Abstract
Flower color is a charming phenotype with very important ornamental and commercial values. Anthocyanins play a critical role in determining flower color pattern formation, and their biosynthesis is typically regulated by R2R3-MYB transcription factors (TFs). Paeonia suffruticosa is a famous ornamental plant with colorful flowers. However, little is known about the R2R3-MYB TFs that regulate anthocyanin accumulation in P. suffruticosa. In the present study, two R2R3-MYB TFs, namely, PsMYB114L and PsMYB12L, were isolated from the petals of P. suffruticosa ‘Shima Nishiki’ and functionally characterized. Sequence analysis suggested that PsMYB114L contained a bHLH-interaction motif, whereas PsMYB12L contained two flavonol-specific motifs (SG7 and SG7-2). Subsequently, the in vivo function of PsMYB114L and PsMYB12L was investigated by their heterologous expression in Arabidopsis thaliana and apple calli. In transgenic Arabidopsis plants, overexpression of PsMYB114L and of PsMYB12L caused a significantly higher accumulation of anthocyanins, resulting in purple-red leaves. Transgenic apple calli overexpressing PsMYB114L and PsMYB12L also significantly enhanced the anthocyanins content and resulted in a change in the callus color to red. Meanwhile, gene expression analysis in A. thaliana and apple calli suggested that the expression levels of the flavonol synthase (MdFLS) and anthocyanidin reductase (MdANR) genes were significantly downregulated and the dihydroflavonol 4-reductase (AtDFR) and anthocyanin synthase (AtANS) genes were significantly upregulated in transgenic lines of PsMYB114L. Moreover, the expression level of the FLS gene (MdFLS) was significantly downregulated and the DFR (AtDFR/MdDFR) and ANS (AtANS/MdANS) genes were all significantly upregulated in transgenic lines plants of PsMYB12L. These results indicate that PsMYB114L and PsMYB12L both enhance anthocyanin accumulation by specifically regulating the expression of some anthocyanin biosynthesis-related genes in different plant species. Together, these results provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in P. suffruticosa and for the breeding of tree peony cultivars with novel and charming flower colors.
Highlights
Paeonia suffruticosa is a very popular ornamental flowering plant that was first cultivated more than 1600 years ago in China and is currently distributed worldwide
The expression level of the Flavonol synthase3 (FLS) gene (MdFLS) was significantly downregulated and the DFR (AtDFR/MdDFR) and anthocyanin synthase (ANS) (AtANS/MdANS) genes were all significantly upregulated in transgenic lines plants of PsMYB12L. These results indicate that PsMYB114L and PsMYB12L both enhance anthocyanin accumulation by regulating the expression of some anthocyanin biosynthesis-related genes in different plant species
By conducting GenBank BLAST searches of the amino acid sequences of these two genes, we found that these genes have the highest homology with transcription factor MYB114-like
Summary
The tree peony cultivar P. suffruticosa ‘Shima Nishiki’ was grown in the experimental nursery of Forestry College, Shandong Agricultural University, Tai’an, Shandong, China. Flower samples were collected at five early flower-bud developmental stages (flower bud emerging stage (S1), small bell-like flower-bud stage (S2), large bell-like flower-bud stage (S3), bell-like flower-bud extending stage (S4), and color exposing stage (S5)) (Figure S2) [65]. All these samples were immediately frozen in liquid nitrogen and stored at –80 ◦ C for further experiments. The plants were grown under a 16 h light/ 8 h dark photoperiod at 23 ◦ C/21 ◦ C
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