Identification and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth

molecules

Article Identiﬁcation and Functional Analysis of a Flavonol Synthase Gene from Grape Hyacinth

Hongli Liu 1,2,3, Beibei Su 1,2,3, Han Zhang 1,2,3, Jiaxin Gong 1,2,3, Boxiao Zhang 1,2,3, Yali Liu 1,2,3,* and Lingjuan Du 1,2,3,*

1 College of Landscape Architecture and Arts, Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (H.L.); [email protected] (B.S.); [email protected] (H.Z.); [email protected] (J.G.); [email protected] (B.Z.) 2 State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling 712100, Shaanxi, China 3 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northwest Region), Ministry of Agriculture, Yangling 712100, Shaanxi, China * Correspondence: [email protected] (Y.L.); [email protected] (L.D.); Tel.: +86-029-8708-2522 (Y.L.)

Received: 31 March 2019; Accepted: 19 April 2019; Published: 22 April 2019

Abstract: Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

Keywords: copigmentation; ﬂavonol; ﬂower color; FLS; grape hyacinth

1. Introduction Flower color is a key characteristic of ornamental plants [1]. Anthocyanins are flavonoids that contribute to the pink, red, orange, scarlet, violet, blue, and yellow pigmentation of ornamental plant flowers [2]. Copigmentation with other flavonoids influences the hue and intensity of flower color. Copigmentation can stabilize colored pigments and induce a blue shift in the final visible color [3]. Flavonols are one group of flavonoid that can act as copigments sandwiched between anthocyanins, causing color shifts and an increased color variety [1]. Furthermore, flavonols can themselves impart pale yellow or yellow coloration [4]. Both anthocyanins and flavonols are produced by branches of the flavonoid pathway and are derived from dihydroflavonols (dihydrokaempferol: DHK, dihydroquercetin: DHQ, and dihydromyricetin: DHM). Anthocyanins are synthesized by the sequential reaction of dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and glucosyltransferase (UFGT). In competition with DFR at a crucial branch point, individually acting FLS can catalyze the oxidation of dihydroflavonols to flavonols [4]. FLS is characterized as a soluble 2-ODD that requires cofactors 2-oxoglutarate, ferrous

Molecules 2019, 24, 1579; doi:10.3390/molecules24081579 www.mdpi.com/journal/molecules Molecules 2019, 24, 1579 2 of 15 iron (II), and ascorbate and introduces the double bond between C-2 and C-3 of the C-ring [5,6]. Besides FLS, other 2-ODD enzymes, namely, flavanone 3β-hydroxylase (F3H), flavone synthase I (FNS I), and ANS, also function in flavonoid biosynthesis [7]. The first FLS cDNA was cloned from petunia (Petunia hybrida) and was functionally expressed in yeast and plants [6]. Additional FLS genes have since been identified and characterized in various plant species, such as Arabidopsis thaliana [8–10], Eustoma grandiflorum (lisianthus) [1], Citrus unshiu [11], Glycine max [12], Gentiana triflora [13], Ginkgo biloba [14], Zea mays (maize) [15], Camellia nitidissima [4], Vaccinium corymbosum [16], Allium cepa (onion) [17], Cyclamen purpurascens (cyclamen) [18], and Litchi chinensis [19]. There have been some reports of FLS genes in ornamental plants that function in flower coloration. Holton et al. demonstrated that antisense expression of a FLS gene in petunia reduced flavonol synthesis and changed the light pink coloration of petals and filaments to red. Similarly, constitutive overexpression of a FLS gene from lisianthus produced flowers with a deeper magenta coloration than the wild type (WT) plants [1]. Heterologous expression of CnFLS1 in Nicotiana tabacum (tobacco) changed floral coloration, resulting in white flowers [4]. Akita et al. isolated two FLS genes (CpurFLS1 and CpurFLS2) and discussed their involvement in flower coloration in cyclamen [18]. Furthermore, previous studies also showed that FLS can cooperate with DFR to control the metabolic balance between the anthocyanin and flavonol branches of the flavonoid pathway to ultimately determine the formation of flower color [20], or identified the transcription factor R2R3-MYB, which regulates FLS to affect spatial pattern variation of floral pigments [21]. Grape hyacinth (Muscari spp.), named for its unique grape-like color and shape, is a monocotyledonous ornamental bulbous plant that blossoms in mid-spring [22,23]. Generally, grape hyacinth varieties display a single color, such as white, pink, purple, azure, cobalt, violet, or lavender. Rarely, some species produce upper flowers that are a different color to the lower flowers [22]. Because of its variable flower coloration, grape hyacinth is a good model to study the secondary metabolism of flower coloration in monocots [22,24]. Previous studies of grape hyacinth flower coloration have focused on the chemical basis of anthocyanin-related pigmentation and on the functions of structural and regulatory proteins involved in anthocyanin biosynthesis [22,23,25]. Few studies have examined the compounds involved in copigmentation or analyzed the genes affecting copigmentation. It is valuable to elucidate the mechanism of flower color formation or create the novel color morphs of grape hyacinth via molecular regulation of the key gene (FLS) for flavonol synthesis. In this study, we identified a FLS gene expressed in grape hyacinth petals, MaFLS, and analyzed its spatial and temporal expression patterns. Moreover, we measured flavonol contents at different floral developmental stages and in different organs. Via heterologous expression in tobacco, we examined the function of MaFLS in planta by examining the effect on phenotype, flavonoid content, and expression of endogenous genes in the petals of the transgenic tobacco lines. We discuss the potential role of MaFLS during floral coloration of grape hyacinth. Overall, our study provides further evidence to a growing literature on FLS–DFR competition and provides the basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.

