Identification and Functional Characterization of Anthocyanidin 3-O-Glucosyltransferases OsA3GT1 and OsA3GT2 in Black Rice

Anthocyanins are key pigments that enhance the nutritional value and aesthetic appeal of black rice (Oryza sativa L.), a variety rich in health-promoting compounds. Despite advances in elucidating the anthocyanin biosynthetic pathway, the specific glucosyltransferase responsible for converting anthocyanidins such as cyanidin and peonidin into their 3-O-glucosides remains unidentified in rice. In this study, we identified two isoenzymes of anthocyanidin 3-O-glucosyltransferase, OsA3GT1 and OsA3GT2, in rice and investigated their roles in anthocyanin biosynthesis. Organ-specific expression profiling, subcellular localization, and CRISPR/Cas9-mediated mutagenesis revealed that OsA3GT1 functions as the primary enzyme for anthocyanin biosynthesis in seeds, whereas OsA3GT2 plays a minor role. Loss-of-function mutants of OsA3GT1 exhibited markedly reduced seed pigmentation and anthocyanin content, especially cyanidin 3-O-glucoside and peonidin 3-O-glucoside. Analysis of double mutants further confirmed the pivotal role of OsA3GT1 in anthocyanin biosynthesis. Both OsA3GT1 and OsA3GT2 catalyzed the glucosylation of anthocyanidins and flavonols, showing the highest specificity for the 3-hydroxyl (3-OH) position, consistent with their roles in anthocyanin biosynthesis. Our findings demonstrate that OsA3GT1 plays a major role in anthocyanin biosynthesis, whereas OsA3GT2 contributes to a lesser extent. Functional analysis of OsA3GT1 and OsA3GT2 in black rice, together with in vitro biochemical characterization of recombinant proteins, enhances our understanding of the genetic and biochemical mechanisms underlying anthocyanin biosynthesis in rice grain, providing valuable insights for breeding anthocyanin-enriched rice cultivars.