Molecular mechanism of high L-ascorbic acid accumulation in chestnut rose fruits

The chestnut rose (Rosa roxburghii Tratt.) has a high accumulation of L-ascorbic acid (AsA) (∼1,762 mg/100 g fresh weight) in its fruits, around 40-fold that of sweet orange (Citrus sinensis), which is known for AsA concentration in fruit. However, the molecular mechanisms behind this high accumulation remain unclear. Here, we revealed that the RroxPME, RroxGalUR, and RroxDHAR2 genes play a crucial role in AsA accumulation in chestnut rose fruit. By comparing R. roxburghii with the closely related R. multiflora, which has a low AsA concentration, we identified a 545 bp insertion in the promoter of RroxGalUR. We determined that RroxTTG2, a well-known key regulator of trichome development, could bind to the W-box containing inserted region of the RroxGalUR promoter, as well as the promoters RroxPME and RroxDHAR2. By contrast, in sweet orange, CsTTG2 can only bind to the CsPME. Furthermore, RroxTTG2 retained its conserved role in the regulation of trichome development at the early stage of fruit development, suggesting it has spatiotemporal specificity in regulating both trichome development and AsA biosynthesis. To test the application value of this pathway in other species, we heterologously expressed RroxTTG2, RroxPME, RroxGalUR, and RroxDHAR2 in lettuce (Lactuca sativa L.), which increased the AsA concentration of the transgenic lines by up to 355% (about increase from 2 to 10 mg/100 g FW). Our study provided insights into the mechanisms underlying AsA accumulation in chestnut rose and the spatiotemporal transcriptional regulation of AsA biosynthesis and trichome development.