2. Results

2.1. MaFLS Gene Cloning and Sequence Analysis We searched for FLS homologs in the transcriptome of M. armeniacum ﬂowers [24] by local BlastP querying with the characterized A. thaliana FLS1 [9,26,27]. One FLS homolog was identiﬁed and designated MaFLS (GenBank accession number MH636605) and its cDNA was isolated by RACE-PCR and PCR. MaFLS consisted of 1418 nucleotides with a poly(A) tail, of which 993 bp represented an ORF encoding 330 amino acid residues, with a molecular weight of 36.45 kDa and a theoretical pI of 6.33. Comparison of the deduced MaFLS amino acid sequence and that of other FLS proteins with known functions revealed that it shared 72% and 71% identity with monocot Allium cepa AcFLS-HRB (AY647262) and AcFLS-H6 (AY221247), respectively; 67% identity with Citrus unshiu CuFLS (AB011796); Molecules 2019, 24, 1579 3 of 15 Molecules 2019, 24, x FOR PEER REVIEW 3 of 14

64%(AB011796); identity 64% with identityGentiana with triflora GentianaGtFLS triflora (AB587658); GtFLS (AB587658); and 63% identity and 63% with identityCamellia with nitidissima Camellia CnFLSnitidissima (ADZ28516). CnFLS (ADZ28516). Moreover, MaFLS Moreover, harbored MaFLS characteristic harbored conservedcharacteristic ferrous conserved iron-binding ferrous residues iron- (His216,binding Asp218,residues and(His216, His272; Asp218, marked and by His272; black arrows; marked Figure by black1) and arrows; the 2-oxoglutarate Figure 1) and binding the 2- residuesoxoglutarate (Arg282 binding and Ser284;residues marked (Arg282 by and gray Ser284; arrows; ma Figurerked by1) ofgray FLS arrows; proteins. Figure This 1) clearly of FLS indicated proteins. 2+ thatThis MaFLSclearly belongedindicated tothat the MaFLS soluble belonged Fe2+/2-ODD to the protein soluble family. Fe /2-ODD Furthermore, protein MaFLS family. possessed Furthermore, five keyMaFLS amino possessed acids (Tyr127, five key Phe129,amino acids Lys197, (Tyr127, Phe288, Phe129, and Ser290;Lys197, markedPhe288, byand black Ser290; dots; marked Figure by1) black that weredots; identifiedFigure 1) that as potential were identified active DHQ as potential binding active site residues, DHQ binding highly site conserved residues, in highly FLSs ofconserved various speciesin FLSs [26 ].of Invarious addition, species MaFLS contained[26]. In theadditi FLS-specificon, MaFLS motifs contained “PxxxIRxxxEQP” the FLS-specific and “SxxTxLVP”, motifs which“PxxxIRxxxEQP” can be used and to distinguish“SxxTxLVP”, FLSs whic fromh can other be used plant to 2-ODDs,distinguish like FLSs F3H, from ANS, other and plant FNS. 2-ODDs, MaFLS possessedlike F3H, ANS, the residues and FNS. responsible MaFLS possessed for the proper the residu foldinges responsible of the FLS for polypeptide the proper (Gly65 folding and of Gly256;the FLS markedpolypeptide by asterisks; (Gly65 and Figure Gly256;1), which marked are conservedby asterisks; in allFigure 2-ODD 1), proteinswhich are [17 conserved]. in all 2-ODD proteins [17].

Figure 1.1. MultipleMultiple alignment alignment of of the predicted amino acid sequence of MaFLS with that of other flavonolflavonol synthase (FLS) proteins. MaFLS is compared with FLS sequences from Eustoma grandiflorumgrandiflorum (EgFLS),(EgFLS), Gentiana trifloratriflora (GtFLS),(GtFLS), PetuniaPetunia hybridahybrida (PhFLS),(PhFLS), NicotianaNicotiana tabacumtabacum (NtFLS),(NtFLS), PetroselinumPetroselinum crispum (PcFLS),(PcFLS),Camellia Camellia nitidissima nitidissima(CnFLS), (CnFLS),Cyclamen Cyclamen purpurascens purpurascens(CpurFLS1), (CpurFLS1),Citrus unshiu Citrus(CuFLS), unshiu Allium(CuFLS), cepa Allium(AcFLS-H6 cepa (AcFLS-H6 and AcFLS-HRB), and AcFLS-HRB),Arabidopsis thalianaArabidopsis(AtFLS1), thalianaAcacia (AtFLS1), confusa Acacia(AcFLS), confusa and Ginkgo(AcFLS), biloba and(GbFLS). Ginkgo biloba The alignment (GbFLS). wasThe generatedalignment usingwas generated CLC sequence using viewer CLC sequence 8.0. The FLS-specific viewer 8.0. motifsThe FLS-specific “PxxxIRxxxEQP” motifs and“PxxxIRxxxEQP” “SxxTxLVP” are and indicated. “SxxTxLVP” Black are arrows indicated. indicate Black the ferrousarrows iron-bindingindicate the residuesferrous iron-binding (His216, Asp218, residues and (His His272).216, Asp218, Gray arrowsand His272). represent Gray the arrows 2-oxoglutarate represent the binding 2-oxoglutarate residues (Arg282binding residues and Ser284). (Arg282 Black and dots Ser284). show Black the putative dots sh DHQ-bindingow the putative residues DHQ-binding (Tyr127, residues Phe129, (Tyr127, Lys197, Phe288, and Ser290). Asterisks indicate the residues responsible for the proper folding of the FLS Phe129, Lys197, Phe288, and Ser290). Asterisks indicate the residues responsible for the proper polypeptide (Gly65 and Gly256). folding of the FLS polypeptide (Gly65 and Gly256). To determine the relationship between the putative MaFLS protein and other plant FLSs, we To determine the relationship between the putative MaFLS protein and other plant FLSs, we performed a phylogenetic analysis with the functionally characterized plant FLSs as well as various performed a phylogenetic analysis with the functionally characterized plant FLSs as well as various putative FLSs (Figure2). MaFLS was grouped with fellow monocots close to Narcissus tazetta NtaFLS putative FLSs (Figure 2). MaFLS was grouped with fellow monocots close to Narcissus tazetta NtaFLS (AFS63900), Allium cepa AcFLS-H6 (AY221247), and AcFLS-HRB (AY647262) in the order Asparagales, (AFS63900), Allium cepa AcFLS-H6 (AY221247), and AcFLS-HRB (AY647262) in the order and Lilium regale LrFLS (ASV46329) in the order Liliales (Figure2). MaFLS showed a marked separation Asparagales, and Lilium regale LrFLS (ASV46329) in the order Liliales (Figure 2). MaFLS showed a from enzymes of dicot plants and of monocot Poaceae grasses. These results indicate that the marked separation from enzymes of dicot plants and of monocot Poaceae grasses. These results phylogenetic analysis fits well with the genetic relationships among the species. indicate that the phylogenetic analysis fits well with the genetic relationships among the species.

