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.

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Main conclusion:A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia.

Butein is one of flavonoids conferring bright yellow flower color and is a precursor of aurone in some species. Butein is synthesized by two steps, 3-malonyl CoA and 4-coumaloyl CoA are converted to isoliquiritigenin in the first step, and then isoliquiritigenin is converted to butein in the second step. In the first step, chalcone synthase (CHS) and chalcone reductase (CHR) catalyze this reaction, however, CHR has been reported for the isoflavone biosynthesis pathway in legumes, and CHR for butein biosynthesis has not yet been isolated. In this study, we report CHR that is evolutionally different gene from legume species is involved in isoliquiritigenin biosynthesis in dahlia. To isolate CHR gene, we conducted comparative RNA-seq analysis between ‛Shukuhai' and its butein-loss lateral mutant ‛Rinka'. We found DvCHR showed significant difference in expression levels that encodes an aldo-keto reductase (AKR) 13 family protein, which was phylogenetically different from legume CHRs belonging to AKR4A family. Gene expression levels and genotype of DvCHR were correlated with butein accumulation among various dahlia cultivars. Though single over expression of DvCHR was not able to accumulate isoliquiritigenin in tobacco, co-overexpression of DvCHR with a chalcone glucosyltransferase Am4'CGT and a MYB transcription factor CaMYBA successfully induced isoliquiritigenin accumulation. In addition, DvCHR homologous gene expression was detected from butein or aurone accumulating Coreopsideae species but not from non-butein or non-aurone accumulating Asteraceae species. These results indicated DvCHR functions as chalcone reductase for butein biosynthesis in dahlia, and isoliquiritigenin biosynthesis in Coreopsideae species has been developed independently from legume species.