Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism

Coexpression of two anthocyanin modification genes elicits blue flower coloration through interaction with colorless flavonoids.
Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)–glucose:anthocyanin 3′,5′-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3′5′H. Newly synthesized 3′,5′-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed “copigmentation,” with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.