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FigureFigure 2.2. PhylogeneticPhylogenetic analysisanalysis ofof MaFLSMaFLS andand other other FLS FLS proteins. proteins. TheThe maximum-likelihoodmaximum-likelihood phylogeneticphylogenetic treetree was generated generated using using MEGA MEGA 6.0 6.0 soft software.ware. Numbers Numbers at each at interior each interior branch branchindicate indicatethe bootstrap the bootstrap values of values1000 replicates. of 1000 The replicates. bar indicates The a bargenetic indicates distance a of genetic 0.1. GenBank distance accession of 0.1. GenBanknumbers accessionare as follows: numbers Muscari are asaucheri follows: MaFLSMuscari (MH636605, aucheri markedMaFLS with (MH636605, red triangle), marked Nicotiana with redtabacum triangle), NtFLS1Nicotiana (ABE28017) tabacum andNtFLS1 NtFLS (ABE28017)(AB289451), andPetunia NtFLS hybrida (AB289451), PhFLS (CAA80264),Petunia hybrida PetroselinumPhFLS (CAA80264),crispum PcFLSPetroselinum (AAP57395), crispum EustomaPcFLS grandiflorum (AAP57395), EgFLSEustoma (AAF64168), grandiflorum GentianaEgFLS triflora (AAF64168), GtFLS Gentiana(AB587658), triflora ScutellariaGtFLS (AB587658), baicalensis SbFLSScutellaria (KC404852), baicalensis AntirrhinumSbFLS (KC404852), majus AmFLSAntirrhinum (ABB53382), majus AmFLS Camellia (ABB53382),nitidissima CamelliaCnFLS (ADZ28516), nitidissima CnFLS Vaccinium (ADZ28516), corymbosumVaccinium VcFLS corymbosum (KP334105),VcFLS Cyclamen (KP334105), purpurascensCyclamen purpurascensCpurFLS1(LC210072)CpurFLS1(LC210072) and CpurFLS2 and CpurFLS2(LC210073), (LC210073), Glycine maxGlycine GmFLS1 max GmFLS1(AB246668), (AB246668), Malus domesticaMalus domesticaMdFLS MdFLS(AY965343), (AY965343), Rosa rugosaRosa rugosaRrFLSRrFLS (KM099095), (KM099095), FagopyrumFagopyrum tataricum tataricum FtFLS1FtFLS1 (JF274262) (JF274262) and andFtFLS2 FtFLS2 (JX401285), (JX401285), CitrusCitrus unshiu unshiu CuFLSCuFLS (AB011796), (AB011796), ArabidopsisArabidopsis thaliana thaliana AtFLS1AtFLS1 (AAB41504), (AAB41504), Allium Alliumcepa AcFLS-H6 cepa AcFLS-H6 (AY221247) (AY221247) and AcFLS-HRB and AcFLS-HRB (AY647262), (AY647262), Lilium regaleLilium LrFLS(ASV46329), regale LrFLS(ASV46329), Narcissus Narcissustazetta NtaFLS tazetta (AFS63900),NtaFLS (AFS63900), BrachypodiumBrachypodium distachyon distachyon BdFLS1BdFLS1 (XM_003570514), (XM_003570514), Oryza sativaOryza OsFLS1 sativa OsFLS1(NP_001048230), (NP_001048230), SorghumSorghum bicolor bicolor SbFLS1SbFLS1 (XP_002454608) (XP_002454608) and and SbFLS2 SbFLS2 (EES00073), (EES00073), ZeaZea maysmays ZmFLS1(BT039956)ZmFLS1(BT039956) and and ZmFLS2 ZmFLS2 (XP_008646309), (XP_008646309),Ginkgo Ginkgo biloba bilobaGbFLS GbFLS (ACY00393), (ACY00393),Camellia Camellia sinensis sinensis CsFLSCsFLS (ABM88786), (ABM88786), and andAcacia Acacia confusa confusaAcFLS AcFLS (JN812062). (JN812062). 2.2. Correlation Analysis of MaFLS Expression Levels and Total Flavonol Content 2.2. Correlation Analysis of MaFLS Expression Levels and Total Flavonol Content We extracted metabolites from roots, bulbs, leaves, and petals at the five floral developmental stagesWe (S1–S5) extracted of “White metabolites Beauty” from and roots, “Dark bulbs, Eyes” leav (Figurees, and3A). petals HPLC at the analysis five floral revealed developmental that total flavonolstages (S1–S5) content of in “White all organs Beauty” of “White and Beauty”“Dark Eyes” was significantly (Figure 3A). higher HPLC than analysis that ofrevealed “Dark Eyes”. that total In “Whiteflavonol Beauty”, content total in all flavonol organs content of “White increased Beauty” during was flowersignificantly development, higher than peaked that before of “Dark blooming, Eyes”. andIn “White then decreased Beauty”, gradually total flavonol after petalcontent expansion increased (Figure during3B). flower However, development, in “Dark Eyes”, peaked the before total flavonolblooming, content and then was decreased higher in S2gradually buds than after in petal the buds expansion at other (Figure developmental 3B). However, stages. in “Dark In addition, Eyes”, totalthe total flavonols flavonol also content accumulated was higher in other in S2 vegetative buds than organs, in the buds especially at other in leavesdevelopmental of the two stages. grape In hyacinthaddition, cultivars. total flavonols Recently, also Louaccumulated et al. reported in other that vegetative the level organs, of total especially anthocyanins in leaves in violet-blue of the two flowersgrape hyacinth such as thosecultivars. of “DarkRecently, Eyes” Lou was et al. substantially reported that higher the level than of that total in anthocyanins the white flowers in violet- of blue flowers such as those of “Dark Eyes” was substantially higher than that in the white flowers of “White Beauty” [22]. Therefore, these results likely reflect relatively higher flavonol accumulation

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Molecules 2019, 24, x FOR PEER REVIEW 5 of 14 “White Beauty” [22]. Therefore, these results likely reflect relatively higher flavonol accumulation and extremelyand extremely low anthocyanin low anthocyanin accumulation accumulation during duri the formationng the formation of white of flowers white in flowers “White in Beauty”. “White Beauty”.

