Abstract
Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in β-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in β-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.
Highlights
Carotenoids are natural lipophilic pigments produced by plant plastids that play important roles in light harvesting during photosynthesis and in protecting the photosynthetic apparatus against excessive light radiation
PUC57:pPG:DcPSY2:nopaline synthase terminator (NosT) was digested with SpeI, pUC57:pPG:DcLCYB1:NosT with AvrII and pPG:tp:XdCrtI:NosT with XhoI, and each cassette was cloned into the Multicloning Site (MCS) of pCP producing the single, double and triple vectors, collectively called pCP-CG vectors (Figure 2)
We have established that the increment in carotenoids correlates with a significant increase in the expression of almost all carotenogenic genes, especially DcPSY2, DcZDS2, DcPDS, DcLCYB1, DcLCYE, DcZEP, and DcNCED1 (Simpson et al, 2016)
Summary
Carotenoids are natural lipophilic pigments produced by plant plastids that play important roles in light harvesting during photosynthesis and in protecting the photosynthetic apparatus against excessive light radiation. These secondary metabolites normally accumulate in fruits, flowers and seeds, providing them yellow, orange, and red colors to facilitate pollination and seed dispersal (Rosas-Saavedra and Stange, 2016). The condensation of two molecules of GGPP, carried out by PSY, yields phytoene (C40) This is the first committed step in carotenoid synthesis, which is highly regulated. Β-carotene presents the highest provitamin A activity since each molecule produces two retinal molecules that are reduced to vitamin A (retinol). 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)
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