LcCDF3-LcTPS39 Regulatory Module Positively Regulates Caryophyllene Biosynthesis to Shape the Floral-Fruity Aroma of Litchi Aril

Volatile terpenoids are pivotal to fruit aroma and serve as the major aroma-donating volatiles in litchi aril, yet their biosynthetic mechanisms remain largely unknown. By profiling 24 litchi cultivars, we identified 14 key terpenoids (e.g., caryophyllene, geraniol, and d-limonene) that classify cultivars into light and strong aroma groups. A genome-wide screening identified 46 <i>LcTPS</i> genes, with <i>LcTPS39</i> showing the highest aril expression and functioning as a caryophyllene synthase. Transcriptomic and promoter analyses revealed a strong correlation between transcription factors <i>LcCDF3</i> and <i>LcTPS39</i>. Subsequent in vitro and in vivo assays confirmed that LcCDF3 directly binds and activates the <i>LcTPS39</i> promoter. Furthermore, overexpression of <i>LcCDF3</i> in litchi callus and tomato fruits significantly enhanced caryophyllene production. Collectively, this study comprehensively characterizes litchi aril volatile terpenoids and elucidates a novel LcCDF3-LcTPS39 regulatory module controlling caryophyllene biosynthesis, offering valuable insights for the genetic improvement of fruit aroma.