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(B)

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FigureFigure 3.3. TheThe flavonolflavonol contentcontent and expressionexpression profilesprofiles ofof MaFLSMaFLS inin M.M. aucheriaucheri “Dark“Dark eyes”eyes” andand M.M. aucheriaucheri“White “White Beauty”. Beauty”. ( A(A)) The The inflorescence inflorescence and and the the petals petals at at five five flower flower developmental developmental stages stages of M. of aucheriM. aucheri“Dark “Dark eyes” eyes” and M.and aucheri M. aucheri“White “White Beauty”. Beauty”. Bars, 5Bars, mm. 5 (mm.B) The (B total) The flavonol total flavonol content content of petals. of FW:petals. Fresh FW: weight. Fresh ( Cweight.) The expression (C) The profileexpression of MaFLS. profileFlavonol of MaFLS. content Flavonol and MaFLS contentexpression and MaFLS were determinedexpression were in roots determined (R), bulbs in (B), roots leaves (R), bulbs (L), and (B), petals leaves at (L), five and flower petals developmental at five flower developmental stages (S1–S5). MaActinstages (S1–S5).was used MaActin as the was internal used expression as the internal control. expression Each column control. represents Each column means representsSD from means three ± ± technicalSD from replicates.three technical Different replicates. letters Different above the letters bars indicate above the significantly bars indicate different significantly values ( Pdifferent< 0.05) calculatedvalues (P < using 0.05) one-waycalculated ANOVA using one-way followed ANOVA by a Duncan’s followed multiple by a Duncan’s range test. multiple range test.

Furthermore,Furthermore, wewe determineddetermined thethe expressionexpression levelslevels ofof MaFLSMaFLS inin didifferentﬀerent organsorgans andand ﬂowerflower developmentdevelopment stagesstages byby qRT-PCRqRT-PCR analysis.analysis. MaFLSMaFLS transcripttranscript levelslevels werewere highesthighest inin S1S1 buds,buds, whereaswhereas MaFLS expression was undetectable in S2–5 petals and in the roots, bulbs, and leaves of M. aucheri “White Beauty” (Figure 3C). Similarly, in M. aucheri “Dark eyes”, MaFLS expression was

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MoleculesMaFLS expression2019, 24, x FOR was PEER undetectable REVIEW in S2–5 petals and in the roots, bulbs, and leaves of M. aucheri6 of 14 “White Beauty” (Figure3C). Similarly, in M. aucheri “Dark eyes”, MaFLS expression was extraordinarily extraordinarilyhigh in S1 buds, high markedly in S1 buds, lower inmarkedly S2 petals, lower and in undetectable S2 petals, and in S3–5 undetectable petals. Moreover, in S3–5 MaFLSpetals. Moreover,expression MaFLS was barely expression detectable was barely in the detectable roots and in bulbs, the roots and and slightly bulbs, detectable and slightly in thedetectable leaves in of theM. aucherileaves “Darkof M. aucheri eyes” (Figure“Dark 3eyes”C). Thus, (FigureMaFLS 3C). isThus, predominantly MaFLS is predominantly expressed in the expressed early stages in the of earlyflower stages development of flower in grapedevelopment hyacinth, in which grape is consistenthyacinth, withwhich reports is consistent in other ornamental with reports plants, in other such ornamentalas carnation (plants,Dianthus such caryophyllus as carnation), petunia, (Dianthus lisianthus, caryophyllus and cyclamen), petunia, [1,6 ,18lisianthus,,28]. Together, and ourcyclamen results [1,6,18,28].show that MaFLS Together,likely our plays results a key show role inthat flower MaFLS coloration, likely plays alongside a key other role copigmentation-related in flower coloration, alongsideFLS genes. Nevertheless,other copigmentation-relatedMaFLS expression isFLS not concomitantgenes. Nevertheless, with the accumulation MaFLS expression of total flavonols is not concomitantin grape hyacinth. with the accumulation of total flavonols in grape hyacinth.

2.3. In In Vivo Vivo Localization Localization of of MaFLS MaFLS To detect the subcellular localization of MaFLS, the positive control 35S:GFP (pBI221) and the recombinant plasmid pBI221-MaFLS-GFP pBI221-MaFLS-GFP were were transformed transformed into into A.A. thaliana thaliana mesophyllmesophyll protoplasts. protoplasts. As shown in Figure 44,, ﬂuorescencefluorescence fromfrom 35S:GFP35S:GFP waswas disperseddispersed throughoutthroughout A. thaliana mesophyll protoplasts, whereas fluorescence ﬂuorescence from the MaFLS:GFP fusion protein was mainly localized in the cytosol and cell periphery. Therefore, MaFLS is both a cytoplasmic and cellcell peripheryperiphery protein.protein.

Figure 4. Subcellular localization of MaFLS proteins. Transient expression of the MaFLS-GFP Figurefusion protein4. Subcellular and the localization 35S:GFP control of MaFLS in A. proteins. thaliana mesophyllTransient expression protoplasts. of GFP,the MaFLS-GFP GFP fluorescence; fusion proteinautofluorescence, and the 35S:GFP chloroplast control autofluorescence; in A. thaliana merge mesophyll is an overlay protoplasts. of chloroplast GFP, GFP autofluorescence, fluorescence; autofluorescence,GFP fluorescence, chloroplast and bright-field autofluorescence; images. Bars, merg 25 mm.e is an overlay of chloroplast autofluorescence, GFP fluorescence, and bright-field images. Bars, 25 mm. 2.4. Heterologous Expression of MaFLS in Tobacco Alters Petal Color 2.4. HeterologousTo ascertain Expression the function of MaFLS of MaFLS in Tobacco in flower Alters coloration, Petal ColorMaFLS was transformed into tobacco (N. tabacumTo ascertain“NC89”) underthe function the control of MaFLS of the 35S-CaMV in flower coloration, promoter for MaFLS heterologous was transformed expression into experiments. tobacco (WeN. identifiedtabacum “NC89”) 11 transformant under the lines control by PCR of the analysis, 35S-CaMV using promoter the vector-specific for heterologous primers 2300-Fexpression and experiments.2300-R (Table We S1). identified Compared 11 with transformant the deep pinklines flowers by PCR of analysis, wild-type using tobacco, the vector-specific the transgenic primers tobacco 2300-Flines heterologously and 2300-R (Table expressing S1). MaFLSComparedshowed with reduced the deep levels pink of petalflowers pigmentation of wild-type (Figure tobacco,5A). Five the transgeniclines displayed tobacco a severe lines phenotype heterologously (S) of pale expressing pink to completely MaFLS whiteshowed petals; reduced three lineslevels displayed of petal a pigmentationmedium phenotype (Figure (M) 5A). of pinkFive petals,lines displayed whereas thea se remainingvere phenotype lines displayed (S) of pale a weak pink phenotypeto completely (W) whiteof pale petals; pink petals three (Figurelines displayed5A). a medium phenotype (M) of pink petals, whereas the remaining lines displayed a weak phenotype (W) of pale pink petals (Figure 5A).

