavonoid 3-O-glycosyltransferase, flavonoid 3, 5-O-glucosyltransferase 와같이플라보노이드아글리콘 (aglycone) 352

3 Fig. 1. Flavonoid biosynthetic pathway in plants. ANS, anthocyanidin synthase; AS, aureusidin synthase; AT, acyltransferase; C4 GT, chalcone 4'-O-glucosyltransferase; CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol 4-reductase; F3H, flavanone 3-hydroxylase; F3'H, flavonoid 3'-hydroxylase; F3'5'H, flavonoid 3',5'-hydroxylase; FLS, flavonol synthase; OMT, O- methyltransferase; UGT, UDP-glycosyltransferase. Painted colors indicated each compound. 과그유도체에직접적으로당을첨가하는효소그룹으로서, 지금까지페튜니아의 PGT와 PH1, 포도의 UFGT, 장미의 RhGT1, 애기장대의 UGT73C6 등이보고되었다 (Ford et al. 1998, Jones et al. 2003, Ogata et al. 2005, Yamazaki et al. 2002). 두번째유형은다이글리코사이드 (diglycoside) 를생성하기위해플라보노이드모노글리코사이드 (monoglycosides) 의당원 (glycogen) 에추가적으로당을결합하는효소그룹으로서, 대표적으로나팔꽃의 3GGT와페튜니아의 3RT 등이이에해당된다 (Kroon et al. 1994, Morita et al. 2005). 식물의메틸전이효소는수산기와카르복실기부분을메틸화하는 O-methyltransferases (OMTs) 가대부분이다 (Du et al. 2015). 안토시아닌은 B링의 C3 위치또는 C5 위치의수산기에서메틸화될수있다 (Fig. 2C). 시아니딘의 C3 위치수산기의메틸화로페오니딘 (peonidin) 이생성되며, 델피니딘의 C3 위치수산기의메틸화로페튜니딘 (petunidin) 이, C3 위치수산기와 C5 위치수산기의메틸화로말비딘 (malvidin) 이생성된다. 이러한메틸화는안토시아닌분자의수용성에영향을미쳐물질의기능적 특성을변화시킨다. 식물의메틸전이효소는서열상동성과기질특이성에따라두그룹으로나누어진다 (Lam et al. 2007, Noel et al. 2003). 첫번째그룹은플라보노이드와아이소플라보노이드 (isoflavonoid) 를메틸화하는효소로, 양이온에독립적이며, kda 분자량을지니고있다 (Du et al. 2015). 안토시아닌의메틸화는이그룹에속하는 anthocyanin O-methyltransferase (AOMT) 에의해이루어진다 (Ibrahim et al. 1998). 두번째그룹은리그닌생합성에관여하는효소군으로양이온에의존적이며, kda의분자량을지니고있다. 그러나이그룹의구성원들중일부는플라보노이드를기질로하여 OMT 활성을보이는것으로보고되었다 (Hugueney et al. 2009, Lee et al. 2008, Ye et al. 1994). 아실화는활성화된공여체분자를통해수용체분자로아실기를전달하는것으로다양한물질생합성과정에서일어난다. 페놀화합물은아실그룹의공여체또는수용체가될수있으며, 플라보노이드도아실전이효소에의해아실화될수있다 (Bontpart et al. 2015, Mugford & Milkowski 2012). 아실화는수용성, 353

4 韓育誌 (Korean J. Breed. Sci.) 50(4), 2018 Fig. 2. Modification of anthocyanin. (A) Basic structure of an anthocyanidin. Numbering indicate different carbon groups. (B) Major glycosyl units in anthocyanin modification. (C) Main anthocyanidins and their methylation pattern. (D) Acyl units involved in anthocyanin modification. The upper panel is an aromatic amino acid group and the lower panel is an aliphatic amino acid group. 안정화, 색변화와같은안토시아닌의분자적특성변화에중요한영향을미친다 (Nakayama et al. 2003). 