Background More than 20,000 cultivars of garden dahlia ( Dahlia variabilis hort.) are available showing flower colour from white, yellow and orange to every imaginable hue of red and purple tones. Thereof, only a handful of cultivars are so-called black dahlias showing distinct black-red tints. Flower colour in dahlia is a result of the accumulation of red anthocyanins, yellow anthochlors (6’-deoxychalcones and 4-deoxyaurones) and colourless flavones and flavonols, which act as copigments. White and yellow coloration occurs only if the pathway leading to anthocyanins is incomplete. Not in all cultivars the same step of the anthocyanin pathway is affected, but the lack of dihydroflavonol 4-reductase activity is frequently observed and this seems to be based on the suppression of the transcription factor DvIVS . The hitherto unknown molecular background for black colour in dahlia is here presented. Results Black cultivars accumulate high amounts of anthocyanins, but show drastically reduced flavone contents. High activities were observed for all enzymes from the anthocyanin pathway whereas FNS II activity could not be detected or only to a low extent in 13 of 14 cultivars. cDNA clones and genomic clones of FNS II were isolated. Independently from the colour type, heterologous expression of the cDNA clones resulted in functionally active enzymes. FNS II possesses one intron of varying length. Quantitative Real-time PCR showed that FNS II expression in black cultivars is low compared to other cultivars. No differences between black and red cultivars were observed in the expression of transcription factors IVS and possible regulatory genes WDR1, WDR2, MYB1, MYB2, 3RMYB and DEL or the structural genes of the flavonoid pathway. Despite the suppression of FHT expression, flavanone 3-hydroxylase (FHT, synonym F3H) enzyme activity was clearly present in the yellow and white cultivars. Conclusions An increased accumulation of anthocyanins establishes the black flowering phenotypes. In the majority of black cultivars this is due to decreased flavone accumulation and thus a lack of competition for flavanones as the common precursors of flavone formation and the anthocyanin pathway. The low FNS II activity is reflected by decreased FNS II expression.

In contrast to the well-studied B-ring hydroxylation of several flavonoid classes, knowledge on the establishment of the B-ring hydroxylation pattern in chalcones is still limited. Previous studies using enzyme preparations from Dahlia variabilis petals showed that a NADPH-dependent, membrane bound enzyme is responsible, but the possible involvement of the common flavonoid 3′-hydroxylase (F3′H) remained unclear. Therefore, we tested for the first time recombinant F3′Hs from eleven ornamental plant species, mostly Asteraceae, for their acceptance of chalcones as substrates. Ten of the 11 enzymes tested were not able to hydroxylate the 6′-deoxychalcone isoliquiritigenin at position 3, indicating the involvement of a specific enzyme in the hydroxylation of chalcones in ring B. Recombinant F3′H from Tagetes erecta accepted isoliquiritigenin as a substrate but low conversion rates and kinetic data clearly indicate that flavonoids are the preferred substrates. As microsomal preparations from Tagetes erecta petals do not hydroxylate chalcones at position 3, the observed low chalcone 3-hydroxylase activity of recombinant Tagetes F3′H seems to be a result of the use of a heterologous overexpression system. Cytochrome P450 reductase-specific antibodies provided final evidence that the CH3H reaction is catalyzed by a cytochrome-P450-dependent monooxygenase. This supports future cloning strategies based on respective conserved regions.

Dahlia ( Dahlia variabilis) exists in a dazzling array of cultivars, showing red, orange, magenta, lilac, yellow and white flower color, which is exclusively based on the presence of flavonoids and biochemically related compounds. Red hues (red, orange, magenta, lilac) are a result of anthocyanin accumulation in varying concentration and composition, while a yellow color is based on the formation of 6′-deoxychalcones in the petals. Red dahlia pigments are all derived from pelargonidin and cyanidin. Delphinidin derivatives are not formed due to the absence of flavonoid 3′,5′-hydroxylase in dahlia petals, which provides an explanation for the lack of blue dahlia flowers. Orange, lilac and rose cultivars are characterized by a lower anthocyanin content compared to many red cultivars. We investigated 198 cultivars for the presence of flavonoid enzymes. The activities of chalcone isomerase (CHI), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (FHT), flavone synthase II (FNSII), flavonol synthase (FLS) and flavonoid 3′-hydroxylase (F3′H) were demonstrated in enzyme preparations of dahlia petals. CHI accepted 6′-hydroxychalcones as substrates, but did not catalyze the conversion of 6′-deoxychalcones to the corresponding flavanones. White cultivars were frequently characterized by the lack of DFR activity, whereas in many yellow cultivars neither FHT nor DFR activity could be shown.