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Figure 5. 5. ChangesChanges in in tobacco tobacco flowers flowers induced induced by by heterologous heterologous expression expression of of MaFLS.MaFLS. ((AA)) Heterologous Heterologous expression of MaFLS inin transgenic tobacco tobacco flowers flowers resulted resulted in in a clear phenotypic change in in petal coloration. Shown Shown are are non-transformed non-transformed controls controls (WT) (WT) and three MaFLS-expressing lines exhibiting differentdifferent phenotypicphenotypic characteristics characteristics (S, (S, strong; strong; M, medium;M, medium; W, weak). W, weak). Bars, 5Bars, cm. (5B )cm. qRT-PCR (B) qRT-PCR analysis analysisof MaFLS ofrelative MaFLS expression relative expression levels in the levels petals in of the three petals independent of three transgenicindependent tobacco transgenic lines. NtTubA1tobacco lines.was used NtTubA1 as the was internal used expression as the internal control. expression (C) The colorcontrol. of the(C) extractionsThe color of is the shown extractions in the centrifugal is shown intubes. the centrifugal HPLC analysis tubes. of anthocyaninHPLC analysis and of flavonol anthocyanin levels and in tobacco flavonol petals levels in mgin tobacco/g fresh weightpetals in (FW) mg/g of freshthe non-transformed weight (FW) of control the non-transformed (wild type, WT) contro and thel (wild transgenic type, lineWT) with and a the strong transgenic phenotype line (S). with Each a strongcolumn phenotype represents (S). means Each SDcolumn from represents three technical mean replicates.s ± SD from Diff threeerent technical letters above replicates. the bars Different indicate ± letterssignificantly above dithefferent bars valuesindicate (P significantly< 0.05) calculated different using values one-way (P < ANOVA0.05) calculated followed using by Duncan’sone-way ANOVAmultiple rangefollowed test. by Duncan’s multiple range test.

To further investigate MaFLS expression levels in the transgenic tobacco lines, we carried out qRT-PCR analysisanalysis usingusing MaFLSMaFLS-speciﬁc-specific qRT-PCR qRT-PCR primers primers (Table (Table S1). S1). We We discovered discovered that that the the extent extent of ofchange change in petalin petal coloration coloration was consistentwas consistent with thewith relative the relative expression expression levels of levelsMaFLS ofin MaFLS the transgenic in the transgenictobacco lines, tobacco in that lines, the linesin that with the a lines severe with and a a severe weak phenotypeand a weak had phenotype the highest had and the lowest highestMaFLS and lowestexpression MaFLS levels, expression respectively levels, (Figure respectively5B). Therefore, (Figure 5B). the Therefore, changes inthe petal changes color in in petal the color transgenic in the transgenictobacco lines tobacco appear lines to be appear the result to be of the heterologous result of heterologousMaFLS expression. MaFLS expression. In addition, we further investigated the changes in anthocyanin and ﬂavonol levels in petals from transgenic tobacco lines with a severe phenotype by HPLC. The color of the extractions indicated that the petals of lines with a severe phenotype contained less anthocyanins than the non-

Molecules 2019, 24, x FOR PEER REVIEW 8 of 14 transformed control (Figure 5C). This was confirmed by HPLC analysis, which showed that the severe phenotype lines had a clear reduction in total anthocyanin levels; however, they accumulated higher levels of total flavonols, compared to the non-transformed control (Figure 5C). These results strongly suggest that high-level MaFLS expression in transgenic tobacco lines causes a remarkable decrease in anthocyanin content and an increase in flavonol accumulation in vivo, which results in a severe reduction in petal pigmentation. Molecules 2019, 24, 1579 8 of 15 2.5. Heterologous Expression of MaFLS in Tobacco Affects the Expression of Anthocyanin Pathway Genes Subsequently,In addition, we we further conducted investigated a qRT-PCR the changes expression in anthocyanin analysis of and endogenous flavonol levels genes in involved petals from in thetransgenic flavonoid tobacco biosynthetic lines with pathway a severe in phenotypethe petals of by MaFLS HPLC.-expressing The color of transgenic the extractions tobacco indicated (Figure that6A, primersthe petals are of listed lines in with Table a severeS1). The phenotype analyzed containedgenes included lessanthocyanins twelve structural than genes the non-transformed (NtPAL, NtC4H, Nt4CLcontrol, (FigureNtCHS,5 C).NtCHI This, NtF3H was confirmed, NtF3’H, by NtF3’5’H HPLC analysis,, NtFLS, whichNtDFR showed, NtANS that, and the NtUFGT severe) phenotype and three regulatorylines had a cleargenes reduction (NtAN2, inNtAN1a total anthocyanin, and NtAN1b levels;), which however, are highlighted they accumulated in red (Figure higher 6A). levels of total flavonols,Based compared on the one-way to the ANOVA non-transformed statistical control results (Figurepresented5C). in These Figure results 6, only strongly the expression suggest level that ofhigh-level NtCHI MaFLSwas notexpression significantly in transgenic different tobacco between lines the causes MaFLS a remarkable-expressing decrease lines inand anthocyanin the non- transformedcontent and ancontrol; increase all the in flavonolother genes accumulation showed thein decreased vivo, which expressions. results in aIn severe particular, reduction NtCHS in, NtF3Hpetal pigmentation., NtDFR, NtANS, and NtAN2 expression was remarkably lower in the MaFLS-expressing lines than in the non-transformed control (Figure 6B–D). Particularly, the transcript levels of two endogenous2.5. Heterologous genes Expression (NtDFR of and MaFLS NtANS in Tobacco) and A ffoneects regulatory the Expression gene of Anthocyanin (NtAN2), closely Pathway related Genes to anthocyaninSubsequently, biosynthesis, we conducted showed aremarkable qRT-PCR expressionreductions in analysis three MaFLS of endogenous-expressing genes lines, involved compared in withthe flavonoid the non-transformed biosynthetic pathwaycontrol (Figure in the petals6C,D). of Conversely,MaFLS-expressing the expression transgenic of endogenous tobacco (Figure NtFLS6A, wasprimers markedly are listed upregulated in Table S1). in Thethe MaFLS analyzed-expressing genes included lines. These twelve results structural suggest genes that (NtPAL heterologous, NtC4H, expressionNt4CL, NtCHS of MaFLS, NtCHI in, tobaccoNtF3H, NtF3’Hpetals substantially, NtF3’5’H, NtFLS inhibited, NtDFR expression, NtANS of, andthe keyNtUFGT structural) and genes three (regulatoryNtDFR and genes NtANS (NtAN2) and ,theNtAN1a vital regulatory, and NtAN1b gene), which(NtAN2 are) that highlighted have been in implicated red (Figure in6A). anthocyanin biosynthesis and upregulated the expression of endogenous NtFLS.

