Combined enhancement of ascorbic acid, β‐carotene and zeaxanthin in gene‐edited lettuce
Yarin Livneh, Ehud Leor‐Librach, Dor Agmon, Tal Makov‐Bouaniche, Vivekanand Tiwari, Ekaterina Shor, Yelena Yeselson, Tania Masci, Arthur A. Schaffer, Dana Charuvi, Joseph Hirschberg, Alexander Vainstein
Abstract
Summary Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral‐based CRISPR/Cas9 system in the commercial lettuce cultivar ‘Noga’. Knockout of lycopene ε‐cyclase ( LCY‐ε ), the enzymatic gatekeeper opposing production of β‐branch carotenoids, increased β‐carotene (provitamin A) levels up to 2.7‐fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY‐ε mutants, though their non‐photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m 2 s ‐1 . However, the gene‐edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP‐L‐galactose phosphorylase 1 and 2 (u GGP1 and u GGP2 ), the rate‐limiting enzyme in AsA production, resulted in an average 6.9‐fold increase in AsA levels. The mutation in u GGP2 was found to dominantly influence AsA over‐accumulation. Knockout lines that combined the mutations in LCY‐ε , u GGP1 , u GGP2 and in carotenoid cleavage dioxygenase 4a ( CCD4a ), an isozyme involved in β‐carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β‐carotene and zeaxanthin. Our results demonstrate the potential of multi‐pathway gene editing to ‘supercharge’ economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.