Cycling Dof Factor 3 mediates light-dependent ascorbate biosynthesis by activating GDP-L-galactose phosphorylase in Rosa roxburghii fruit

Abstract

Light plays an important role in determining the L-ascorbate (AsA) pool size in plants, primarily through the transcriptional regulation of AsA metabolism-related genes. However, the specific mechanism of transcriptional induction responsible for light-dependent AsA biosynthesis remains unclear. In this study, we used a promoter sequence containing light-responsive motifs from GDP-L-galactose phosphorylase 2 (RrGGP2), a key gene involved in AsA overproduction in Rosa roxburghii fruits, to identify participating transcription factors. Among these factors, Cycling Dof Factor 3 (RrCDF3) was highly responsive to variations in light intensity, quality, and photoperiod, leading to alterations in RrGGP2 expression. Further yeast one-hybrid and dual-luciferase assays confirmed that RrCDF3 acts as a transcriptional activator of RrGGP2 by binding specifically to its promoter. Modulating the expression of RrCDF3 in fruits through transient overexpression and silencing resulted in significant changes in RrGGP2 expression and AsA synthesis. Additionally, stable overexpression of RrCDF3 in R. roxburghii calli and Solanum lycopersicum plants resulted in a significant increase in AsA content. Notably, the well-known photo-signal transcription factor ELONGATED HYPOCOTYL5 (RrHY5) directly interacted with the RrCDF3 promoter, enhancing its transcription. These findings reveal a special mechanism involving the RrHY5-RrCDF3-RrGGP2 module that mediates light-induced AsA biosynthesis in R. roxburghii fruit.