Abstract
Common wheat (Triticum aestivum L.) is a staple food crop for humans, yet it primarily accumulates the non-provitamin A carotenoid lutein and exhibits limited natural variation in provitamin A β-carotene among its various accessions. This characteristic necessitates the development of alternative strategies for provitamin A biofortification. To address this challenge, we targeted key control points in the carotenoid biosynthetic pathway using the CRISPR-Cas9 system in a wheat cultivar Fielder. Specifically, we knocked out the gene encoding lycopene ε-cyclase (LCYE), an enzyme that acts as a gatekeeper opposing the production of β-branch carotenoids. Biochemical analysis of homozygous transgene-free mutant endosperms at 20 days post-anthesis (DPA) revealed marked metabolic rerouting of carotenoid biosynthesis, characterized by differential, line-specific accumulation patterns. Provitamin A carotenoids-specifically β-carotene-increased by 26.1-34.5% relative to wild-type controls, concomitant with elevated 22.9-125.4% for zeaxanthin, 41.6-73.9% for violaxanthin, and 26.2-186.5% for antheraxanthin. However, these gains were offset by drastic lutein reduction in lines 1-4 and 5-1. Consequently, total carotenoid levels displayed non-uniform responses, with line 5-1 exhibiting a modest decrease relative to wild-type. Moreover, the mutant lines exhibited elevated levels of amylose and soluble sugar, and the seed coats and endosperms of the triple homozygous transgene-free mutant lines exhibited an orange-yellow hue. In conclusion, we have successfully developed novel carotenoids biofortified wheat lines through a gene-editing approach. This study demonstrates targeted redirection of carotenoid biosynthesis via gene editing as an effective strategy to enhance the nutritional value of commercial wheat and mitigate micronutrient deficiencies in modern food systems.