Overexpression of alfalfa Orange gene in tobacco enhances carotenoid accumulation and tolerance to multiple abiotic stresses

The multifunctional Orange (Or) protein plays crucial roles in carotenoid homeostasis, photosynthesis stabilization, and antioxidant activity in plants under various abiotic stress conditions. The Or gene has been cloned in several crops but not in alfalfa (Medicago sativa L.). Alfalfa is widely cultivated across the world; however, its cultivation is largely limited by various abiotic stresses, including drought. In this study, we isolated the Or gene from alfalfa (MsOr) cv. Xinjiang Daye. The amino acid sequence of the deduced MsOr protein revealed that the protein contained two trans-membrane domains and a DnaJ cysteine-rich zinc finger domain, and showed a high level of similarity with the Or protein of other plants species. The MsOr protein was localized in leaf chloroplasts of tobacco. The expression of MsOr was the highest in mature leaves and was significantly induced by abiotic stresses, especially drought. To perform functional analysis of the MsOr gene, we overexpressed MsOr gene in tobacco (Nicotiana benthamiana). Compared with wild-type (WT) plants, transgenic tobacco lines showed higher carotenoid accumulation and increased tolerance to various abiotic stresses, including drought, heat, salt, and methyl viologen-mediated oxidative stress. Additionally, contents of hydrogen peroxide and malondialdehyde were lower in the transgenic lines than in WT plants, suggesting superior membrane stability and antioxidant capacity of TOR lines under multiple abiotic stresses. These results indicate the MsOr gene as a potential target for the development of alfalfa cultivars with enhanced carotenoid content and tolerance to multiple environmental stresses.