Modulation of anthocyanin accumulation in storage roots of sweetpotato by transcription factor IbMYB1-2 through direct binding to anthocyanin biosynthetic gene promoters

The storage roots of purple-fleshed sweetpotato rich in anthocyanins are considered nutrient-rich foods with health effects. However, the molecular mechanism underlying anthocyanin biosynthesis and regulation remains to be revealed. In this study, IbMYB1-2 was isolated from purple-fleshed sweetpotato "Xuzishu8". The phylo-genetic and sequence analysis indicated that IbMYB1-2 belongs to the SG6 subfamily with a conserved bHLH motif. Subcellular localization analysis and transcriptional activity assay revealed that IbMYB1-2 is a key tran-scriptional activator and is specific to the nucleus. Agrobacterium rhizogenes-mediated overexpression of IbMYB1-2 in sweetpotato through in vivo root transgenic system led to an increase in anthocyanins in the root of sweetpotato. qRT-PCR and transcriptome analysis depicted that the transcript levels of IbMYB1-2, IbbHLH42, and eight structural genes that are associated with the synthesis of anthocyanin were upregulated in overexpressed IbMYB1-2 transgenic roots. Dual-luciferase reporter (DLR) assay and yeast one-hybrid (Y1H) assay demonstrated IbMYB1-2 binding to the promoter regions of IbbHLH42 and other anthocyanin biosynthetic genes, including IbCHS, IbCHI, IbF3H, IbDFR, IbANS, IbGSTF12, IbUGT78D2, and IbUF3GT. Moreover, IbbHLH42 was shown to be an active enhancer for the formation of MYB-bHLH-WD40 (MBW) complex, which strongly supports the pro -moter activities of the IbCHS, IbANS, IbUGT78D2, and IbGSTF12 genes to induce anthocyanin accumulation. Taken together, our findings not only revealed the underlying regulatory molecular mechanism of IbMYB1-2 for anthocyanin accumulation in the storage roots of sweetpotato but also uncovered a potential mechanism by which IbbHLH42 modulated anthocyanin biosynthesis through a positive feedback regulatory loop.