(A)

(B)

Figure 6. Cont.

Molecules 2019, 24, 1579 9 of 15 Molecules 2019, 24, x FOR PEER REVIEW 9 of 14

(C)

(D)

FigureFigure 6. A6. schematicA schematic diagram diagram of flavonoid of flavonoid biosynthetic biosynth pathwayetic andpathway expression and profilesexpression of anthocyanin profiles of biosyntheticanthocyanin endogenous biosynthetic TF endogenous genes in petals. TF genes (A) Ain schematicpetals. (A) diagram A schematic of flavonoid diagram biosynthetic of flavonoid pathway.biosynthetic EBGs, pathway. the early EBGs, biosynthetic the early genes; biosynthetic LBGs, the genes; late biosynthetic LBGs, the late genes; biosynthetic PAL, phenylalanine genes; PAL, ammonia-lyase;phenylalanine ammonia-lyase; C4H, cinnamate C4H, 4-hydroxylase; cinnamate 4-hydroxylase; 4CL, 4-coumarate: 4CL, 4-coumarate: CoA ligase; CoA CHS, ligase; chalcone CHS, synthase;chalcone CHI, synthase; chalcone CHI, isomerase; chalcone F3H,isomerase; flavanone F3H, 3-hydroxylase; flavanone 3-hydroxylase; F3’H, flavonoid F3’H, 3’-hydroxylase; flavonoid 3’- F3’5’H,hydroxylase; flavonoid F3’5’H, 3’5’-hydroxylase; flavonoid 3’5’-hydroxylase; FLS, flavonol synthase; FLS, flavonol DFR, dihydroflavonol synthase; DFR, 4-reductase;dihydroflavonol ANS, 4- anthocyanidinreductase; ANS, synthase; anthocyanidin UFGT, uridine synthase; diphosphate-sugar: UFGT, uridine Flavonoid diphosphate-sugar: glycosyltransferases; Flavonoid MYBs, MYBglycosyltransferases; transcription factors; MYBs, bHLHs, MYB transcription basic helix-loop-helix factors; bHLHs, proteins; basic WDR, helix-loop-helix WD repeat proteins; protein. WDR, The analyzedWD repeat genes protein. are highlighted The analyzed in red. genes (B )Theare highlighted expression in patterns red. (B of)TheNtPAL expression, NtC4H patterns, Nt4CL ,ofNtCHS NtPAL, , NtCHINtC4H, and, Nt4CLF3H in, NtCHS petals., (NtCHIC) The, expressionand F3H in patterns petals. ( ofC)NtF3’H The expression, NtF3’5’H ,patternsNtFLS, NtDFR of NtF3’H, NtANS, NtF3’5’H, and , NtUFGTNtFLS, inNtDFR petals., NtANS (D) The, and expression NtUFGT patterns in petals. ofthree (D) The regulatory expression genes patterns (NtAN2 of, NtAN1a three regulatory, and NtAN1b genes) in( petals.NtAN2, Data NtAN1a are for, and the NtAN1b non-transformed) in petals. control Data are (WT) for andthe threenon-transformedMaFLS-expressing control lines (WT) exhibiting and three diMaFLSfferent-expressing phenotypic characteristicslines exhibiting (S, di strong;fferent M, phenotypic medium; W, characteristics weak). Eachcolumn (S, strong; represents M, medium; means W, ± SDweak). from threeEach technicalcolumn represents replicates. means Different ± SD letters from above three thetechnical bars indicate replicates. significantly Different dilettersfferent above values the (Pbars< 0.05) indicate calculated significantly using one-way different ANOVA values ( followedP < 0.05) bycalculated Duncan’s using multiple one-way range ANOVA test. followed by Duncan’s multiple range test. Based on the one-way ANOVA statistical results presented in Figure6, only the expression level of3.NtCHI Discussionwas not significantly different between the MaFLS-expressing lines and the non-transformed control; all the other genes showed the decreased expressions. In particular, NtCHS, NtF3H, NtDFR, NtANSAnthocyanins, and NtAN2 andexpression flavonols was are remarkably important flavonoi lower ind thephytochemicalsMaFLS-expressing that contribute lines than to in flower the non-transformedcoloration. Understanding control (Figure the 6molecularB–D). Particularly, and bioc thehemical transcript controls levels of of anthocyanin two endogenous and flavonol genes (NtDFRpathways,and orNtANS studying) and related one regulatory genes, is genean import (NtAN2ant), focus closely in relatedthe ornamental to anthocyanin plant industry, biosynthesis, since showednovel color remarkable morphs reductions can be profitable. in three MaFLSHere, we-expressing describe lines,the cloning compared and withmolecular the non-transformed characterization controlof MaFLS (Figure, the6 C,D).first FLS Conversely, characterized the expression from ornamental of endogenous monocots.NtFLS Sequencewas markedly alignments upregulated revealed that in theMaFLSMaFLS possesses-expressing the lines. HxDx ThesenH and results RxS suggest motifs thatfor heterologousbinding ferrous expression iron and of 2-OG,MaFLS respectively.in tobacco petalsMoreover, substantially MaFLS inhibited has the expressionfive amino ofacid the residues key structural (Tyr127, genes Phe129, (NtDFR Lys197,and NtANS Phe288,) and and the Ser290; vital Figure 1) responsible for substrate binding [26]. It is noteworthy that Tyr127 and Ser290 are not conserved. Similar to Tyr132 in AcFLSs and Phe137 in GbFLS [14,17], the presence of Tyr127 in MaFLS (instead of the His, residue commonly present in other FLSs at this position) might impart improved enzyme catalytic activity for quercetin production [26]. Because the previous study showed

Molecules 2019, 24, 1579 10 of 15 regulatory gene (NtAN2) that have been implicated in anthocyanin biosynthesis and upregulated the expression of endogenous NtFLS.

