Inhibition of DCL4 activity by maternally supplied flavonoid aglycons induces a bicolor pattern in the saddle soybean seed coat

Domesticated soybean (Glycine max) varieties can be divided into three major groups based on seed coat color: yellow (colorless), bicolored (saddle), and black. In yellow cultivars, the expression of the gene encoding chalcone synthase, essential for flavonoid biosynthesis, is inhibited by post-transcriptional gene silencing (PTGS). In saddle cultivars, PTGS is spatially inhibited in the central region around the hilum, which is black. However, the molecular mechanism of this region-specific inhibition of PTGS remains unclear. This study examined the relationship between Dicer-like 4 (DCL4), essential for PTGS, and flavonoid aglycons, which can inhibit DCL4. In the immature seed coat, DCL4 activity was specifically detected in the region that becomes colorless after seed maturation, but it was not detected in the region that becomes black after seed maturation, although its expression level had no difference. By contrast, phenolic compounds, including flavonoids, accumulated specifically in the region that becomes black. Especially, quercetin accumulated specifically in the central region of immature saddle seed coat and inhibited the dicing activity of DCL4. Furthermore, flavonoids highly accumulated in the funiculi of saddle and black but not yellow cultivars, and immature seeds cultured in vitro had reduced phenolic compounds in their seed coats. These results indicate that flavonoid aglycons including quercetin transported from the maternal tissues via the funiculus, accumulated in the central region of the immature seed coat, and induced bicolor pigmentation in the saddle seed coat by region-specific inhibition of DCL4 (PTGS).