MsRH53 is involved in brassinolide biosynthesis and positively regulates the salt tolerance in alfalfa

Genetic transformation is a critical way to generate new salt tolerant alfalfa (Medicago sativa L.) germplasm adapted to increasingly serious soil salinization. Here, the expression of an alfalfa DEAD-box helicase gene MsRH53 was found remarkably induced under salt stress. We speculated that MsRH53 could play an important role in alfalfa salt tolerance. By genetic transformation, overexpression of MsRH53 (OE) transgenic alfalfa plants were generated. Under salt stress, the selected OE lines with different MsRH53 expression level showed enhanced salt tolerance, reduced accumulation of reactive oxygen species (ROS), decreased membrane lipid peroxidation (MDA content), and increased peroxidase (POD) activity compared to that of wild type (WT) plants. Transcriptomic analysis comparing WT and OE lines before and after salt treatment revealed that differentially expressed genes (DEGs) were significantly enriched in the brassinosteroid (BR) biosynthesis and signaling pathways. The BRs content remained significantly higher in OE lines than in WT plants before and after salt treatment. Exogenous application of BRs also improved alfalfa salt tolerance. Collectively, our results reveal that overexpression of MsRH53 affects BR biosynthesis and promotes alfalfa salt tolerance and provides a new molecular tool for breeding of salt-tolerant alfalfa cultivars.