3. Discussion Anthocyanins and flavonols are important flavonoid phytochemicals that contribute to flower coloration. Understanding the molecular and biochemical controls of anthocyanin and flavonol pathways, or studying related genes, is an important focus in the ornamental plant industry, since novel color morphs can be profitable. Here, we describe the cloning and molecular characterization of MaFLS, the first FLS characterized from ornamental monocots. Sequence alignments revealed that MaFLS possesses the HxDxnH and RxS motifs for binding ferrous iron and 2-OG, respectively. Moreover, MaFLS has the five amino acid residues (Tyr127, Phe129, Lys197, Phe288, and Ser290; Figure1) responsible for substrate binding [ 26]. It is noteworthy that Tyr127 and Ser290 are not conserved. Similar to Tyr132 in AcFLSs and Phe137 in GbFLS [14,17], the presence of Tyr127 in MaFLS (instead of the His, residue commonly present in other FLSs at this position) might impart improved enzyme catalytic activity for quercetin production [26]. Because the previous study showed that it is a potentially attractive substrate binding site for modification to engineer a FLS with improved activity [26]. Some progress has recently been made in analyzing the anthocyanin profiles of Muscari [22,24,29–31]. However, few reports have focused on the flavonol profiles in Muscari. Our previous work revealed that flavonols in grape hyacinth petals are mainly derivatives of kaempferol and quercetin, in accordance with flavonol accumulation in lisianthus petals [32]. Here, we determined the levels of total flavonols in two grape hyacinth cultivars, one with white flowers and the other with blue. We found that total flavonol content of the white-flower cultivar “White Beauty” was significantly higher than that of blue-flower cultivar “Dark Eyes”. How flavonols regulate flower color in these cultivars is unclear, but one possibility is that they act as copigments that participate in petal coloration. In addition, we observed little correlation between the expression levels of MaFLS and the accumulation of total flavonols in grape hyacinth. Conversely, positive correlations between flavonoid concentration and mRNA levels of FLS genes have been reported in other plant species [19]. First, this correlation depends on the availability of primary substrate (dihydroflavonols) flux in the pathway, because only some of them can be catalyzed by MaFLS to the corresponding flavonols, while others may synthesize many other molecules that were not measured in this analysis. Second, this phenomenon perhaps indicates that other MaFLS isogenes may be expressed to produce the appropriate flavonols in grape hyacinth, as is the case in A. thaliana, lisianthus, and onion [1,9,17]. However, only one putative MaFLS sequence has been identified thus far. Due to a lack of sufficient genomic resources, we are unaware of other FLS genes that exist in grape hyacinth. Nevertheless, we speculate that the MaFLS characterized here promotes flavonol production to act as UV-B sunscreens and protect young developing floral tissue and that these colorless flavonols may act as copigments sandwiched between anthocyanin molecules to induce a blue shift [1,12]. Furthermore, these colorless flavonols might act as UV-spectrum flower pigments, contributing to the attractive and defensive functions for insects [33], or they might be important for male fertility and auxin metabolism and transport [34,35]. At the subcellular level, MaFLS appears to localize to the cytoplasm and cell periphery, but not to the nucleus, in accordance with the observation that flavonoids are synthesized and localized to the cytoplasm in Arabidopsis [36]. However, there are many flavonoid metabolic enzymes for which dual cytoplasmic/nuclear localization has been observed, such as CHS, CHI, and FLS [35]. We have shown that constitutive heterologous expression of MaFLS influenced flower color in transgenic tobacco lines. HPLC analysis showed a dramatic decrease in the levels of total anthocyanins and an increase in the levels of total flavonols in the resulting tobacco flowers. The severity of the flower color phenotype was highly consistent with the MaFLS transgene expression level. It is noteworthy that over-expression of MaFLS in tobacco inhibited expression of the genes involved in the early step of flavonoid biosynthesis, particularly dampening the expression of the key structural genes Molecules 2019, 24, 1579 11 of 15

(NtDFR and NtANS) and the vital regulatory gene (NtAN2) in the anthocyanin synthesis pathway. This phenomenon may be explained as a feedback mechanism existing in the flavonoid pathway that affected the expression of genes in the early step of flavonoid biosynthesis [20]. The result also further demonstrated that the competition between FLS and DFR genes ultimately determines the flower coloration.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions Grape hyacinth (M. aucheri “White Beauty” and M. aucheri “Dark Eyes”) plants were grown in the experimental field of the Northwest A&F University at Yangling Distruct in Shaanxi Province, China. The petals of grape hyacinth were divided into five floral development stages (S1–S5), according to the previous description (Figure3A) [ 22]. To obtain RNA and metabolite samples, healthy tissues and flowers were collected, frozen immediately in liquid nitrogen, and stored at 80 C. Tobacco plants − ◦ (Nicotiana tabacum “NC89”) for transformation were grown aseptically from seed on Murashige and Skoog medium, supplemented with 3% (w/v) sucrose, and transgenic tobacco flowers were harvested at the full-bloom stage.

4.2. Cloning of Full-Length MaFLS cDNAs A FLS homolog was identiﬁed in the transcriptome of grape hyacinth [24], designated MaFLS. To obtain the full-length cDNA sequence of MaFLS, we performed both 50- and 30-RACE experiments, using the ﬂowers of M. aucheri “White Beauty”, as in the previous method [23]. The primers used for 501 and 30-RACE PCR and full-length gene cloning are listed in Table S1. The obtained MaFLS cDNA sequence was submitted to the NCBI GenBank database (accession number MH636605).

4.3. Sequence Alignment and Phylogenetic Analysis Full-length amino acid sequences of MaFLS and other FLS proteins were retrieved from the GenBank database. Multiple sequence alignments were performed with CLC sequence viewer 8.0 software. FLS-speciﬁc motifs and conserved amino acid residues were indicated by diﬀerent colored symbols. The phylogenetic tree was constructed by the maximum likelihood method with 1000 bootstrap replicates using MEGA 6.0.

