Functional Validation of Phytoene Synthase and Lycopene ε-Cyclase Genes for High Lycopene Content in Autumn Olive Fruit (Elaeagnus umbellata)

Abstract

Lycopene is the most potent antioxidant among all carotenoids and is beneficial to human health. A ripe fruit of autumn olive (Elaeagnus umbellata Thunb.) accumulates a high level of lycopene, which is 5-20 times higher than that in an ordinary tomato fruit. During fruit ripening of autumn olive, only phytoene synthase (EutPSY) expression pattern shows a tight positive correlation with the increased lycopene content observed at four ripening stages, while the lycopene ε-cyclase (EutLCYe) transcript could not be detected throughout fruit ripening. Here, we investigated whether the two genes are important targets for engineering lycopene biosynthesis. The full-length cDNAs of EutPSY and EutLCYe were first isolated. Fruit-specific overexpression of EutPSY in tomato fruits resulted in elevated contents of lycopene and β-carotene through feedforward regulation of carotenogenic genes, i.e., downregulation of SlLCYe and upregulation of SlLCYb and SlCYCB. These fruits were decreased in ethylene production throughout ripening. Transcript levels of genes for system-2 ethylene synthesis (SlACS2, SlACS4, SlACO1, and SlACO3), perception (SlNR/ETR3 and SlETR4), and response (SlE4 and SlE8) were also inhibited in EutPSY-overexpressing fruits. Repressing ethylene synthesis and signaling transduction delayed fruit climacteric ripening of transgenic tomato plants. Additionally, RNAi suppression of SlLCYe enhanced β-carotene but not lycopene accumulation through altered expression of carotenogenic genes in transgenic tomato fruits by both feedforward and feedback regulatory mechanisms. Ethylene production in SlLCYe-RNAi fruits decreased, thereby delaying fruit ripening. Collectively, these results confirmed that transcriptional regulation of EutPSY and EutLCYe plays a crucial role and a part in massive lycopene accumulation in autumn olive fruits, respectively. EutPSY overexpression enhanced lycopene accumulation in tomato fruits independently of the ethylene pathway but did not influence the size and weight of tomato fruits. EutPSY can be used as an effective strategy capable of elevating the lycopene content in fruits for improving quality.

Keywords: Elaeagnus umbellata; EutLCYe; EutPSY; fruit ripening; gene characteristic; lycopene; transgenic tomato.