The MADS-box Transcription Factor <i>OfDEFA</i> Orchestrates Floral Aroma Biosynthesis in <i>Osmanthus fragrans</i> Flowers through a Dual-Layer Transcriptional Regulatory Network

Floral scent, primarily comprised of volatile organic compounds such as the monoterpene linalool, serves as a critical medium for plant-pollinator communication and determines ornamental value. Linalool is catalyzed by terpene synthases (TPSs) in plants through Methylerythritol 4-phosphate (MEP) pathway. In sweet osmanthus (Osmanthus fragrans), linalool is a characteristic aroma compound, yet the transcriptional regulation of its biosynthesis remains largely unknown. Although MADS-box transcription factors (TFs) are known as master regulators of floral organ identity, their role in controlling terpenoid volatile production is largely unexplored. In this study, we elucidated the OfDEFA, a nucleus-localized MADS-box TF, centrally regulates linalool biosynthesis through a sophisticated dual-layer mechanism. Using the key enhancer region of the linalool synthase gene OfTPS1a promoter as bait, we identified OfDEFA via yeast-one-hybrid screening. Transient overexpression and virus-induced gene silencing of OfDEFA in O. fragrans flowers confirmed its role as a positive regulator of linalool biosynthesis. We demonstrated that OfDEFA can directly bind to CArG-box motifs in the promoters of two linalool synthase genes, OfTPS1a and OfTPS7, to activate their expression. Furthermore, we uncovered OfDEFA can also directly activate OfMYB21, a known positive regulator of OfTPS1a, thereby regulating the production of linalool. Our findings reveal a novel, elaborate regulatory module in which a floral homeotic MADS-box factor coordinates floral scent production by directly engaging structural genes and simultaneously reinforcing the regulatory network of MEP pathway, providing new insights into the complex regulatory network underlying linalool biosynthesis in sweet osmanthus.