AcMYB96 promotes anthocyanin accumulation in onion (Allium cepa L) without forming the MBW complex.

Anthocyanins are major flavonoid compounds with established health benefits. Although the molecular mechanisms of MYB transcription factors (TFs) within the MYB-basic helix-loop-helix (bHLH)-WD-repeat protein (MBW) complex in anthocyanin biosynthesis have been revealed, the functions of other MYB TFs that are unable to form the MBW complex in this process remain unclear. In this study, we uncovered and extensively characterized an R2R3-MYB TF in onion (Allium cepa L.), named AcMYB96, which was identified as a potential anthocyanin activator. AcMYB96 was classified into subgroup 1 of the R2R3-MYB TF family and lacked the conserved sequences required for interactions with bHLH IIIf TFs. Consistently, yeast two-hybrid assays showed that AcMYB96 did not interact with any bHLH IIIf TFs examined, including AcB2 and AtTT8. The transcription pattern of AcMYB96 correlated with the level of anthocyanin accumulation, and its role in activating anthocyanin biosynthesis was confirmed through overexpression in the epithelial cells of onion bulbs and Arabidopsis. Yeast one-hybrid, electrophoretic mobility shift, and promoter transactivation assays further demonstrated that AcMYB96 promoted anthocyanin biosynthesis by binding to the promoters of the chalcone synthase (AcCHS1), anthocyanidin synthase (AcANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (AcUFGT) genes, thereby activating their expression independent of bHLH IIIf TFs. These results demonstrate that AcMYB96 activates anthocyanin biosynthesis without forming the MBW complex, providing a theoretical foundation to further enrich the gene resources for promoting anthocyanin accumulation and breeding red onions with high anthocyanin content.

Transcriptome Sequencing and Metabolism Analysis Reveals the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulbs.

Onion (Allium cepa L.) is an important bulbous vegetable crop that possesses important properties related to health as well as extraordinary colors. Naturally white onion bulbs were used in this study to reveal the complex metabolic mechanisms that underlie phenotypic traits, especially bulb pigmentation. Six libraries (three dark-red and three white) were constructed and analyzed to elucidate differences in cyanidin (Cy) metabolism between dark-red and white onion bulbs. Libraries were screened using RNA-sequencing (RNA-seq) to reveal the differentially expressed genes (DEGs) involved in anthocyanin biosynthesis at the transcriptional level. Comparison with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database shows that a total of 27 unigenes participate in onion anthocyanin biosynthesis and 16 DEGs perform critical roles in flavonoid biosynthesis. Expression patterns of color-related flavonoid compounds associated with the onion anthocyanin biosynthesis pathway (ABP) show that flavonoid 3',5'-hydroxylase (F3'5'H) and dihydroflavonol 4-reductase (DFR) genes play crucial roles in the biosynthesis of dark-red bulbs, the expression levels of flavonol synthase (FLS) and DFR genes may act to block blue pigmentation, and the loss of Cy from white onion bulbs might explain multibranching in the synthesis of this compound. Positive variation in the F3'5'H/F3'H ratio also affects onion bulb color diversity. The transcriptome presented here provides a basis for future onion molecular breeding based on variations in the diversity of ornamental plant pigmentation.