Exogenous application of signaling molecules to enhance the resistance of legume-rhizobium symbiosis in Pb/Cd-contaminated soils

Being signaling molecules, nitric oxide (NO) and hydrogen sulfide (H₂S) can mediate a wide range of physiological processes caused by plant metal toxicity. Moreover, legume-rhizobium symbiosis has gained increasing attention in mitigating heavy metal stress. However, systematic regulatory mechanisms used for the exogenous application of signaling molecules to alter the resistance of legume-rhizobium symbiosis under metal stress are currently unknown. In this study, we examined the exogenous effects of sodium nitroprusside (SNP) as an NO donor additive and sodium hydrosulfide (NaHS) as a H₂S donor additive on the phytotoxicity and soil quality of alfalfa (Medicago sativa)-rhizobium symbiosis in lead/cadmium (Pb/Cd)-contaminated soils. Results showed that rhizobia inoculation markedly promoted alfalfa growth by increasing chlorophyll content, fresh weight, and plant height and biomass. Compared to the inoculated rhizobia treatment alone, the addition of NO and H₂S significantly reduced the bioaccumulation of Pb and Cd in alfalfa-rhizobium symbiosis, respectively, thus avoiding the phytotoxicity caused by the excessive presence of metals. The addition of signaling molecules also alleviated metal-induced phytotoxicity by increasing antioxidant enzyme activity and inhibiting the level of lipid peroxidation and reactive oxygen species (ROS) in legume-rhizobium symbiosis. Also, signaling molecules improved soil nutrient cycling, increased soil enzyme activities, and promoted rhizosphere bacterial community diversity. Both partial least squares path modeling (PLS-PM) and variation partitioning analysis (VPA) identified that using signaling molecules can improve plant growth by regulating major controlling variables (i.e., soil enzymes, soil nutrients, and microbial diversity/plant oxidative damage) in legume-rhizobium symbiosis. This study offers integrated insight that confirms that the exogenous application of signaling molecules can enhance the resistance of legume-rhizobium symbiosis under metal toxicity by regulating the biochemical response of the plant-soil system, thereby minimizing potential health risks.