4.4. RNA Isolation and qRT-PCR Analysis Total RNA was extracted from frozen tissue of the flowers, roots, bulbs, and leaves of grape hyacinth (M. aucheri “White Beauty” and M. aucheri “Dark eyes”) as well as flowers of tobacco (NC89), using the Omega Total RNA Kit (Omega, Norcross, GA, USA). Purified RNA was assessed using agarose electrophoresis and measured on a Nanodrop 2000 (Thermo Scientific). Then, 1-µg RNA aliquots were used for reverse transcription to cDNA, using the PrimeScript™ RT Reagent Kit (TaKaRa Biotechnology, Dalian, China). The cDNA was diluted five-fold and used as the template for qRT-PCR. NovoStart®SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China) was used as the fluorochrome for the qRT-PCR assay. The assay was conducted using the iQ5 RT-PCR detection system (Bio-Rad, Hercules, CA, USA). Each reaction mixture consisted of NovoStart®SYBR qPCR SuperMix Plus with 0.8 µL of forward and reverse primers each, 1 µL of cDNA, and 7.4 µL of ddH2O in a final volume of 20 µL. The amplification protocol was 95 ◦C for 1 min, followed by 40 cycles of 95 ◦C for 20 s, 58 to 62 ◦C for 20 s, and 72 ◦C for 30 s. The qRT-PCR primers of grape hyacinth and tobacco are listed in Table S1. MaActin and NtTubA1 were used as the internal control genes in each grape hyacinth and tobacco sample, respectively. All analyses were conducted in technical triplicate. Molecules 2019, 24, 1579 12 of 15

4.5. Subcellular Localization of MaFLS To investigate the subcellular localization of MaFLS, the ORF of MaFLS without a termination codon was inserted between the XbaI and KpnI sites of the pBI221-GFP vector, using the Seamless Cloning and Assembly Kit (Novoprotein, China; primers are listed in Table S1) to generate the recombinant plasmid pBI221-MaFLS-GFP. Using the PEG-calcium mediated transfection method [37], the recombinant plasmid pBI221-MaFLS-GFP was transformed into A. thaliana mesophyll protoplasts. After 16–18 h of cultivation, the transformed protoplasts were observed with a confocal laser scanning microscope (TCS SP8, Leica, Wetzlar, Germany).

4.6. Heterologous Expression Vector Construction and Stable Tobacco Transformation For constructing the MaFLS heterologous expression vector, the MaFLS ORF sequence without a termination codon was inserted between KpnI and XbaI of the pCAMBIA2300 vector, using the Seamless Cloning and Assembly Kit (Novoprotein, China; primers are listed in Table S1) to produce the recombinant plasmid p2300-MaFLS. Then, the recombinant plasmid p2300-MaFLS was introduced into Agrobacterium tumefaciens Gv3101 (MP) by electroporation. Tobacco leaf disk transformation was conducted using a previously described protocol [38]. T0 generation of transgenic tobacco lines heterologously expressing MaFLS were used for qPCR analysis, and the lines showing a severe phenotypic change in petal pigmentation were used for further HPLC analysis.

4.7. HPLC Analysis Petals from the five floral developmental stages; roots, bulbs, and leaves of grape hyacinth; and the fresh petals of transgenic tobacco were ground to powder in liquid nitrogen and extracted with methanol to H2O to formic acid to trifluoroacetic acid (70:27:2:1, v/v/v/v) for analysis of total anthocyanins [39,40] or extracted with methanol for analysis of total flavonols [41]. The supernatant was filtered through 0.22-µm Millipore filters. Total anthocyanin content of tobacco flowers was measured using a UV-visible spectrophotometer (UV2600, Shimadzu, Kyoto, Japan) and calculated according to the equation: QAnthocyanins = (A530 0.25 A657) M 1. − × × − Flavonol was detected at 360 nm using reverse HPLC analysis, as in the previous descriptions [22,23]. The results were expressed as milligrams of anthocyanins or flavonols per gram of fresh weight (mg/g FW). All samples were analyzed in three biological replicates.

4.8. Statistical Analysis Analysis of variance (one-way ANOVA) was performed using the SAS program (version 8.0, SAS Institute, Cary, NC, USA). The statistical diﬀerence was compared by the Duncan’s multiple range test (P < 0.05 was considered signiﬁcant).

5. Conclusions In this study, we identified a FLS gene, MaFLS, which is predominantly expressed in the early stages of flower development in grape hyacinth. Heterologous expression of MaFLS in tobacco showed a reduction in pigmentation in the petals because of a remarkable decrease in anthocyanin content and an increase in flavonol accumulation. Moreover, our study demonstrates the vital role of MaFLS, to promote flavonol production in young developing floral tissue, and that these flavonols may play a key role in flower coloration in grape hyacinth. However, MaFLS-based regulation does not seem to fully explain the flower coloration of grape hyacinth. Future research should be conducted to determine the following: (1) How MaFLS and MaDFR gene products compete for common substrates to regulate flavonoid biosynthesis and coloration, (2) how flavonol and anthocyanin biosynthesis is regulated under the control of transcriptional regulators, and (3) how the precise ratio of flavonol and anthocyanin metabolites affects flower coloration. Molecules 2019, 24, 1579 13 of 15

Supplementary Materials: The following are available online. Author Contributions: L.D. and Y.L. conceived and designed the research. H.L., B.S., H.Z., J.G., and B.Z. conducted the experiments. H.L. analyzed the data. L.D. and H.L. contributed to the writing of the manuscript. All authors read and approved the manuscript. Funding: The authors would like to thank the anonymous reviewers for their comments. This work was supported by the National Natural Science Foundation of China (Grant No. 31700625 and No. 31471905), Shaanxi Province Science and Technology Research and Development Program (2017JQ3019), and the Fundamental Research Funds for the Central Universities (Z109021603). Conﬂicts of Interest: The authors declare that they have no conﬂict of interests.

Abbreviations

FLS ﬂavonol synthase. DFR dihydroﬂavonol 4-reductase ANS anthocyanidin synthase qRT-PCR quantitative real-time PCR DHK dihydrokaempferol DHQ dihydroquercetin ORF open reading frame PCR polymerase chain reaction UFGT glucosyltransferase 2-ODD 2-oxoglutarate-dependent dioxygenase

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Sample Availability: Samples of the compounds analyzed in the study are unavailable from the authors due to their isolation on a small scale.

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