OfWRKY33 binds to the promoter of key linalool synthase gene OfTPS7 to stimulate linalool synthesis in Osmanthus fragrans flowers

Volatile aroma compounds make significant contributions to human perception of flowers. Osmanthus fragrans is a famous aroma plant, and linalool is proved to be the dominant aroma active compound. Although some terpene synthases have been characterized, a comprehensive study of the hub metabolic gene and its transcriptional regulation remain to be revealed. Here, we selected a specific cultivar Boyeyingui with the highest content of linalool among 20-wide-cultivated cultivars for genome and transcriptome sequencings. Among the 25 new putative OfTPSs, only OfTPS6, OfTPS7 could exclusively produce linalool in planta. Biochemical analysis demonstrated that OfTPS6, OfTPS7 were able to catalyze geranyl diphosphate into linalool and a small proportion of other monoterpenes in vitro. Spatial and temporal correlation analysis further confirmed the expression level of OfTPS7 was strongly correlated with linalool content in a panel of 20 cultivars, suggesting OfTPS7 was the essential linalool synthase gene. Combined with yeast one-hybrid screen and weighted correlation network analysis, a nucleus-localized transcriptional factor OfWRKY33 was identified as a prospective modulator. Y1H, LUC, and EMSA demonstrated that OfWRKY33 directly bound to the W-box of OfTPS7 promoter to stimulate its transcription. OfWRKY33 could coordinately induce the expressions of OfTPS7 and 1-deoxy-d-xylulose 1, thereby promoting the linalool formation. The results first identified the key linalool synthase gene OfTPS7 and a novel transcription factor playing a role in the complex regulatory network of linalool biosynthesis in O. fragrans flowers.