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

<i>Litsea cubeba</i> 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 <i>L. cubeba</i> chromosome can promote efficient terpene synthesis through an interconnected gene cascade, typically incorporating terpene synthase (TPS) genes and <i>CYP450</i>. However, the function of the <i>TPS-CYP450</i> cascade in citral synthesis and the regulatory mechanism of upstream transcription factors (TFs) remain unclear. In this study, we identified a species-specific <i>TPS28-CYP94A1</i> cascade in <i>L. cubeba</i>. LcTPS28 was found to catalyze the synthesis of geraniol, nerol, d-limonene, 3-carene, β-ocimene, and β-myrcene <i>in vitro</i>, while LcCYP94A1 catalyzed the conversion of nerol into neral. Transient overexpression of <i>TPS28-CYP94A1</i> in <i>L. cubeba</i> resulted in the accumulation of neral and geranial. Furthermore, overexpression of <i>TPS28-CYP94A1</i> endowed transgenic tomatoes with a lemon flavor. To further investigate TFs that specifically regulate citral synthesis via <i>CYP94A1</i>, 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 <i>LcCYP94A1</i> promoter. Our research has constructed a regulatory network for citral synthesis centered on the <i>TPS28-CYP94A1</i> cascade and offers novel genetic resources for metabolic engineering to enhance citral yield in plants.