The dicing activity of DCL3 and DCL4 is negatively affected by flavonoids.

KEY MESSAGE: The dicing activities of DCL3 and DCL4 are inhibited by accumulated metabolites in soybean leaves. Epicatechin and 7,4'-dihydroxyflavone inhibited Arabidopsis DCL3 and DCL4 in vitro. Flavonoids are major secondary metabolites in plants, and soybean (Glycine max L.) is a representative plant that accumulates flavonoids, including isoflavonoids, to high levels. Naturally-occurring RNA interference (RNAi) against the chalcone synthase (CHS) gene represses flavonoid (anthocyanin) biosynthesis in an organ-specific manner, resulting in a colorless (yellow) seed coat in many soybean cultivars. To better understand seed coat-specific naturally-occurring RNAi in soybean, we characterized soybean Dicer-like (DCL) 3 and 4, which play critical roles in RNAi. Using a previously established dicing assay, two dicing activities producing 24- and 21-nt siRNAs, corresponding to DCL3 and DCL4, respectively, were detected in soybean. Dicing activity was detected in colorless seed coats where RNAi against CHS genes was found, but no dicing activity was detected in leaves where CHS expression was prevalent. Biochemical analysis revealed that soybean leaves contained two types of inhibitors effective for Arabidopsis Dicers (AtDCL3 and AtDCL4), one of which was a heat-labile high molecular weight compound of 50 to 100 kD while another was a low molecular weight substance. We found that some flavonoids, such as epicatechin and 7,4'-dihydroxyflavone, inhibited both AtDCL3 and AtDCL4, but AtDCL4 was more sensitive to these flavonoids than AtDCL3. These results suggest that flavonoids inhibit the dicing activity of DCL4 and thereby attenuate RNAi in soybean leaves.