Exogenous proline enhances salt acclimation in soybean seedlings: Modifying physicochemical properties and controlling proline metabolism through the ornithine-glutamate dual pathway

Soil salinization has emerged as a major factor negatively affecting soil quality and plant productivity. Proline, functioning as an osmotic regulator, has been proposed as an effective strategy for enhancing plant tolerance to salt stress. This study aimed to investigate the effects of exogenous proline on salt tolerance in soybeans. A hydroponic experiment was conducted with different salt treatments (without NaCl, -NaCl; with 100 mM NaCl, +NaCl) and with or without 150 mM proline (+Pro, -Pro). The results showed that proline application alleviated salt stress-induced reductions in plant growth, photosynthetic parameters, and chlorophyll content while aiding recovery from leaf chlorosis. Proline treatment improved ion homeostasis by reducing Na⁺ levels and increasing K⁺ and Ca²⁺ contents in the leaves. Salt stress increased malondialdehyde (MDA) and reactive oxygen species (ROS) levels, along with leaf peroxidase (POD) and catalase (CAT) activities, while decreasing superoxide dismutase (SOD) activity. Moreover, salt stress obviously enhanced proline accumulation, accompanied by increases in glutamate (Glu), glutamate-1-semialdehyde (GSA), and pyrroline-5-carboxylate (P5C) content, as well as the activities of pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) in the glutamate pathway, while reducing proline dehydrogenase (ProDH) activity. Exogenous proline treatment further elevated proline content and increased key substances and enzyme activities in both the glutamate (Glu and P5C content, P5CS and P5CR activity) and ornithine (Orn content and OAT activity) pathways while also reducing ProDH activity. Collectively, our results revealed that exogenous proline contributed to an attenuation of salt stress in soybeans by regulating both the glutamate and ornithine pathways to stimulate endogenous proline accumulation, mediate Na⁺/K⁺ homeostasis, and inhibit oxidative damage.