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The yellow pigments of dahlia flowers are derived from 6′-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo–keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, ‘Shukuhai’ and its butein-deficient lateral mutant ‘Rinka’, was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6′-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4′-O-glucosyltransferase (Am4′CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6′-deoxychalcone, but not in Asteraceae species lacking 6′-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6′-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.

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 yell

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