Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase

Flavonoids, in particular the anthocyanins, are responsible for flower colour in many species. The dihydroflavonols represent a branch point in flavonoid biosynthesis, being the intermediates for production of both the coloured anthocyanins, through the action of the enzyme dihydroflavonol 4-reductase (DFR), and the colourless flavonols, produced by flavonol synthase (FLS). In this study the white-flowered, flavonol accumulating Mitchell line of petunia was used as a model to examine the interaction between DFR and FLS enzyme activities and possibilities for redirecting flavonoid biosynthesis away from production of flavonols and towards anthocyanins. Introduction of a 35S CaMV-DFR sense transgene construct caused the production of anthocyanins, resulting in a pink-flowered phenotype. Furthermore, inhibition of FLS production through introduction of an FLS antisense RNA construct also led to anthocyanin production and a pink-flowered phenotype. A combination of both transgenes gave the highest level of anthocyanin formation. Anthocyanins were produced in the DFR-sense and FLS-antisense transgenic lines in spite of the greatly reduced levels of gene expression in the Mitchell line for three enzymes late in anthocyanin biosynthesis, anthocyanindin synthase, UDP-glucose: flavonoid 3-O-glucosyltransferase and UDP-rhamnose: anthocyanidin-3-glucoside rhamnosyltransferase. Thus, the level of gene activity required for visible anthocyanin formation is much lower than the high levels normally induced during petal development. Altering the balance between the DFR and FLS enzyme activities, using genetic modification, may be a useful strategy for introducing or increasing anthocyanin production in target ornamental species.