Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium 'Siberia'

Terpenoids are the main components contributing to the fragrance of Lilium 'Siberia', and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids. Although the major terpene synthases in Lilium 'Siberia' have been identified, how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower. This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium, and disclose the related underlying transcriptional regulation mechanism. In this study, we identified three R2R3-MYB TFsdLiMYB1, LiMYB305 and LiMYB330, which were involved in regulating the biosynthesis of terpenes in Lilium 'Siberia'. Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds. When LiMYB1, LiMYB305 and LiMYB330 were overexpressed in flowers, the release of some main monoterpenes, such as linalool and ocimene, as well as the expression of TPS genes, especially for LiTPS2, were enhanced. A virus-induced gene silencing (VIGS) assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes. The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2. Moreover, the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330, indicating their synergistic roles in the regulation of floral terpene biosynthesis. In general, these results indicated that LiMYB1, LiMYB305, and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.