A single amino acid change at position 96 (Arg to His) of the sweetpotato Orange protein leads to carotenoid overaccumulation.

KEY MESSAGE: IbOr-R96H resulted in carotenoid overaccumulation and enhanced abiotic stress tolerance in transgenic sweetpotato calli. The Orange (Or) protein is involved in the regulation of carotenoid accumulation and tolerance to various environmental stresses. Sweetpotato IbOr, with strong holdase chaperone activity, protects a key enzyme, phytoene synthase (PSY), in the carotenoid biosynthetic pathway and stabilizes a photosynthetic component, oxygen-evolving enhancer protein 2-1 (PsbP), under heat and oxidative stresses in plants. Previous studies of various plant species demonstrated that a single-nucleotide polymorphism (SNP) from Arg to His in Or protein promote a high level of carotenoid accumulation. Here, we showed that the substitution of a single amino acid at position 96 (Arg to His) of wild-type IbOr (referred to as IbOr-R96H) dramatically increases carotenoid accumulation. Sweetpotato calli overexpressing IbOr-WT or IbOr-Ins exhibited 1.8- or 4.3-fold higher carotenoid contents than those of the white-fleshed sweetpotato Yulmi (Ym) calli, and IbOr-R96H overexpression substantially increased carotenoid accumulation by up to 23-fold in sweetpotato calli. In particular, IbOr-R96H transgenic calli contained 88.4-fold higher levels of β-carotene than those in Ym calli. Expression levels of carotenogenesis-related genes were significantly increased in IbOr-R96H transgenic calli. Interestingly, transgenic calli overexpressing IbOr-R96H showed increased tolerance to salt and heat stresses, with similar levels of malondialdehyde to those in calli expressing IbOr-WT or IbOr-Ins. These results suggested that IbOr-R96H is a useful target for the generation of efficient industrial plants, including sweetpotato, to cope with growing food demand and climate change by enabling sustainable agriculture on marginal lands.