ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed 1[OPEN] Sabrina Gonzalez-Jorge, Payam Mehrshahi 2 , Maria Magallanes-Lundback 2 , Alexander E. Lipka, Ruthie Angelovici, Michael A. Gore, and Dean DellaPenna* Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824 (S.G.-J., P.M., M.M.-L., R.A., D.D.P.); Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA United Kingdom (S.G.-J., P.M.); Department of Crop Sciences, College of Agricultural, Consumer, and Environmental Sciences, University of Illinois, Urbana-Champaign, Illinois 61801 (A.E.L.); Division of Biological Sciences, University of Missouri, Columbia, Missouri 65201 (R.A.); and Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853 (M.A.G.) ORCID IDs: 0000-0002-0783-1929 (S.G.-J.); 0000-0002-7192-0942 (P.M.); 0000-0001-6896-8024 (M.A.G.). Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP- mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species. Carotenoids are a group of more than 700 lipid- soluble pigments synthesized by all photosynthetic organisms and some nonphotosynthetic bacteria and fungi (Ruiz-Sola and Rodríguez-Concepción, 2012). In leaves of higher plants, carotenoids are essential struc- tural and functional components of the photosynthetic machinery with roles in light harvesting, in non- photochemical quenching, and in limiting membrane damage by reactive oxygen species and singlet oxygen species (DellaPenna and Pogson, 2006; Howitt and Pogson, 2006; Cuttriss et al., 2011). Carotenoids also are essential for seed maturation and dormancy as bio- synthetic precursors for the synthesis of the plant hor- mones abscisic acid (ABA) and strigolactones (Koornneef et al., 2002; Frey et al., 2006; Gomez-Roldan et al., 2008; Alder et al., 2012). Carotenoids also are essential for animal growth, development, and maintenance, primarily through their roles as biosynthetic precursors for vitamin A synthesis. As animals cannot de novo synthesize ca- rotenoids, they must be obtained through dietary in- take, most often from plant-derived foods (Krinsky and Johnson, 2005). Such dietary carotenoids are divided into two classes: the nonprovitamin A carotenoids, which include the majority of carotenoids; and the provitamin A carotenoids, which are limited in number and include a-, b-, and g-carotenes and b-cryptoxanthin. Provitamin A carotenoids are distinguished by the presence of a b-ionone ring, which allows their enzy- matic conversion to vitamin A in the body. Although nonprovitamin A carotenoids by definition cannot 1 This work was supported by the National Science Foundation (grant no. DBI–4 0922493 to D.D.P.). 2 These authors contributed equally to the article. * Address correspondence to dellapen@msu.edu. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de- scribed in the Instructions for Authors (www.plantphysiol.org) is: Dean DellaPenna (dellapen@msu.edu). S.G.-J. designed and performed research and wrote the article; P.M. designed research and wrote the article; M.M.-L., A.E.L., and R.A. performed data analysis; M.A.G. designed and performed data analysis; D.D.P. designed research and wrote the article. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.16.00604 Plant Physiology Ò , July 2016, Vol. 171, pp. 1837–1851, www.plantphysiol.org Ó 2016 American Society of Plant Biologists. All Rights Reserved. 1837