International Journal of Molecular Sciences Article Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanin Biosynthesis of Chrysanthemum Sun-Hyung Lim 1,*, Bora Park 2,3, Da-Hye Kim 2, Sangkyu Park 2, Ju-Hee Yang 2, Jae-A Jung 4 , JeMin Lee 3 and Jong-Yeol Lee 2,* 1 Division of Horticultural Biotechnology, School of Biotechnology, Hankyong National University, Anseong 17579, Korea 2 National Institute of Agricultural Sciences, Rural Development Administration, Jeonju 54874, Korea; [email protected] (B.P.); [email protected] (D.-H.K.); [email protected] (S.P.); [email protected] (J.-H.Y.) 3 Department of Horticultural Science, Kyungpook National University, Daegu 41566, Korea; [email protected] 4 Floriculture Research Division, National Institute of Horticultural & Herbal Science, Rural Development Administration, Wanju 55365, Korea; [email protected] * Correspondence: [email protected] (S.-H.L.); [email protected] (J.-Y.L.); Tel.: +82-31-670-5105 (S.-H.L.); +82-63-238-4616 (J.-Y.L.) Received: 30 September 2020; Accepted: 25 October 2020; Published: 27 October 2020 Abstract: Dihydroflavonol 4-reductase (DFR) catalyzes a committed step in anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonols to leucoanthocyanidins. However, the role of this enzyme in determining flower color in the economically important crop chrysanthemum (Chrysanthemum morifolium Ramat.) is unknown. Here, we isolated cDNAs encoding DFR from two chrysanthemum cultivars, the white-flowered chrysanthemum “OhBlang” (CmDFR-OB) and the red-flowered chrysanthemum “RedMarble” (CmDFR-RM) and identified variations in the C-terminus between the two sequences. An enzyme assay using recombinant proteins revealed that both enzymes catalyzed the reduction of dihydroflavonol substrates, but CmDFR-OB showed significantly reduced DFR activity for dihydrokaempferol (DHK) substrate as compared with CmDFR-RM. Transcript levels of anthocyanin biosynthetic genes were consistent with the anthocyanin contents at different flower developmental stages of both cultivars. The in planta complementation assay, using Arabidopsis thaliana dfr mutant (tt3-1), revealed that CmDFR-RM, but not CmDFR-OB, transgenes restored defective anthocyanin biosynthesis of this mutant at the seedling stage, as well as proanthocyanidin biosynthesis in the seed. The difference in the flower color of two chrysanthemums can be explained by the C-terminal variation of CmDFR combined with the loss of CmF3H expression during flower development. Keywords: anthocyanin; chrysanthemum; dihydroflavonol 4-reductase; flavonoid; flower color 1. Introduction Flower color influences the commercial value of ornamental plants. In general, floral coloration involves the accumulation of pigments such as flavonoids (including anthocyanins), carotenoids, and betalains. The flavonoid class anthocyanins are responsible for flower colors ranging from pale pink to blue in many ornamental plants including chrysanthemum (Chrysanthemum morifolium), carnation (Dianthus caryophyllus), lily (Lilium longiflorum), gerbera (Gerbera hybrida), petunia (Petunia hybrida), and rose (Rosa hybrida). The mechanism of anthocyanin biosynthesis has been extensively investigated, and most structural and regulatory genes involved in this process have been identified in several Int. J. Mol. Sci. 2020, 21, 7960; doi:10.3390/ijms21217960 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 16 Int. J. Mol. Sci. 2020, 21, 7960 2 of 17 investigated, and most structural and regulatory genes involved in this process have been identified in several plant species [1–3]. Dihydroflavonol 4-reductase (DFR) plays an important role in the plantformation species of anthocyanins [1–3]. Dihydroflavonol and proanthocyanidins, 4-reductase (DFR) as shown plays anin the important Figure role1. DFR in thecatalyzes formation the ofconversion anthocyanins of andthe proanthocyanidins, colorless dihydroflavonols, as shown in theincluding Figure1. DFRdihydrokaempferol catalyzes the conversion (DHK), ofdihydroquercetin the colorless dihydroflavonols, (DHQ), and dihydromyricetin including dihydrokaempferol (DHM), into leucoanthocyanidins. (DHK), dihydroquercetin These (DHQ), three andcolorless dihydromyricetin dihydroflavonols (DHM), are intosubs leucoanthocyanidins.trates of flavonol biosynthesis These three that colorless are catalyzed dihydroflavonols by flavonol are substratessynthase (FLS). of flavonol Competition biosynthesis between that areFLS catalyzed and DFR by can flavonol modify synthase the metabolic (FLS). Competitionflux and alter between flower FLScolor and [4–6]. DFR can modify the metabolic flux and alter flower color [4–6]. Figure 1.1. Schematic representation of flavonols,flavonols, proanthocyanidins,proanthocyanidins, and