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.