Identification of Chalcones and their Contribution to Yellow Coloration in Dahlia (Dahlia variabilis) Ray Florets

Yellow color in dahlia flowers is conferred from chalcones, butein and isoliquiritigenin. The color intensity of yellow dahlia cultivars is diverse, but a detailed study on this has not yet been performed. In this study, we first identified structures of flavonoids by nuclear magnetic resonance imaging in ray florets of the red-white bicolor ‘Shukuhai’, which contains chalcones, flavones and anthocyanins. Four anthocyanins, four flavone derivatives, five isoliquiritigenin derivatives and five butein derivatives were identified. Among the identified compounds, butein 4'-malonylsophoroside is considered to be the final product for butein derivatives and the presence of chalcone 4'-glucosyltransferase, chalcone 4'-glucoside glucosyltransferase, and chalcone 4'-glucoside malonyltransferase for isoliquiritigenin and butein modification was predicted. Also, the biosynthetic pathway of butein and isoliquiritigenin derivatives in dahlia with butein 4'-malonylsophoroside as the final product was predicted from the identified compounds. Next, we used nine yellow cultivars and lines with different color intensities and analyzed the correlation between the b* value, an indicator of yellow color, and level of chalcones. There was no difference in the presence or absence of major peaks among the cultivars and lines. Peak area per fresh weight measured by HPLC was high in butein 4'-malonylglucoside, butein 4'-sophoroside and isoliquiritigenin 4'-malonylglucoside, suggesting these three compounds were accumulated abundantly. Among the identified chalcones, the highest correlation coefficient was detected between the b* value and butein 4'-malonylglucoside (r = 0.86), butein 4'-sophoroside (r = 0.82) or isoliquiritigenin 4'-malonylglucoside (r = 0.76). These results suggest that these three chalcones confer yellow color in dahlia ray florets. The findings in this study will contribute not only to efforts at breeding new yellow dahlia cultivars, but also to molecular breeding of yellow flowers in other species by introducing the butein biosynthetic pathway

Endogenous post-transcriptional gene silencing of flavone synthase resulting in high accumulation of anthocyanins in black dahlia cultivars.

Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping

Petal Color Is Associated with Leaf Flavonoid Accumulation in a Labile Bicolor Flowering Dahlia (Dahlia variabilis) ‘Yuino’

Bicolor flowering dahlias generally produce inflorescences with bicolor petals characterized by a colored basal part and a white tip; however, they frequently produce single-colored petals. This petal color lability prevents uniform production of cut or pot flowers of bicolor dahlias and reduces the economic value of bicolor cultivars. In this study, to reveal the underlying mechanism and control color lability, the pattern of occurrence of single-colored petals was characterized in a red–white bicolor flowering cultivar ‘Yuino’. ‘Yuino’ produced inflorescences with bicolor petals, red petals, and both red and bicolor petals. Red petals occurred almost always at the outer whorls or sectorally in a mixed inflorescence, similar to a chimera or a lateral mutant. The occurrence of red petals was higher in field experiments during May to December than in greenhouse experiments during October to next July. We identified the “R-line” plant, which produced red petals with high frequency during the winter to spring cultivation; this characteristic to produce red petals with high frequency was retained through vegetative propagation. There were strong relationships between inflorescence color and leaf phenotype; red petal-producing plants accumulated flavonoids in leaves, whereas only bicolor petal-producing plants tended not to accumulate flavonoid in leaves. This suggests that petal color of ‘Yuino’ is associated with flavonoid synthetic potential in shoot. Therefore, a phenotypic difference is observed not only in petal colors but also at the whole plant level