anthocyaninsanthocyanins biosyntheticbiosynthetic pathway in in plants plants The The abbreviations abbreviations of ofenzyme enzyme names names are areas follows: as follows: CHS, CHS, chalcone chalcone synthase; synthase; CHI, CHI,chalcone chalcone isomerase; isomerase; F3H, F3H,flavanone flavanone 3-hydroxylase; 3-hydroxylase; FLS, FLS,flavonol flavonol synthase; synthase; F3′H, F3 0flavonoidH, flavonoid 3′- 3hydroxylase;0-hydroxylase; F3 F3′5′H,050 H,flavonoid flavonoid 3′ 3,50′,5-hydroxylase;0-hydroxylase; DFR, DFR, dihydroflavonol dihydroflavonol 4-reductase; 4-reductase; LAR, leucoanthocyanidin reductase; reductase; ANR, ANR, anthocyanidi anthocyanidinn reductase; reductase; ANS, ANS, anthocyanidin anthocyanidin synthase; synthase; and andUFGT, UFGT, UDP-glucose: UDP-glucose: flavonoid flavonoid 3-O-glucosyltransferase. 3-O-glucosyltransferase. Several studies have revealed that the substrate specificityspecificity of DFR plays an important role in the determination ofof anthocyaninanthocyanin types types [ 7[7–10].–10]. According According to to di differencesfferences at at the the 134th 134th amino amino acid acid residues residues in theirin their substrate substrate binding binding domains, domains, DFRs DFRs are divided are divi intoded three into types,three thetypes, asparagine the asparagine (Asn)-type, (Asn)-type, aspartic acidaspartic (Asp)-type, acid (Asp)-type, and non-Asn and non-Asn/Asp-type./Asp-type. Previous Prev studiesious have studies reported have reported that Asn-type that Asn-type DFRs catalyze DFRs thecatalyze conversion the conversion of all three of dihydroflavonolsall three dihydrofla (DHK,vonols DHQ, (DHK, and DHQ, DHM), and whereas DHM), Asp-typewhereas DFRsAsp-type are highlyDFRs are specific highly for specific DHQ and for DHMDHQ ratherand DHM than rather for DHK than as for a substrate DHK as [a8– substrate10]. However, [8–10]. not However, all Asn-type not DFRsall Asn-type can catalyze DFRs all can three catalyze dihydroflavonols all three dihydroflavonols as substrates. DFRs as substrates. from sweet DFRs potato from (IbDFR) sweet and potato from freesia(IbDFR) (FeDFR2) and from are freesia classified (FeDFR2) into the are Asn-type classified DFR, into duethe toAsn-type their 134th DFR, amino due to acid their residues 134th [amino10,11]. IbDFRacid residues catalyzes [10,11]. only IbDFR the DHK catalyzes but not only DHQ the and DHK DHM, but not while DHQ FeDFR2 and DHM, utilizes while both FeDFR2 the DHQ utilizes and DHMboth the but DHQ not the and DHK DHM as a substrate.but not the Other DHK studies as a havesubstrate. shown Other that thestudies neighboring have shown residues that of the substrateneighboring binding residues sites of and the NADPsubstrate binding binding regions sites and of DFRs NADP play binding pivotal regions roles inof proteinDFRs play activity pivotal in buckwheatroles in protein (Fagopyrum activity esculentumin buckwheat), gerbera, (Fagopyrum and grape esculentum hyacinth), gerbera, (Muscari and armeniacum grape hyacinth)[11–13 (].Muscari These resultsarmeniacum imply) [11–13]. that other These factors results can imply influence that the other substrate factors specificity can influence of DFR the enzyme. substrate specificity of DFR Chrysanthemum,enzyme. a perennial herbaceous flowering plant, is an important floricultural crop worldwide.Chrysanthemum, The ray florets a perennial of chrysanthemum herbaceous flowers flowering can beplant, pink, is red,an important yellow, white, floricultural purple, green, crop orworldwide. various bicolored The ray florets forms of due chry tosanthemum the accumulation flowersof can di befferent pink, proportions red, yellow,of white, two pigments,purple, green, i.e., anthocyaninsor various bicolored and carotenoids forms due [14 ].to Therethe accumulation are three basic of types different of anthocyanidins, proportions of namely, two pigments, pelargonidin, i.e., cyanidin,anthocyanins and delphinidin.and carotenoids Although [14]. the There ray florets are three of chrysanthemum basic types onlyof anthocyanidins, accumulate the cyanidinnamely, derivatives,pelargonidin, the cyanidin, substrate and feeding delphinidin. assay revealed Although that CmDFRthe ray couldflorets utilize of chrysanthemum DHK and DHM only as a substrateaccumulate for the pelargonidin cyanidin derivatives, and delphinidin the substrat biosynthesis,e feeding respectively assay revealed [14–16 that]. CmDFR could utilize DHKIn and the DHM current as study,a substrate we identified for pelargonidin two DFR and genes, delphinidinCmDFR-OB biosynthesis,and CmDFR-RM, respectivelywith [14–16]. different C-terminus, from chrysanthemum flowers with white and red colors, respectively, and demonstrated that these genes function in anthocyanin