Multi-omics reveals the molecular regulatory mechanism of exogenous glutathione in alleviating cadmium stress in alfalfa

Soil and water cadmium (Cd) pollution, intensified by industrial activities and excessive usage of cadmium-containing phosphate fertilizers, poses severe threats to crop growth. Glutathione (GSH), a key antioxidant, widely used in plants to maintain the homeostasis of reactive oxygen species under abiotic stress, including Cd stress, but its molecular mechanism in alfalfa was unclear. This study integrated transcriptomics, metabolomics, growth and physiological indicators to reveal GSH's role in alleviating Cd stress in alfalfa. Our results revealed that Cd stress significantly hindered alfalfa growth, which GSH application partially restored, evidenced by increased fresh weight and plant height. GSH reduced oxidative damage markers MDA content, superoxide anion level) and elevated antioxidant defenses (GSH content, superoxide dismutase (SOD) activity, POD activity, total antioxidant capacity (T-AOC)). Multi-omics analysis revealed that GSH alleviates Cd stress by enhancing the expression of genes and accumulation of metabolites associated with plant hormone signal transduction. Meanwhile, GSH stimulates amino acid and soluble sugar biosynthesis, notably increasing tryptophan and rhamnose levels, while concurrently upregulating genes like Acetyl s erotonin O-methyltransferase (ASMT), proline dehydrogenase (PRODH), and Trehalose-6-phosphate Phosphatase (otsB) to boost reactive oxygen species (ROS) clearance and osmotic regulation. Additionally, it upregulates lignin biosynthesis genes to fortify cell wall barriers against Cd. Moreover, GSH enhances gene expression of metal efflux protein and restricts Cd ion transport to the shoots, thereby reducing toxicity. In summary, this study provides novel molecular insights into GSH-mediated Cd tolerance in alfalfa, offering significant potential for developing Cd-tolerant varieties and advancing sustainable agriculture. • Cadmium stress enhanced oxidative stress, thereby inhibiting its growth, and exogenous glutathione effectively restored it. • Glutathione reduced cadmium transport and absorption by inhibiting the expression of metal transporters YSL, NRAMP, and HMA. • Glutathione activated tryptophan and glutathione metabolism to alleviate oxidative stress caused by cadmium stress.