Despite ongoing research on carotenoid biosynthesis in model organisms, there is a paucity of information on pathway regulation operating in the grasses (Poaceae), which include plants of world-wide agronomic importance. As a result, efforts to either breed for or metabolically engineer improvements in carotenoid content or composition in cereal crops have led to unexpected results. In comparison to maize (Zea mays), rice (Oryza sativa) accumulates no endosperm carotenoids, despite having a functional pathway in chloroplasts. To better understand why these two related grasses differ in endosperm carotenoid content, we began to characterize genes encoding phytoene synthase (PSY), since this nuclear-encoded enzyme appeared to catalyze a rate-controlling step in the plastid-localized biosynthetic pathway. The enzyme had been previously associated with the maize Y1 locus thought to be the only functional gene controlling PSY accumulation, though function of the Y1 gene product had never been demonstrated. We show that both maize and rice possess and express products from duplicate PSY genes, PSY1 (Y1) and PSY2; PSY1 transcript accumulation correlates with carotenoid-containing endosperm. Using a heterologous bacterial system, we demonstrate enzyme function of PSY1 and PSY2 that are largely conserved in sequence except for N-and C-terminal domains. By database mining and use of ortholog-specific universal PCR primers, we found that the PSY duplication is prevalent in at least eight subfamilies of the Poaceae, suggesting that this duplication event preceded evolution of the Poaceae. These findings will impact study of grass phylogeny and breeding of enhanced carotenoid content in an entire taxonomic group of plant crops critical for global food security.Carotenoids, a class of over 600 structures derived from isoprenoids, are synthesized by all photosynthetic organisms, some bacteria, and fungi. In plants, carotenoids are essential for plant growth and development; mutations blocking carotenoid accumulation have pleiotropic effects on chloroplast biogenesis and seed development (Robertson et al., 1978;Wurtzel, 1992). Carotenoids function as accessory pigments in photosynthesis, as photoprotectors preventing photooxidative damage, and as precursors to the plant hormone, abscisic acid (Hirschberg, 2001). The presence of carotenoids in plant endosperm tissue adds nutritional value; in humans and animals, dietary carotenoids are essential precursors to vitamin A and to retinoid compounds needed in development (Lee et al., 1981;Bendich and Olson, 1989). Nonprovitamin A carotenoids, such as lycopene, lutein, zeaxanthin, and others, also play beneficial roles in human health (Giovannucci et al., 1995;Kohlmeier et al., 1997;Sommerburg et al., 1998;Krinsky et al., 2003). The various roles of carotenoids affecting plant yield and nutritional potential has made them targets for breeding and metabolic engineering (Shewmaker et al., 1999;Matthews and Wurtzel, 2000;Ye et al., 2000;Davison, 2002;Blott et al., 2003;Gallagher et al., ...

Vitamin A deficiency, a global health burden, can be alleviated through provitamin A carotenoid biofortification of major crop staples such as maize (Zea mays) and other grasses in the Poaceae. If regulation of carotenoid biosynthesis was better understood, enhancement could be controlled by limiting b-carotene hydroxylation to compounds with lower or no nonprovitamin A activity. Natural maize genetic diversity enabled identification of hydroxylation genes associated with reduced endosperm provitamin A content. A novel approach was used to capture the genetic and biochemical diversity of a large germplasm collection, representing 80% of maize genetic diversity, without having to sample the entire collection. Metabolite data sorting was applied to select a 10-line genetically diverse subset representing biochemical extremes for maize kernel carotenoids. Transcript profiling led to discovery of the Hydroxylase3 locus that coincidently mapped to a carotene quantitative trait locus, thereby prompting investigation of allelic variation in a broader collection. Three natural alleles in 51 maize lines explained 78% of variation and approximately 11-fold difference in b-carotene relative to b-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of b-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide.