| Summary: | Drought is a major abiotic stress that limits the growth and yield of alfalfa, a vital forage legume. The plant metalloproteinase Filamentation temperature-sensitive H (FtsH) is an ATP- and Zn<sup>2+</sup>-dependent enzyme that plays a significant character in the plant’s response to environmental stress. However, its functional role in drought resistance remains largely unexplored. This study investigates the drought tolerance role of alfalfa <i>MsFtsH8</i> by analyzing the growth, physiology, and gene expression of overexpressing plants under drought conditions. The results demonstrated that both <i>MsFtsH8</i>-overexpressing <i>Arabidopsis</i> and alfalfa plants exhibited superior growth condition and enhanced membrane stability. The overexpressing alfalfa plants also showed reduced MDA levels, higher proline content, lower H<sub>2</sub>O<sub>2</sub> accumulation, an increased activity of antioxidant-related enzymes (SOD, POD, and CAT) activity, and an elevated expression of antioxidant-related genes. These results indicated that the overexpression of <i>MsFtsH8</i> enhanced growth, improved osmotic regulation, reduced ROS levels, and increased antioxidative capacity, ultimately leading to greater drought tolerance in alfalfa. Our findings suggest that <i>MsFtsH8</i> mitigates oxidative damage caused by drought by modulating the plant’s antioxidant system, thus improving drought tolerance in alfalfa. This study provides a molecular basis and candidate genes for enhancing drought resistance in alfalfa through genetic engineering.
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