LcMYB46, a R2R3-MYB Transcription Factor, Promotes the Accumulation of Citral by Activating TPS-CYP Cascade in Litsea cubeba

Litsea cubeba is a crucial species of Lauraceae family, and its fruit is rich in essential oils, mainly composed of monoterpenes, among which citral is a major component. Synthase genes on the L. cubeba chromosome can promote efficient terpene synthesis through an interconnected gene cascade, typically incorporating terpene synthase (TPS) genes and CYP450 . However, the function of the TPS-CYP450 cascade in citral synthesis and the regulatory mechanism of upstream transcription factors (TFs) remain unclear. In this study, we identified a species-specific TPS28-CYP94A1 cascade in L. cubeba . LcTPS28 was found to catalyze the synthesis of geraniol, nerol, d -limonene, 3-carene, β-ocimene, and β-myrcene in vitro, while LcCYP94A1 catalyzed the conversion of nerol into neral. Transient overexpression of TPS28-CYP94A1 in L. cubeba resulted in the accumulation of neral and geranial. Furthermore, overexpression of TPS28-CYP94A1 endowed transgenic tomatoes with a lemon flavor. To further investigate TFs that specifically regulate citral synthesis via CYP94A1, we employed expression profiling clustering analysis and identified a key regulator-LcMYB46. Yeast one-hybrid, dual-luciferase, and Electrophoretic Mobility Shift Assay analysis indicated that LcMYB46 directly binds to and activates the LcCYP94A1 promoter. Our research has constructed a regulatory network for citral synthesis centered on the TPS28-CYP94A1 cascade and offers novel genetic resources for metabolic engineering to enhance citral yield in plants.