Transcription factor CitERF71 activates the terpene synthase gene CitTPS16 involved in the synthesis of E-geraniol in sweet orange fruit

The unique flavor of Citrus fruit depends on complex combinations of soluble sugars, organic acids, and volatile compounds. The monoterpene E-geraniol is an important volatile, contributing to flavor in sweet orange (Citrus sinensis Osbeck). Moreover, antifungal activity of E-geraniol has also been observed. However, the terpene synthase (TPS) responsible for its synthesis has not been identified in sweet orange. Terpene synthase 16 (CitTPS16) was shown to catalyze synthesis of E-geraniol in vitro, and transient overexpression of CitTPS16 in fruits and leaves of Newhall sweet orange resulted in E-geraniol accumulation in vivo. Having identified the responsible enzyme, we next examined transcriptional regulation of CitTPS16 in the fruit. Among cloned members of the AP2/ERF transcription factor gene family, CitERF71 showed a similar expression pattern to CitTPS16. Moreover, CitERF71 was able to activate the CitTPS16 promoter based on results from transient dual-luciferase assays and yeast one-hybrid assays. EMSAs showed that CitERF71 directly binds to ACCCGCC and GGCGGG motifs in the CitTPS16 promoter. These results indicate an important role for CitERF71 in transcriptional regulation of CitTP16 and, therefore, in controlling production of E-geraniol in Citrus fruit.