Abstract
INTRODUCTION: Sweetpotato (Ipomoea batatas (L.) Lam.) is a genetically intricate hexaploid crop. The purple-fleshed variety, enriched with anthocyanin pigments, is an outstanding source for creating high-value functional products. Previous research on anthocyanin biosynthesis has primarily focused on the above-ground plant parts at the transcriptional level. However, the regulatory mechanisms underlying anthocyanin accumulation in underground tuberous roots of sweetpotato remain largely unexplored. OBJECTIVES: This study aimed to elucidate the post-transcriptional and post-translational mechanisms of Ib-miR2111 and its target gene IbKFB in anthocyanin synthesis in sweetpotato. METHODS: Genetic manipulation techniques were used to validate the function of Ib-miR2111 and IbKFB in anthocyanin biosynthesis in sweetpotato. To investigate how IbKFB works, a series of protein interaction assays, including yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), GST pull-down, co-immunoprecipitation (Co-IP), and ubiquitination, were conducted. Additionally, the impact of anthocyanin extracts from the genetically modified sweetpotato lines on inflammatory cells morphology, cytokine expression, and cell proliferation were evaluated using in vitro assays. RESULTS: Purple-fleshed sweetpotato (PFSP) varieties exhibited elevated Ib-miR2111 expression compared to white-fleshed sweetpotato (WFSP) varieties, with an inverse expression pattern in IbKFB. Genetic manipulations, including overexpression, CRISPR/Cas9 knockouts, and targeted mutations, confirmed their critical roles in anthocyanin modulation. Furthermore, IbKFB's interactions and ubiquitination with phenylalanine ammonia-lyase 1 (IbPAL1) and glyceraldehyde-3-phosphate dehydrogenase 1 (IbGAPCp1) were elucidated, revealing intricate regulatory mechanisms. Enhanced anthocyanin content showed significant effects on inflammatory cell morphology, cytokine expression, and cell proliferation. CONCLUSION: This study provides new insights into the regulatory mechanisms of Ib-miR2111 and IbKFB in anthocyanin biosynthesis and suggests potential health benefits of anthocyanin-rich sweetpotatoes.