The Mutations in RcMYB114 Affect Anthocyanin Glycoside Accumulation in Rose

In plants, the R2R3-MYB transcription factors are one of the largest MYB gene families. These MYB transcription factors are very important for regulating plant growth and development. RcMYB114, RcbHLH, and RcWD40 promote anthocyanin accumulation by forming the MBW (MYB-bHLH-WD40) complex and determine the rose flower's color. <i>RcMYB114</i> genomic sequences differ between the red petal and white varieties. Two non-synonymous substitutions were found in the open reading frame. It leads to a change in amino acids. Here, the anthocyanin content showed that there was no anthocyanin in white petals, while the anthocyanin content in red petals increased firstly at stage 2, decreased slightly at stage 4, and then increased again at stage 5. The spatiotemporal expression pattern analysis showed that <i>RcMYB114</i> was not expressed in all petals and tissues of white petals at different flower development stages. In red petal varieties, <i>RcMYB114</i> was highly expressed in petals, followed by styles, and not expressed in stems, young leaves, and stage 1 of flower development. However, <i>RcMYB114</i> has the highest expression level at the blooming stage. The <i>RcMYB114</i> sequence contains 9 SNPs in the coding region, 7 of which were synonymous substitutions that had no effect on the translation product and 2 of which were non-synonymous substitutions that resulted in amino acid alteration at positions 116 and 195, respectively. The <i>RcMYB114</i> gene in red rose was named <i>RcMYB114a</i>, and in white rose was <i>RcMYB114b</i>. <i>RcMYB114c</i> was mutated into leucine via artificial mutation; it was valine at position 116 of <i>RcMYB114a</i>, and Glycine mutated into Arginine at position 195 of <i>RcMYB114a</i> was <i>RcMYB114d</i>. <i>RcMYB114b</i> was the double mutation at positions 116 and 195 of <i>RcMYB114a</i>. The results of yeast two-hybrid experiments showed that RcMYB114a and its missense mutations RcMYB114b, RcMYB114c, and RcMYB114d could both interact with RcbHLH and RcWD40 to form the MYB-bHLH-WD40 complex. A transient transformation experiment in tobacco confirmed that <i>RcMYB114a</i> and its missense mutations <i>RcMYB114b</i>, <i>RcMYB114c</i>, and <i>RcMYB114d</i> could significantly promote the high expression of related structural genes in tobacco, together with the <i>RcbHLH</i> gene, which led to the accumulation of anthocyanins and produced the red color of the leaves. The <i>RcMYB114a</i> gene and its missense mutations <i>RcMYB114b</i>, <i>RcMYB114c</i>, and <i>RcMYB114d</i> interacted with the <i>RcbHLH</i> gene and significantly regulated the accumulation of anthocyanins. The two non-synonymous mutations of <i>RcMYB114</i> do not affect the function of the gene itself, but the content of the anthocyanins accumulated was different. This study should provide clues and references for further research on the molecular mechanism underlying the determination of rose petal color.