안토시아닌의당잔기는방향족산 (p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, gallic acid) 또는지방산 (malonic acid, acetic acid, succinic acid) 을공여체로하여아실화된다 (Fig. 2D). 방향족산을이용한 아실화는안토시아닌의파란색조를강화시키는반면, 지방산을이용한아실화는색변화를일으키지않으나, 안토시아닌의안정성을증가시킨다 (Kondo et al. 1987, Luo et al. 2007). 식물에서검출되는안토시아닌의대부분은아실화된상태이며액포내에존재한다 (Andersen & Markham 2006, Nakayama et al. 2003). 354

5 아실전이효소들은 BAHD군과 serine carboxypeptidase-like (SCPL) 군으로분류된다 (Bontpart et al. 2015). BAHD군은 BEAT (benzylalcohol O-acetyltransferase), AHCT (anthocyanin O-hydroxycinnamoyltransferase), HCBT (anthranilate N-hydroxycinnamoyl/benzoyltransferase), DAT (deacetylvindoline 4-O-acetyltransferase) 와같은 4개의아실전이효소들을포함하며그룹명은생화학적으로특성화된각각의효소명으로부터유래하여명명하였다. 플라보노이드를아실화하는 BAHD군은 acyl-coa 를공여체로사용하는효소군으로서, 기질과공유결합하는 HxxxD 모티프, 효소의구조적인특성을지니는 DFGWG, NYFGNC의모티프를공유하고있지만, 단백질유사성은 10-30% 로낮으며, 단량체로서기능하고있음이보고되었다 (Nakayama et al. 2003, St Pierre & De Luca 2000, Unno et al. 2007). SCPL군은클로로겐산 (chlorogenic acid), 시나포일에스테르 (sinapoylester), 갈로탄닌 (gallotannin) 과같은다양한페놀릭화합물의생합성에관여하며, serine, histidine, aspartic acid의세가지비연속아미노산으로구성된공통모티프를지니고있다 (Bontpart et al. 2015, Dahlbender & Strack 1984, Gross 1983, Kojima & Uritani 1972). 그러나 BAHD군과비교하여특성규명된효소들이많지않으며, 현재까지오직 9개의효소에서만특성이분석되었다 (Milkowski & Strack 2004, St Pierre & De Luca 2000). 전사인자에의한플라보노이드생합성조절기작플라보노이드생합성유전자들의발현은 R2R3 MYB, basic helix-loop-helix (bhlh), WD40 전사인자로구성된 MBW 중합체에의해서조절되는것으로알려져있으며 (Davies 2008, Grotewold 2006, Koes et al. 2005, Quattrocchio et al. 2006), 페튜니아, 다알리아, 나리등과같은화훼작물꽃잎의색소발달에이러한전사인자군들이관여하는것으로보고되고있다 (Albert et al. 2014, Ohno et al. 2011, Yamagishi et al. 2010). 분홍꽃색을지니는국화품종에서분리된 CmMYB6 유전자의경우, 꽃잎에서만특이적으로발현되며꽃잎에서의 CmMYB6 유전자의발현양상과플라보노이드생합성유전자의발현양상이일치하였다 (Liu et al. 2015). CmbHLH2 유전자는꽃잎전사체분석을통하여선발되었고, 담배잎에서의일시발현검정실험을통해서 CmMYB6 유전자와 CmbHLH2 유전자가안토시아닌생합성에양성조절자로작용함이확인되었다 (Xiang et al. 2015). 플라보노이드계물질생성에양성조절자인 MBW 중합체는다양한음성조절자에의해서조절되는것으로보고되고있다 (Albert et al. 2014, Xu et al. 2015). 애기장대의 MYB-LIKE 2 (MYBL2) 와 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9) 은 MBW 중합체형성을방해하는음성조절자로작용하는것으로알려져있다 (Dubos et al. 2008, Gou et al. 2011, Matsui et al. 2008). MYBL2는약광조건에서높은발현을나타내어안토시아닌축적을억제하는것으로보고되었다 (Dubos et al. 2008). SPL9 유전자의발현은 mir156에의해서영향을받는것으로보고되었다. SPL9 유전자의발현이높은조직에서는안토시아닌의축적이억제되는반면, mir156의강한발현을나타내는조직에서는 SPL9 유전자의발현감소와함께안토시아닌의축적이증가되었다 (Gou et al. 2011). 