Boosting carotenoid content inMalus domesticavar. Fuji by expressingAtDXRthrough anAgrobacterium‐mediated transformation method

Abstract

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.

Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits

“…The constructs were transiently expressed by agroinfiltration in Nicotiana tabacum (tobacco) leaves and tomato fruits at breaker stage. A. tumefaciens suspensions containing the pPG:DcPSY2:GFP, pPG:tp:XdCRTI:GFP and both control constructs were infiltrated into 2 months old tobacco leaves and into the tomato fruit flesh following previously described protocols ( Moreno et al, 2013 ; Arcos et al, 2020 ). Plants and fruits were maintained at 20–22°C, 30–50% humidity and while tobacco plants were kept under a 16 h white light photoperiod, tomato fruits were kept in the darkness for 4 days.…”

“…Transient transformation of apples fruits was performed according to Arcos et al (2020) with modifications. Fuji apple fruits at the mature green stage were infiltrated using the Agrobacterium -injection strategy containing the genetic construct of interest (pPSY2, pLCBY1, pPSY2-CRTI, or pPSY2-CRTI-LCYB1) and controls (WT).…”

“…Genetic engineering also allows nutritional traits to be targeted to specific organs (e.g., cereal seeds) and multiple traits can be combined in the same plants without complex breeding programs ( Naqvi et al, 2009 , 2010 ). Other examples of efficient enhancement of carotenoids through metabolic engineering have been obtained in agronomical relevant crops ( Alós et al, 2016 ), such as rice ( Paine et al, 2005 ), tomato ( D’Ambrosio et al, 2004 ), potato ( Diretto et al, 2006 ), carrot ( Jayaraj et al, 2008 ), canola ( Shewmaker et al, 1999 ; Ravanello et al, 2003 ), cassava ( Failla et al, 2012 ), sorghum ( Lipkie et al, 2013 ), orange ( Pons et al, 2014 ), and apple ( Arcos et al, 2020 ).…”

“…However, commercial varieties such as “Royal Gala” and “Fuji” present low levels of carotenoids in the flesh reaching about 2-6 μg/g fresh weight (FW; Ampomah-Dwamena et al, 2012 ; Cerda et al, 2020 ). Specifically, the flesh of Fuji fruits accumulates around 1.8 μg/g FW in total carotenoids ( Arcos et al, 2020 ), 0.04–0.22 μg/g dry weight (DW) of lutein and 0.79–1.14 μg/g DW of β-carotene ( Delgado-Pelayo et al, 2014 ). Low carotenoid levels can be ascribed to reduced biosynthesis, deficient accumulation, and/or efficient degradation.…”

“…Previous studies determined that the PSY enzyme in apple is codified by a family of genes with different levels of expression in fruit flesh ( Ampomah-Dwamena et al, 2012 ; Cerda et al, 2020 ) and that the most expressed PSY genes are functional ( Ampomah-Dwamena et al, 2015 ; Cerda et al, 2020 ) suggesting that the synthesis pathway is not the limiting step for carotenoid accumulation. In addition, the ectopic expression of the Arabidopsis 1-deoxy-D-xylulose 5-phosphate reductoisomerase ( AtDXR) in Fuji apple, a gene participating in the synthesis of carotenoid precursors, produces a threefold rise in total and individual carotenoids in leaves ( Arcos et al, 2020 ). This information led to the proposal that apple is a potential target to become a functional food with high pro-vitamin A and antioxidant components, when genes that codify for biosynthetic enzymes are expressed.…”