Abstract

Carotenoids are vital to numerous physiological processes in plants and animals as structural components of the photosynthetic apparatus, hormone precursors, photoprotectants and antioxidants. Animals depend on dietary intake of carotenoids, especially of vitamin A precursors such as α- and β-carotene, to meet their daily health demands. Vitamin A deficiency is prevalent in much of the developing world, mainly because diets are based on staple crop seeds with low carotenoid or provitamin A content. A fully elucidated plant carotenoid pathway has allowed successful engineering of carotenoid-enriched crops (e.g. rice), but these efforts have been limited to plant carotenoid biosynthesis genes, though QTL analysis makes clear that in addition to biosynthetic genes other novel loci significantly contribute to the natural variation of carotenoids in crops. To identify such loci, the natural variation of Arabidopsis seed carotenoids was explored by quantitative trait loci (QTL) analysis and genome wide association studies (GWAS). Using two recombinant inbred line (RIL) populations, a carotenoid cleavage dioxygenase 4, CCD4 , with LOD scores >10 and explaining >40% phenotypic variation was identified through QTL fine mapping. Concurrently, a genome wide significant association for seed β-carotene was mapped to the coding region of CCD4 . Functional characterization showed CCD4 to be highly expressed during late embryogenesis and desiccation stages (18-21 DAP ) of seed development. The Arabidopsis ccd4 null mutant resulted in a 13.3-fold increased mature-seed β-carotene content relative to wild type and complementation of this line with superior and inferior CCD4 alleles confirmed the QTL is this gene. These data coupled with the identification of CCD4 by both QTL and GWAS , highlight the functional significance of this enzyme as a novel and important regulator of carotenoid homeostasis in Arabidopsis seeds. The identification of such regulatory components of seed provitamin A content will be critical to enhancing crop breeding and genetic engineering efforts tasked with tackling provitamin A deficiency.