Boosting carotenoid content in Malus domestica var. Fuji by expressing AtDXR through an Agrobacterium-mediated transformation method.

Apple (Malus domestica) fruits accumulate negligible levels of carotenoids, antioxidant pigments that are precursors for vitamin A in humans. As vitamin A deficiency is an important public health issue, we aimed at increasing carotenoids in apple by constitutively expressing the Arabidopsis thaliana DXR gene, one of the key regulatory steps in the plastidial isoprenoid pathway. For this purpose, we optimized an Agrobacterium-mediated transformation method in the commercial Fuji Raku Raku variety. This resulted in a shoot establishment efficiency of 0.75% at 20 weeks after infection. Molecular and microscopical analyses revealed that 80% of the hygromycin resistant shoots contained and expressed AtDXR:eGFP and that the AtDXR:eGFP fusion protein located in plastids. Transgenic seedlings displayed up to 3-fold increase in total carotenoids and in individual carotenoids compared to the WT, correlating with an increased transcript abundance of endogenous carotenogenic genes such as MdDXS, MdPSY1, MdPSY2, MdPSY3, MdLCYB1, and MdLCYB2. In addition, buds of 2-year-old transgenic dormant trees showed an increment up to 3-fold in lutein, and transient transformation of fruits revealed that AtDXR induced a 2-fold increment in total carotenoids. Thus, these results suggest that DXR may be a good candidate for increasing carotenoid levels in apple fruits through metabolic engineering.