Yuan, H., Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY, United States, Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Owsiany, K., Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Sheeja, T.E., Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Zhou, X., Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY, United States, Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Rodriguez, C., Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Li, Y., Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, China
Welsch, R., University of Freiburg, Freiburg, Germany
Chayut, N., Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel
Yang, Y., Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY, United States
Thannhauser, T.W., Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY, United States
Parthasarathy, M.V., Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States
Xu, Q., Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, China
Deng, X., Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, China
Fei, Z., Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY, United States
Schaffer, A., Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel
Katzir, N., Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel
Burger, J., Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel
Tadmor, Y., Newe Ya’ar Research Center, Agricultural Research Organization, Ramat Yishay, Israel
Li, L., Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY, United States, Plant Breeding Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States

Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtORHis (R90H) or SbORHis (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtORAla (R90A) functioned similarly to AtORHis to promote carotenoid overproduction. Neither AtOR nor AtORHis greatly affected carotenogenic gene expression. AtORHis exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHis triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastidtype changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtORHis in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates ORHis/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying ORHis-regulated carotenoid accumulation. © 2015 American Society of Plant Biologists. All rights reserved.