Enrichment of rice endosperm with anthocyanins by endosperm-specific expression of rice endogenous genes

A diet rich in anthocyanins can benefit human health against a broad spectrum of human diseases due to the high antioxidant activities of anthocyanins. Enrichment of anthocyanins in the starchy endosperm of rice is an effective solution to provide nutritional food in human diets. However, previous attempts failed to engineer anthocyanin biosynthesis in the rice endosperm by transgenic expression of rice endogenous genes. In this study, four rice endogenous genes, OsDFR (encoding dihydroflavonol 4-reductase), OsRb (encoding a bHLH family transcription factor), OsC1 (encoding an R2R3-MYB-type transcription factor) and OsPAC1 (encoding a WD40 class protein), were employed to rebuild the anthocyanin biosynthesis pathway in the rice endosperm. Endosperm-specific expression of OsDFR-OsRb-OsC1 (DRC) or OsDFR-OsPAC1-OsRb-OsC1 (DPRC) resulted in transgenic rice germplasm with dark purple grains. The expression of endogenous anthocyanin biosynthesis- related genes was significantly upregulated in the transgenic lines. Metabolomics analysis revealed a substantial increase in flavonoids flux, including 12 anthocyanins, in the polished grains of these transgenic lines. Our findings demonstrated that ectopic expressing a minimal set of three rice endogenous genes enabled de novo anthocyanin biosynthesis in the rice endosperm. This study contributes valuable insights into the molecular mechanisms underlying rice organ coloration and provides valuable guidance for future anthocyanin biofortification in crops.