이외에도, subgroup 4에속하는 MYB 유전자들이안토시아닌생합성의음성조절자로작용한다는결과들이보고되고있다. 페튜니아의 PhMYB27 유전자를 RNA 간섭기술로발현을억제함으로써꽃잎과잎조직에서안토시아닌생성이증가되었다 (Albert et al. 2014). 또한, 딸기의 FaMYB1 유전자를담배에과발현했을때꽃잎에서안토시아닌의생성이감소되었고, RNA 간섭기술로발현을억제함으로딸기과실에서안토시아닌생성이증가되었다 (Aharoni et al. 2001, Kadomura-Ishikawa et al. 2015). 국화의경우에는감마선처리로인해보라색이생성된꽃잎에서 CmMYB1 유전자의발현이크게감소됨이확인되었으며, CmMYB1의아미노산서열이애기장대의안토시아닌생합성의음성조절인자인 AtMYB4와높은유사성을나타냈다 (Sung et al. 2013). 이러한결과들은국화꽃잎에서 CmMYB1이안토시아닌생합성의음성조절자로써기능함을시사한다. 국화의안토시아닌생합성수준을전반적으로조절하기위해서는이와같은양성조절자와음성조절자들간의상호작용을보다폭넓게이해해야할것이다. 따라서국화의 MBW 중합체구성원들및음성조절자들에대한분자적특성규명을위한체계적인연구들이요구된다. 다양한환경조건의안토시아닌생합성조절안토시아닌의생성은온도에영향을받는다. 애기장대의경우, 고온조건하에서 MBW 중합체를양성조절하는것으로알려진 ELONGATED HYPOCOTYL 5 (HY5) 단백질이분해되었으며음성조절자인 MYBL2 유전자의발현이크게증가하였다 (Kim et al. 2017). 또한고온조건하에서의 HY5 단백질의감소와안토시아닌함량감소가유의한상관관계를나타내는것으로보고되었다. 국화의경우에는 30 C 이상의고온재배시꽃잎에서의안토시아닌함량이크게감소하였다 (Nozaki et al. 2006). 이러한 355

6 韓育誌 (Korean J. Breed. Sci.) 50(4), 2018 현상은온대지역의여름철과열대지역에서의국화품질에심각한악영향을줄수있다. 분홍꽃색을지니는국화품종을고온조건에재배했을때품종별로안토시아닌감소반응은다르게나타났다 (Nozaki et al. 2006). 따라서고온조건에서안정적인꽃색을유지하는국화를개발하기위해서는고온반응과관련한유전자들에대한분자적인기작연구가필요하다. 안토시아닌생성은빛의영향을받으며, 강광조건에서는애기장대의안토시아닌생성이촉진된다 (Maier & Hoecker 2015). 일반광조건하에서는 MBW 복합체의 MYB 단백질에특이적 E3 ligase 인 COP1/SPA 복합체가작용하여안토시아닌생성을억제하였다 (Maier et al. 2013). 국화꽃잎에차광처리 (shading treatment) 시안토시아닌함량이급격히감소하였으며안토시아닌생합성유전자인 CmF3H, CmDFR, CmANS 및 Cm3GT 유전자의발현과양성조절자인 CmMYB6, CmbHLH24, CmCRY1a, CmCOP1 및 CMHY5의발현이크게감소되었다 (Hong et al. 2015). 이는국화의 MBW 중합체가빛에반응하여안토시아닌생합성조절에관여할수있음을시사한다. 따라서온도와빛과같은다양한환경조건하에안토시아닌의생성기작에대한연구가필요하며, 이러한연구결과를바탕으로불량환경조건하에서도안정적인꽃색을나타내는국화를개발하는것이가능하리라생각된다. 다양한꽃색생성을위한플라보노이드대사공학전략플라보노이드대사공학을통하여새로운꽃색을생성한다양한연구들이보고되었다. 현재까지제시된꽃색이변경된다양한사례들과전략들을소개하고자한다 (Figs. 3, 4). 흰색꽃개발흰색꽃은일반적으로안토시아닌과카로티노이드계색소물질이모두결여되어있으며, 대부분플라보노이드물질인플라본 Fig. 3. Strategies for modification of flower colors in chrysanthemum. Schematic diagram for obtaining white colored flower (A), yellow colored flower (B), orange colored flower (C), blue flower (D), and pink and red colored flower (E). Red arrows indicate the overexpression of target genes and blue solid lines with blunt end refer to suppress of target genes. Painted colors indicated each compound. Gray letters indicated suppressed enzymes and compounds. bhlh, basic helix loop helix; DHK, dihydrokaempferol; DHM, dihydromyricetin; DHQ, dihydroquercetin; LC, leucocyanidin; LD, leucodelphinidin; LP, leucopelargonidin. 356

