Bidirectional feedforward regulatory loop of Dicer-like 4 and flavonoid aglycons causes floral bicolor patterning in petunia and dahlia

Floral bicolor pigmentation in some cultivars of petunia and dahlia is caused by naturally occurring RNA interference (RNAi). In both species, the chalcone synthase gene is highly expressed only in the pigmented regions of bicolor petals. However, the mechanism by which RNAi is specifically induced in the unpigmented regions remains unknown. To elucidate the mechanism underlying this bicolor pattern formation, we analyzed the dicing activity of Dicer-like 4 (DCL4), an essential enzyme in the RNAi pathway. Crude extracts prepared from the pigmented regions strongly inhibited DCL4 activity, whereas this inhibition was abolished when flavonoids were removed from the extracts. Further analyses revealed that the inhibitory activity was attributable to flavonoid aglycons. In vivo dicing activity was detected only in colorless protoplasts prepared from the unpigmented regions of bicolor dahlia petals. These results indicate that in the unpigmented regions, flavonoid aglycons that inhibit DCL4 are not synthesized, allowing RNAi to remain active. In contrast, in the pigmented regions of mature petals, DCL4-and consequently RNAi-is inhibited by flavonoid aglycons, maintaining anthocyanin biosynthesis. Exogenous application experiments of flavonoid aglycons to floral apexes with small flower buds support this conclusion. Therefore, during bicolor flower development, at the stage when petals mature, the clear bicolor pattern is established through a bidirectional feedforward loop involving mutual antagonism between DCL4 and flavonoid aglycons.