Genome-Wide Identification, Classification, and Expression Profiling Reveals R2R3-MYB Transcription Factors Related to Monoterpenoid Biosynthesis in Osmanthus fragrans

Abstract

<i>Osmanthus fragrans</i> is widely grown for the purpose of urban greening and the pleasant aroma emitted from its flowers. The floral scent is determined by several monoterpenoid volatiles, such as linalool and its oxides, which are a few of the most common volatiles and the main components of the essential oils in most sweet osmanthus cultivars. In addition, the relative contents of <i>cis</i>- and <i>trans</i>-linalool oxide (furan) may affect the aromas and quality of the essential oils. MYB proteins represent the largest family of transcription factors in plants and participate in regulating secondary metabolites. Several <i>cis</i>-elements, especially AC-rich regions, are known to be bound by 2R-MYBs and could be found in the promoter of the enzyme genes in the terpenoid metabolic pathway. However, there has to date been no investigation into the 2R-MYB family genes involved in regulating terpenoid biosynthesis in <i>O. fragrans</i>. Here, 243 non-redundant 2R-MYB proteins were grouped into 33 clusters based on the phylogeny and exon-intron distribution. These genes were unevenly distributed on 23 chromosomes. Ka/Ks analysis showed that the major mode of 2R-MYB gene evolution was purifying selection. Expression analysis indicated that 2R-MYB genes in <i>O. fragrans</i> exhibited varied expression patterns. A total of 35 OfMYBs representing the highest per kilobase per million mapped reads in the flower were selected for quantitative real-time PCR analysis. The correlation analysis between the expression level and the contents of fragrant compounds at different flowering stages suggested that <i>OfMYB19</i>/<i>20</i> exhibited remarkably positive correlation with the accumulation of <i>cis</i>-linalool oxides. <i>OfMYB51</i>/<i>65</i>/<i>88</i>/<i>121</i>/<i>137</i>/<i>144</i> showed significantly negative correlations with one or more linalool oxides. Characterization of these proteins revealed that OfMYB19 and OfMYB137 were localized in the nuclei, but did not show transcriptional activation in the yeast system, which suggested that they may be bound to other transcription factors to exert regulatory functions. These findings provide useful information for further functional investigation of the 2R-MYBs and offer a foundation for clarifying the 2R-MYB transcription factors involved in the molecular mechanism of the regulation of monoterpenoid biosynthesis in <i>Osmanthus fragrans</i>.