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.

Onion bulb color is a key breeding trait. The red bulb color is caused by the presence of anthocyanins, which are products of the flavonoid synthesis pathway. Research on flavonoid regulation in onion is lagging compared with that in other crops. AcB2 encodes a bHLH transcription factor, and its transcription is positively associated with anthocyanin accumulation and correlated with the expression of AcMYB1, which is an activator in the flavonoid biosynthetic pathway in onion. Phylogenetic analysis showed that AcB2 was grouped into the TRANSPARENT TESTA 8 (TT8) clade of the bHLH IIIf subgroup. The AcB2 protein contained an MYB-interacting region and physically interacted with AcMYB1 in yeast and tobacco leaves. AcMYB1 directly bound to the promoters of anthocyanidin synthase (AcANS) and flavonoid 3-hydroxylase 1 (AcF3H1) and activated their expression. The coexpression of AcB2 with AcMYB1 in Arabidopsis thaliana protoplasts dramatically increased the expression of AcANS and AcF3H1 compared with that under the expression of AcMYB1 alone. Transient co-overexpression of AcB2 with AcMYB1 induced anthocyanin accumulation in the epithelial cells of onion bulbs. Complementation of the Arabidopsis tt8-1 mutant with AcB2 restored pigmentation defects in tt8-1. In addition, AcB2 physically interacted with AtTT2 in yeast cells and tobacco leaves, indicating that the functions of AcB2 were similar to those of AtTT8. Together, these results demonstrated that AcB2 enhanced the function of AcMYB1 in upregulating anthocyanin biosynthesis in onion, which provided a theoretical basis for breeding onions with higher anthocyanin contents.

S -spicas of P. vulgaris sown in autumn; BUSCO -Benchmarking Universal Single-Copy Orthologs; GO -Gene Ontology; GC -numbers of bases G and C in the total number of bases; KEGG -Kyoto Encyclopedia of Genes and Genomes; COG/KOG -Clusters of Orthologous Groups of proteins; Ref -reference sequence; Nr -nonredundant protein sequences; Nt -nucleotide sequences; Pfam -protein family; Q20 -rate of bases where the quality is greater than 20; Q30 -the rate of bases where the quality is greater than 30; real time RT-qPCR -real-time reverse transcription quantitative PCR; S-S-L -leaf of P. vulgaris sown in spring; SWISS-PROT -protein sequence database.