A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity

ダリア花弁の黒色化に対する主要4種アントシアニンの寄与度の定量的評価

The black flower color of dahlias (Dahlia variabilis) has been suggested to be attributed to a high accumulation of cyanidin (Cy)-based anthocyanins. A possible explanation for this effect is that Cy-based anthocyanins in dahlias contribute more to the black flower color than pelargonidin (Pg)-based anthocyanins by lowering petal lightness (L*) and chroma (C*), but no obvious evidence has been reported. In this study, four major anthocyanins accumulated in dahlia petals, 3, 5-diglucoside (3, 5diG) and 3-(6''-malonylglucoside)-5-glucoside (3MG5G) of Pg and Cy, were purified and their colors were evaluated in vitro at various pHs (3.0, 4.0, 4.5, 5.0, 5.5, 6.0, or 7.0) and various concentrations (0.25, 0.5, 1.0, 2.0, or 3.0 mg·mL−1 at pH 5.0 or pH 3.0). The color of solution of purified anthocyanins varied depending on pH. At pH 5.0, which is approximately the same as pH of dahlia petals, and at pH 3.0, at which anthocyanins are relatively stable, the L* and C* of Cy 3, 5diG were similar to or higher than those of Pg 3, 5diG, suggesting that Cy 3, 5diG did not contribute more to the black flower coloring than Pg 3, 5diG. On the other hand, the L* and C* of Cy 3MG5G were significantly lower than those of Pg 3MG5G, particularly above 2.0 mg·mL−1, suggesting that Cy 3MG5G contributed more than Pg 3MG5G. A similar tendency was observed in the color measurement of mixed anthocyanins in various proportion of Pg and Cy. The L* and C* of Pg 3MG5G were much higher than those of the other three anthocyanins; therefore, its color was considered to be the farthest from black among the four anthocyanins. The accumulated amount of 3MG5G-type anthocyanins was much higher than that of 3, 5diG-type anthocyanins in all nine cultivars, although the proportion of Pg- and Cy-based anthocyanins varied among the cultivars. Considering these results, it was suggested that because 3MG5G-type anthocyanins predominantly accumulate in petals, and Cy 3MG5G has a significantly higher contribution to lowering L* and C* than Pg 3MG5G, the high accumulation of Cy-based anthocyanins is critical for the black flower coloring of dahlias. The contribution of each anthocyanin is considered to depend on the structure; therefore, identifying the anthocyanin with the highest contribution to lowering L* and C* may enable the production of black flowers in various species through the high accumulation of the anthocyanin in petals.ダリア（Dahlia variabilis）の黒色花はシアニジン（Cy）系アントシアニンの高蓄積に起因するものであることが先行研究により示唆されていた．そのため，ダリア花弁に蓄積する Cy 系アントシアニンはペラルゴニジン（Pg）系アントシアニンよりも花弁の明度 L* および彩度 C* を下げるはたらきが強く，花弁黒色化への寄与度が高いことが予想されたが，これまでにそれを示した報告はない．本研究では，ダリア花弁に蓄積する 4種類の主要なアントシアニン，Pg 3, 5-ジグルコシド（Pg 3, 5diG），Cy 3, 5-ジグルコシド（Cy 3, 5diG）Pg 3-（6''-マロニルグルコシド）-5-グルコシド（Pg 3MG5G）および Cy 3-（6''-マロニルグルコシド）-5-グルコシド（Cy 3MG5G）を抽出精製し，異なる pH（3.0，4.0，4.5，5.0，5.5，6.0 あるいは 7.0）あるいは異なる濃度（0.25，0.5，1.0，2.0 あるいは 3.0 mg·mL−1）における溶液の色（CIE L*a*b*C*）を in vitro で評価した．各アントシアニンの色は溶液の pH により変化した．ダリア花弁の pH に近い pH 5.0 およびアントシアニンが比較的安定な構造を保つ pH である pH 3.0 のいずれにおいても，Cy 3, 5diG の L* および C* は Pg 3, 5diG と同様あるいは高かったことから，Cy 3, 5diG は Pg 3, 5diG よりも花弁黒色化への寄与度が高いわけではないと考えられた．一方で，Cy 3MG5G の L* および C* は Pg 3MG5G よりも，特に 2.0 mg·mL−1 以上の高濃度において有意に低く，花弁黒色化への寄与度が高いことが示唆された．同様の傾向が Pg 系アントシアニンと Cy 系アントシアニンを様々な割合で混合した色素の測色でもみられた．Pg 3MG5G の L* および C* は他の 3 種のアントシアニンよりも極めて高かったことから，Pg 3MG5G は 4種のアントシアニンのなかで最も黒色から遠い色を示すことが考えられた．ダリア花弁に蓄積する Pg 系アントシアニンと Cy 系アントシアニンの量比は品種によって様々であったのに対し，いずれの品種においても 3MG5G 型アントシアニンの蓄積量は 3, 5diG 型アントシアニンよりも多かった．これらの結果から，ダリア花弁においては 3MG5G 型アントシアニンが主要に蓄積しており，かつ，Cy 3MG5G が Pg 3MG5G よりも花弁 L* および C* を下げるはたらきが強く花弁黒色化への寄与度が高いために，Cy 系アントシアニンの高蓄積が花弁の黒色化に重要であると示唆された．個々のアントシアニンの花弁黒色化への寄与度は各アントシアニンの構造により決まると考えられたため，L* および C* が最も低いアントシアニンを特定し，それを高濃度で花弁に蓄積させることで，様々な花卉品目において黒花品種を作成することが可能になると考えられた

Tobacco streak virus (strain dahlia) suppresses post-transcriptional gene silencing of flavone synthase II in black dahlia cultivars and causes a drastic flower color change.

Tobacco streak virus suppressed post-transcriptional gene silencing and caused a flower color change in black dahlias, which supported the role of cyanidin-based anthocyanins for black flower appearance. Black flower color of dahlia (Dahlia variabilis) has been attributed, in part, to the high accumulation of cyanidin-based anthocyanins that occurs when flavone synthesis is reduced because of post-transcriptional gene silencing (PTGS) of flavone synthase II (DvFNS). There are also purple-flowering plants that have emerged from a black cultivar 'Kokucho'. We report that the purple color is not caused by a mutation, as previously thought, but by infection with tobacco streak virus (TSVdahlia), which suppresses the PTGS of DvFNS. When TSVdahlia was eliminated from the purple-flowering 'Kokucho' by leaf primordia-free shoot apical meristem culture, the resulting flowers were black. TSVdahlia-infected purple flowers had lower numbers of siRNAs to DvFNS than black flowers, suggesting that TSVdahlia has a silencing suppressor. The graft inoculation of other black cultivars with TSVdahlia altered their flower color drastically except for 'Fidalgo Blacky', a very deep black cultivar with the highest amount of cyanidin-based anthocyanins. The flowers of all six TSVdahlia-infected cultivars accumulated increased amounts of flavones and reduced amounts of cyanidin-based anthocyanins. 'Fidalgo Blacky' remained black despite the change in pigment accumulation, and the amounts of cyanidin-based anthocyanins in its TSVdahlia-infected plants were still higher than those of other cultivars. We propose that black flower color in dahlia is controlled by two different mechanisms that increase the amount of cyanidin-based anthocyanins: DvFNS PTGS-dependent and -independent mechanisms. If both mechanisms occur simultaneously, the flower color will be blacker than if only a single mechanism is active