The spatio-temporal biosynthesis of floral flavonols is controlled by differential phylogenetic MYB regulators in Freesia hybrida.

Floral flavonols play specific pivotal roles in pollinator attraction, pollen germination and fertility besides other functions in vegetative organs. In contrast to early flower developmental stages, flavonol biosynthesis in late flower developmental stages or matured flower tissues remains unclear in many plants, which might be regulated independent of subgroup 7 R2R3 MYB (SG7 MYB) transcription factors. In this study, two FLS genes were shown to be expressed synchronously with the flower development- and tissue- specific biosynthesis of flavonols in Freesia hybrida. FhFLS1 contributed to the flavonol biosynthesis in early flower buds and toruses or calyxes, and was regulated by four well-known SG7 MYB proteins, designated as FhMYBFs, with at least partial regulatory redundancy. FhFLS2 accounted for flavonols in late developed flowers and petal, stamen and pistil, and was targeted directly by non SG7 MYB protein FhMYB21L2. In parallel, AtMYB21 and AtMYB24 also activated AtFLS1, a gene highly expressed in Arabidopsis anther and pollen, indicating the conserved regulatory roles of MYB21 against FLS genes in these two evolutionarily divergent angiosperm plants. Our results reveal a novel regulatory and synthetic mechanism underlying flavonol biosynthesis in floral organs or tissues, and may be exploited to investigate supplementary roles of flavonols in flowers.