7 과플라보놀을함유한다 (Chen et al. 2012). 플라보노이드생합성경로의주요유전자들의발현을억제시켜꽃색을변경시킨다양한결과들이보고되고있다. Lim et al.(2016) 은 RNA 간섭기술을이용하여내재 DFR 유전자의발현을억제하여흰꽃색이생성되게하였다 (Fig. 3A). 형질전환담배의꽃잎에서플라보노이드생합성유전자들의발현분석결과, 내재 DFR 유전자의발현이크게감소됨을확인하였고, 색소물질인안토시아닌과프로안토시아니딘의함량이감소됨을확인하였다. 최근주목받는유전자교정기술인 Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated protein 9 (CRISPR/Cas9) 시스템을이용하여나팔꽃의 DFR 유전자를돌연변이시켜꽃색을변경하였다 (Watanabe et al. 2017). Fig. 4A에서처럼대조구나팔꽃은보라색을나타내는반면, 형질전환체는흰꽃색이생성되었다. 이러한연구결과는유전자교정기술을이용한최초의꽃색변경연구결과이며유전자교정기술을이용하여안정적으로꽃색변경이가능하다는것을증명한다. 국화의흰꽃색을가지는품종 (Keikai, Jinba) 과분홍꽃색을가지는품종 (H5) 에대해플라보노이드물질분석과플라보노이드생합성유전자발현분석을실시하였다 (Chen et al. 2012). 흰꽃색의국화품종은플라본이주요물질이었으며플라보노이 드생합성유전자들의발현이낮은수준으로확인되었다. 반면, 분홍꽃색의국화품종은시아니딘계안토시아닌이주요물질이었으며, DFR과 GT의발현이증가되어있음을확인하였다. 흰꽃색국화품종인 Argus에감마선처리로보라꽃색을나타내는돌연변이개체인 ARTI-purple을개발하였다 (Sung et al. 2013). ARTI-purple 개체는 Argus와는달리꽃잎에서높은함량의안토시아닌이검출되었고, 꽃잎전사체분석결과 Argus 에비해 ARTI-purple개체에서 CmMYB1의발현이현저히감소되었음을확인하였다. 이러한결과는 CmMYB1이안토시아닌생성에음성조절자로작용한다는것을의미하며, CmMYB1을국화에서과발현시킬경우흰꽃색을얻을수있다는것을예측케한다. 노란색꽃개발플라보노이드계물질들중오론은노란색소물질이다 (Schwarz- Sommer et al. 2003). 일반적으로다알리아, 금어초등의노란꽃색은오론축적에의해나타난다. 국화의경우, 노란색꽃의주요색소물질은카로티노이드이며, 국화품종에서의오론계물질존재여부는확인되지않았다 (Berman et al. 2016). 오론은찰콘을기질로하여 C4 GT와 AS의효소활성에의해생성되어액포에저장된다 (Fig. 1). Ono et al.(2006) 은보라색토레니아의 Fig. 4. Flower color modification through genetic engineering of flavonoid biosynthetic pathway. (A) Flower color modification of morning glory. Left; host, right; Flower of CRISPR/Cas9-mediated dfr-b mutants (Watanabe et al. 2017). (B) Flower color was changed by accumulating aurones in torenia. Left; host, right; transgenic torenia with suppression of ThF3H gene and co-expressed AmC4 GT and AmAS genes (Ono et al. 2006). (C) Blue chrysanthemum. Left; host, right; blue chrysanthemum by overexpression of CamF3 5 H and CtUGT (Noda et al. 2017). (D) Enhancing of flower color by simultaneous expression of MYB and bhlh transcription factors. Left; host, right; transgenic tobacco flower (Kim et al. 2018). 357

