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Ectopic Expression of the Grape Hyacinth (Muscari armeniacum) R2R3-MYB Transcription Factor Gene, MaAN2, Induces Anthocyanin Accumulation in Tobacco.

ABSTRACT: Anthocyanins are responsible for the different colors of ornamental plants. Grape hyacinth (Muscari armeniacum), a monocot plant with bulbous flowers, is popular for its fascinating blue color. In the present study, we functionally characterized an R2R3-MYB transcription factor gene MaAN2 from M. armeniacum. Our results indicated that MaAN2 participates in controlling anthocyanin biosynthesis. Sequence alignment and phylogenetic analysis suggested that MaAN2 belonged to the R2R3-MYB family AN2 subgroup. The anthocyanin accumulation of grape hyacinth flowers was positively correlated with the expression of MaAN2. And the transcriptional expression of MaAN2 was also consistent with that of M. armeniacum dihydroflavonol 4-reductase (MaDFR) and M. armeniacum anthocyanidin synthase (MaANS) in flowers. A dual luciferase transient expression assay indicated that when MaAN2 was co-inflitrated with Arabidopsis thaliana TRANSPARENT TESTA8 (AtTT8), it strongly activated the promoters of MaDFR and MaANS, but not the promoters of M. armeniacum chalcone synthase (MaCHS), M. armeniacum chalcone isomerase (MaCHI), and M. armeniacum flavanone 3-hydroxylase (MaF3H). Bimolecular fluorescence complementation assay confirmed that MaAN2 interacted with AtTT8 in vivo. The ectopic expression of MaAN2 in Nicotiana tabacum resulted in obvious red coloration of the leaves and much redder flowers. Almost all anthocyanin biosynthetic genes were remarkably upregulated in the leaves and flowers of the transgenic tobacco, and NtAn1a and NtAn1b (two basic helix-loop-helix anthocyanin regulatory genes) were highly expressed in the transformed leaves, compared to the empty vector transformants. Collectively, our results suggest that MaAN2 plays a role in anthocyanin biosynthesis.

SUBMITTER: Chen K

PROVIDER: S-EPMC5462982 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Ectopic Expression of the Grape Hyacinth (Muscari armeniacum) R2R3-MYB Transcription Factor Gene, MaAN2, Induces Anthocyanin Accumulation in Tobacco.

Chen Kaili K Liu Hongli H Lou Qian Q Liu Yali Y

Frontiers in plant science 20170608

Anthocyanins are responsible for the different colors of ornamental plants. Grape hyacinth (Muscari armeniacum), a monocot plant with bulbous flowers, is popular for its fascinating blue color. In the present study, we functionally characterized an R2R3-MYB transcription factor gene MaAN2 from M. armeniacum. Our results indicated that MaAN2 participates in controlling anthocyanin biosynthesis. Sequence alignment and phylogenetic analysis suggested that MaAN2 belonged ...[more]

PMID: 28642775

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Project description:BackgroundThe 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.ResultsIn 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.ConclusionThe 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.

Dataset Information

Ectopic Expression of the Grape Hyacinth (Muscari armeniacum) R2R3-MYB Transcription Factor Gene, MaAN2, Induces Anthocyanin Accumulation in Tobacco.

Publications

Ectopic Expression of the Grape Hyacinth (<i>Muscari armeniacum</i>) R2R3-MYB Transcription Factor Gene, <i>MaAN2</i>, Induces Anthocyanin Accumulation in Tobacco.

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