Identification of Two Novel R2R3-MYB Transcription factors, PsMYB114L and PsMYB12L, Related to Anthocyanin Biosynthesis in Paeonia suffruticosa

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). <i>Paeonia suffruticosa</i> is a famous ornamental plant with colorful flowers. However, little is known about the R2R3-MYB TFs that regulate anthocyanin accumulation in <i>P. suffruticosa</i>. In the present study, two R2R3-MYB TFs, namely, <i>PsMYB114L</i> and <i>PsMYB12L</i>, were isolated from the petals of <i>P. suffruticosa</i> 'Shima Nishiki' and functionally characterized. Sequence analysis suggested that <i>PsMYB114L</i> contained a bHLH-interaction motif, whereas <i>PsMYB12L</i> contained two flavonol-specific motifs (SG7 and SG7-2). Subsequently, the in vivo function of <i>PsMYB114L</i> and <i>PsMYB12L</i> was investigated by their heterologous expression in <i>Arabidopsis thaliana</i> and apple calli. In transgenic <i>Arabidopsis</i> plants, overexpression of <i>PsMYB114L</i> and of <i>PsMYB12L</i> caused a significantly higher accumulation of anthocyanins, resulting in purple-red leaves. Transgenic apple calli overexpressing <i>PsMYB114L</i> and <i>PsMYB12L</i> also significantly enhanced the anthocyanins content and resulted in a change in the callus color to red. Meanwhile, gene expression analysis in <i>A. thaliana</i> and apple calli suggested that the expression levels of the flavonol synthase (<i>MdFLS</i>) and anthocyanidin reductase (<i>MdANR</i>) genes were significantly downregulated and the dihydroflavonol 4-reductase (<i>AtDFR</i>) and anthocyanin synthase (<i>AtANS</i>) genes were significantly upregulated in transgenic lines of <i>PsMYB114L</i>. Moreover, the expression level of the <i>FLS</i> gene (<i>MdFLS</i>) was significantly downregulated and the <i>DFR</i> (<i>AtDFR</i>/<i>MdDFR</i>) and <i>ANS</i> (<i>AtANS</i>/<i>MdANS</i>) genes were all significantly upregulated in transgenic lines plants of <i>PsMYB12L</i>. These results indicate that <i>PsMYB114L</i> and <i>PsMYB12L</i> 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 <i>P. suffruticosa</i> and for the breeding of tree peony cultivars with novel and charming flower colors.