A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis

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 AtOR His (R90H) or SbOR His (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtOR Ala (R90A) functioned similarly to AtOR His to promote carotenoid overproduction. Neither AtOR nor AtOR His greatly affected carotenogenic gene expression. AtOR His exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtOR His 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 plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtOR His in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates OR His/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying OR His -regulated carotenoid accumulation.