A novel R2R3-MYB from grape hyacinth, MaMybA, which is different from MaAN2, confers intense and magenta anthocyanin pigmentation in tobacco

Abstract

Background The primary pigments in flowers are anthocyanins, the biosynthesis of which is mainly regulated by R2R3-MYBs. Muscari armeniacum is an ornamental garden plant with deep cobalt blue flowers containing delphinidin-based anthocyanins. An anthocyanin-related R2R3-MYB MaAN2 has previously been identified in M. armeniacum flowers; here, we also characterized a novel R2R3-MYB MaMybA, to determine its function and highlight similarities and differences between MaMybA and MaAN2. Results In this study, a novel anthocyanin-related R2R3-MYB gene was isolated from M. armeniacum flowers and functionally identified. A sequence alignment showed that MaMybA contained motifs typically conserved with MaAN2 and its orthologs. However, the shared identity of the entire amino acid sequence between MaMybA and MaAN2 was 43.5%. Phylogenetic analysis showed that they were both clustered into the AN2 subgroup of the R2R3-MYB family, but not in the same branch. We also identified a IIIf bHLH protein, MabHLH1, in M. armeniacum flowers. A bimolecular fluorescence complementation assay showed that MabHLH1 interacted with MaMybA or MaAN2 in vivo; a dual luciferase assay indicated that MaMybA alone or in interaction with MabHLH1 could regulate the expression of MaDFR and AtDFR, but MaAN2 required MabHLH1 to do so. When overexpressing MaMybA in Nicotiana tabacum ‘NC89’, the leaves, petals, anthers, and calyx of transgenic tobacco showed intense and magenta anthocyanin pigments, whereas those of OE-MaAN2 plants had lighter pigmentation. However, the ovary wall and seed skin of OE-MaMybA tobacco were barely pigmented, while those of OE-MaAN2 tobacco were reddish-purple. Moreover, overexpressing MaMybA in tobacco obviously improved anthocyanin pigmentation, compared to the OE-MaAN2 and control plants, by largely upregulating anthocyanin biosynthetic and endogenous bHLH genes. Notably, the increased transcription of NtF3′5′H in OE-MaMybA tobacco might lead to additional accumulation of delphinidin 3-rutinoside, which was barely detected in OE-MaAN2 and control plants. We concluded that the high concentration of anthocyanin and the newly produced Dp3R caused the darker color of OE-MaMybA compared to OE-MaAN2 tobacco. Conclusion The newly identified R2R3-MYB transcription factor MaMybA functions in anthocyanin biosynthesis, but has some differences from MaAN2; MaMybA could also be useful in modifying flower color in ornamental plants.

Genome-Wide Analysis Reveals the Potential Role of MYB Transcription Factors in Floral Scent Formation in Hedychium coronarium

“…Meanwhile, the grape VvMYB4-like gene and the soybean GmMYB100 negatively regulate flavonoid biosynthesis in plants (Yan et al, 2015;Pérez-Díaz et al, 2016). Similarly, R2R3-MybA TFs from Muscari armeniacum was involved in the biosynthesis of anthocyanin and can be used in flower color modification (Chen et al, 2019). Similarly, the characterization of novel litchi R2R3-MYB revealed its involvement in tissue acidification and anthocyanin biosynthesis (Lai et al, 2019).…”

Integrated metabolomic and transcriptomic analysis of the anthocyanin regulatory networks in Salvia miltiorrhiza Bge. flowers

“…The expression of genes coding for enzymes and transcription factors play multiple key roles in regulating anthocyanin biosynthesis [ 13 ]. For example, high expressions of dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and anthocyanidin 3-O-glucosyltransferase (UFGT) commonly increase color accumulation in fruits [ 14 ], and the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 transcription factors can form an MBW complex to regulate the biosynthesis of anthocyanin [ 15 – 17 ]. The formation of pigment relies on hydroxylation, glycosylation, methoxylation, and acylation to maintain stability [ 18 , 19 ].…”

Metabolite and transcriptome analyses provide new insights into the generation of the blue supramolecular pigment in cornflower

“…When co-infiltrated with CcMYB6-1 and CcbHLH1, both the trans-activity of CcF3H and CcDFR promoters as well as the anthocyanin content accumulation were significantly enhanced in tobacco leaves (Fig. 5), similar to the cases in chrysanthemum and grape hyacinth [34,35]. Subgroup 4 of Arabidopsis R2R3 MYBs encodes transcriptional repressors, and AtMYB4 was identified as a repressor in the regulation of phenylpropanoid pathway gene expression [21,36].…”