Boosting pro-vitamin A content and bioaccessibility in leaves by combining engineered biosynthesis and storage pathways with high-light treatments

SUMMARY The relevance of plants as food is expected to grow for a more sustainable diet. In this new context, improving the nutritional quality of plant-derived foods is a must. Biofortification of green leafy vegetables with pro-vitamin A carotenoids such as β-carotene has remained challenging to date. Here we combined two strategies to achieve this goal. One of them (that we call strategy C) involves producing β-carotene in extraplastidial locations of leaf cells to avoid the negative impacts on photosynthesis derived from changing the balance of carotenoids and chlorophylls in chloroplasts. The second approach (that we refer to as strategy P) involves the conversion of chloroplasts into non-photosynthetic, carotenoid-overaccumulating chromoplasts in some leaves, leaving other non-engineered leaves to sustain normal plant growth. Combination of these two strategies resulted in a 5- fold increase in the amount of β-carotene in Nicotiana benthamiana leaves. Following several attempts to further improve β-carotene leaf contents by metabolic engineering, hormone treatments and genetic screenings, it was found that promoting the proliferation of plastoglobules with high-light treatments not only improved β-carotene accumulation but it also resulted in a much higher bioaccessibility. Combination of strategies C and P together with a high-light treatment increased the levels of accessible β-carotene 30-fold compared to controls. We further demonstrate that stimulating plastoglobule proliferation with strategy P but also with high-light alone can also stimulate and hence improve β-carotene contents and bioaccessibility in edible lettuce ( Lactuca sativa ) leaves, unveiling the power of non-GMO approaches for leaf biofortification.