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

• Published in: Biotechnology and bioengineering Vol. 117; no. 7; pp. 2209 - 2222
• Main Authors: Arcos, Yessica, Godoy, Francisca, Flores‐Ortiz, Carlos, Arenas‐M, Anita, Stange, Claudia
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.07.2020
• Subjects: Agrobacterium; Agrobacterium apple transformation; Antioxidants; Apples; AtDXR; Carotenoids; Fruits; fuji variety; Fusion protein; Genetic transformation; Hygromycin; Lutein; Malus domestica; Metabolic engineering; Pigments; Plastids; Protein folding; Public health; Retinene; Seedlings; Transcription; Transformations; Vitamin A; Vitamin deficiency; Agrobacterium apple transformation; AtDXR; Malus domestica; carotenoids; fuji variety
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27358

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. Arcos et al., 2020 revealed that the DXR enzyme presents a relevant role in the carotenoid biosynthesis in apples and can be a good candidate for metabolic engineering, because 1) stable expression of AtDXR:eGFP in Fuji Raku Raku increased the transcript abundance of three MdPSYs members and MdLCYBs, together with a significant increase in total carotenoids, lutein, b‐carotene and a‐carotene in leaves and lutein in dormant buds and 2) transient expression of AtDXR:eGFP in Fuji fruits produced a significant increment in total carotenoids in the flesh.