8 韓育誌 (Korean J. Breed. Sci.) 50(4), 2018 내재 F3H 유전자의발현을억제시키고, 금어초유래의 AmC4'GT 와 AmAS1을동시에발현시켜노란색토레니아를개발하였다 (Fig. 4B). 국화의오론생합성효소들의유무는아직규명되지않았으나, 만일토레니아와같은경우라면, F3H 유전자발현억제와함께오론생합성관련유전자들을도입함으로써노란색국화개발이가능할것으로기대된다 (Fig. 3B). 주황색꽃개발주황색국화개발을위해서는안토시아닌물질중펠라고니딘축적을증가시키는전략을적용할수있을것이다. 빨간꽃색국화의경우, F3 H 유전자의높은발현율에의해서시아니딘계열의안토시아닌이주로축적된다. 페튜니아또한시아니딘계열의안토시아닌을축적하여빨간꽃색을나타낸다. Tsuda et al.(2004) 은내재 F3 H 유전자의발현을억제하고 DHK에기질선호도를보이는장미의 RhDFR 을도입하여펠라고니딘이축적된주황색페튜니아를개발하였다. 이러한전략을적용하여플라보노이드 B-ring의수산화를억제하고, 펠라고니딘축적을유도함으로써주황색국화생성이가능하리라예측한다 (Fig. 3C). 파란색꽃개발파란꽃색의구현을위한핵심색소물질은델피니딘으로, 플라보노이드 B-ring 의삼중수산화에관여하는 F3 5 H 활성이필수적으로요구된다. F3 5 H를암호화하는유전자는페튜니아에서가장먼저동정되었으며, 이후많은식물종에서분리되었다 (Brugliera et al. 1999, Holton et al. 1993). 국화과 (Asteraceae) 에속하는과꽃, 시네나리아등은 F3 5 H 활성을지니며파란꽃색을나타낸다. 계통발생분석에의하면식물의 F3 5 H는대개속씨식물과겉씨식물로나뉘기전에 F3 H로부터진화되었다. 반면, 국화과의 F3 5 H는국화과내의 F3 H로부터독립적으로진화되어서아미노산서열이다른식물의 F3 5 H 보다 F3 H 서열에대해높은유사성을나타낸다 (Ohmiya 2018). 그러나, 국화과식물들중국화속에속하는식물종들에서는 F3 5 H 활성을지니지않는다. 따라서속간교배를통해서는델피니딘계열안토시아닌을생성하는국화를개발하기는어렵다. 파란장미와더불어현재상업적으로판매되는파란색카네이션은흰색꽃에페튜니아의 PhF3 5 H와 PhDFR 유전자를과발현시킴으로써개발되었다 (Tanaka et al. 2009). 국화에서도파란꽃색을개발하기위한다양한연구가진행되었다. He et al.(2013) 은국화의내재 F3 H 유전자의발현을억제시키고국화과식물인세네시오 (Senecio) 의 ScF3 5 H를과발현시켰으나델피니딘은축적되지 않았다. 그러나 Noda et al.(2013) 은초롱꽃의 CamF3 5 H를과발현시켜델피니딘이생성된국화를개발하였으며 Brugliera et al.(2013) 은내재 F3 H를억제시키고팬지의 VtF3 5 H를과발현시켜국화에서델피니딘이축적되었으며자주색혹은보라색의국화가생성되었다. Fig. 4C에서처럼, 최근에초롱꽃의 CamF3'5'H와나비콩 (butterfly pea) 의 anthocyanin 3',5'-O-glucosyltransferase를암호화하는 CtUGT를국화에도입하여보라색또는파란색의꽃잎을지니는국화가개발되었다 (Noda et al. 2017). 유전자발현분석결과, 보라색국화에서는 CamF3 5 H만발현되고, 파란꽃색을나타내는국화에서는 CamF3 5 H와 CtUGT 유전자가모두발현되었다. 이와같은결과는파란색국화를얻기위해서 F3 5 H 외에도 UGT활성이필수적으로요구됨이확인되었다 (Fig. 3D). 분홍색 / 빨간색꽃개발시아니딘계열안토시아닌의축적은분홍색혹은빨간색을나타낸다. 담배에서꽃잎특이프로모터를이용하여애기장대 R2R3-MYB 전사인자 PAP1과옥수수 bhlh 전사인자 B-peru 를동시발현시킴으로써시아니딘계열안토시아닌의함량을증가시킨연구가최근보고되었다 (Fig. 4D, Kim et al. 2018). 담배형질전환체는진한붉은색꽃을생성하였으며, 안토시아닌함량은대조구와비교하여 124배증가되었다. 이러한결과는안토시아닌생합성양성조절자의발현을통해색소의축적을극적으로증가시킬수있음을나타내고, 국화의꽃색강화에있어서전사인자의중요성을시사하는사례라고할수있다 (Fig. 3E). 결론꽃색은화훼작물의관상가치를결정하는산업적으로중요한형질중의하나이며, 생명공학기술을기반으로한꽃색변경은매우매력적인연구분야이다. 성공적인꽃색변경을위해서는식물생리에대한생물학적이해및정교한육종기술과생명공학기술의적용이필요하다. 식물의대표적인이차대사산물군인플라보노이드와카로티노이드계물질들은꽃색결정에관여할뿐아니라식물의생존및번식에있어서중요한역할을수행한다. 또한각각의물질들은독립적인생합성경로를지니고있으면서도서로밀접하게관련되어조절받는것으로알려져있다. 본논문에서는플라보노이드계물질축적에초점을맞추어생합성대사경로및조절기작에대해전반적으로서술하였다. 또한, 생명공학기술을이용하여여러작물에서꽃색변경을달성한 358

