Abstract
Cadmium (Cd) is a highly toxic heavy metal that poses a serious threat to plant growth and the quality of agricultural and animal products. Therefore, elucidating the response and regulatory mechanisms of plants to Cd stress is essential for controlling the safety of agricultural and animal products at the source. In this study, we used a Cd-tolerant alfalfa cultivar, Medicago sativa 'Zhaodong' (ZD), and a Cd-sensitive cultivar, M. sativa 'Zhongmu No.1' (ZM), to investigate their differential responses to Cd stress in terms of morphology, photosynthetic function, and oxidative damage through combined physiological and RNA-Seq analyses. The results indicated that Cd stress inhibited leaf growth and photosynthetic function, and increased oxidative damage in alfalfa. The Cd-tolerant cultivar ZD exhibited greater tolerance compared to the Cd-sensitive cultivar ZM. RNA-Seq analysis revealed that the differentially expressed genes (DEGs) between the two cultivars under Cd stress were predominantly enriched in photosynthesis-related KEGG and GO pathways. Based on RNA-Seq and weighted gene co-expression network analysis (WGCNA), the MEgreen module exhibiting the highest correlation with physiological traits such as biomass and photosynthetic function was identified. The DEGs in this module were predominantly enriched in pathways related to photosynthesis and plant hormone signal transduction. Furthermore, a key gene, MsERF15, which regulates photosynthesis and ROS metabolic homeostasis in alfalfa under Cd stress, was identified. Although heterologous expression of MsERF15 significantly increased plant height in transgenic tobacco, it markedly reduced Cd stress tolerance, particularly by decreasing chlorophyll content and photochemical reaction center activity in the leaves under Cd stress, as well as elevating ROS accumulation. The potent growth-promoting effect of MsERF15 may represent a key factor contributing to its reduced Cd stress tolerance. In summary, our findings demonstrate that MsERF15 negatively regulates Cd stress tolerance in alfalfa. These results provide new insights into the adaptation mechanisms of alfalfa under Cd stress and offer a novel target for genetic improvement of Cd resistance in this important forage crop.