Carotenoids are a complex class of isoprenoid pigments playing diverse roles in plants and providing nutritional value. Metabolic engineering of the biosynthetic pathway has been of interest to specifically address global vitamin A deficiency by breeding cereal crop staples in the Poaceae (Grass family) for elevated levels of provitamin A carotenoids. However, there remain open questions about the rate-controlling steps that limit predictability of metabolic engineering in plants, whether by transgenic or nontransgenic means. We decided to focus on the first committed biosynthetic step which is mediated by phytoene synthase. Our studies revealed that in the Grasses, PSY is encoded by three genes. Maize transcript profiling, together with carotenoid and ABA analysis, revealed that the three PSY copies have subfunctionalized and provide the Grasses with a fine tine control of carotenogenesis in response to various developmental and external cues. Promoter analysis supports subfunctionalization; cis-element analysis of maize PSY1 alleles and comparison with Grass orthologs suggests that man’s selection of yellow maize endosperm has occurred at the expense of a change of gene regulation in photosynthetic tissue as compared to the progenitor white endosperm PSY1 allele.