9 성공적인사례들을제시하고생명공학기술이전통적육종방식의한계를어떻게극복했는지를보여주고, 궁극적으로국화의꽃색변경을달성할수있는전략들을제시하였다. 식물의이차대사물질은식물의종에따라심지어같은개체의서로다른조직에서도생육환경에따라생합성과조절양상이다양하다. 따라서, 지금까지알려진정보들만으로는식물체내의이차대사물질의축적과조절을명확히이해하기에는많은한계를지니고있으며국화의성공적인꽃색변경을달성하기위해서는많은부분들이추가적으로연구되어야한다. 플라보노이드물질중주요색소물질군이외의조색소물질군인플라본의생합성및세포내의축적기작에대한연구는다양한색조의꽃색개발에기여할수있을것이다. 또한, 다양한재배환경에서도안정적꽃색품질을유지하기위해서는환경스트레스에대한세밀한연구도수행되어야할것이다. 플라보노이드대사공학을통해새로운꽃색을지니는작물은많은화훼작물에서개발되고있으나, 실제적인상업화는몇몇화훼작물에국한되고있다. 생명공학기술로개발된작물들의상업화를위해서는연구및개발에소요되는비용과맞먹는안전성평가비용이소요된다. 하지만생명공학작물규제대상에서벗어나는유전자교정기술을활용한다면형질개량된화훼작물이보다빠르게시장에진입할수있으리라판단된다. 뿐만아니라, 본총설에서제시된플라보노이드대사공학의여러가지전략들을카로티노이드대사공학과접목함으로자연계는존재하지않는새로운꽃색의국화작물을개발할수있으리라기대된다. 적요관상용화훼작물에있어서꽃의색깔과형태는중요한형질중하나이다. 일반적으로꽃색은카로티노이드, 플라보노이드, 베타라인에의해결정된다. 그중플라보노이드는보다넓은영역의색을나타낸다. 국화는세계적으로인기가많은관상용화훼작물이며꽃색을바꾸기위한많은연구가진행되어왔다. 국화의경우, 시아니딘계열안토시아닌의축적으로분홍색혹은빨간색의꽃색을나타내며, 카로티노이드계열색소물질의축적으로노란색또는초록색의꽃색을나타낸다. 그러나자연계에는파란꽃색의국화는존재하지않는다. 지금까지플라보노이드계물질생합성을조절함으로써파란색꽃을개발하기위한여러연구가시도되었다. 반면그외의플라보노이드계물질을기반으로한새로운꽃색국화개발연구는거의수행되지않았다. 플라보노이드생합성조절에는다양한전사인자들이관여하고플라 보노이드계물질기반꽃색변경을위해서는구조유전자및전사인자들을이해하는것이중요하다. 따라서본논문에서는화훼작물의플라보노이드생합성및조절에대하여전반적으로서술하였고, 그동안보고된플라보노이드계물질의꽃색변경연구들을검토하였다. 이러한결과들은생명공학기술을기반으로한국화꽃색변경달성을위한중요한길잡이가될수있을것이다. 사사본연구는농촌진흥청차세대바이오그린 21 연구사업 ( 과제번호 : PJ 및 PJ013346) 및국립농업과학원의연구사업 (PJ012458) 의지원에의해수행되었습니다. REFERENCES 1. Aharoni A, De Vos CHR, Wein M, Sun Z, Greco R, Kroon A, Mol JNM, O'connell AP The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J 28: Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolise C, Boase MR, Ngo H, Jameson PE, Schwinn KE A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26: Andersen OM, Markham KR Flavonoid-protein interactions. In: Flavonoids chemistry, biochemistry and applications. CRC Press. pp Anderson NO Chrysanthemum. In: Flower breeding and genetics: Issues, challenges and opportunities for the 21st century. Springer. pp Azadi P, Bagheri H, Nalousi AM, Nazari F, Chandler SF Current status and biotechnological advances in genetic engineering of ornamental plants. Biotechnol Adv 34: Bedon F, Bomal C, Caron S, Levasseur C, Boyle B, Mansfield SD, Schmidt A, Gershenzon J, Grima-Pettenati J, Séguin A, MacKay J Subgroup 4 R2R3-MYBs in conifer trees: Gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses. J Exp Bot 61: Ben Zvi MM, Shklarman E, Masci T, Kalev H, Debener T, Shafir S, Ovadis M, Vainstein A PAP1 transcription 359

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