Glutathione Restores Hg-Induced Morpho-Physiological Retardations by Inducing Phytochelatin and Oxidative Defense in Alfalfa

SIMPLE SUMMARY: An ecofriendly approach to mitigate mercury (Hg) toxicity in alfalfa, one of the important forage crops, is highly desirable for environmental sustainability. In this study, the exogenous glutathione (GSH) substantially improved the morphological hindrance and photosynthesis inefficiency in Hg-exposed alfalfa plants. In addition, the Fe and S status of Cd-toxic alfalfa was restored due to GSH supplementation. Interestingly, GSH applied to Hg-exposed plants showed elevated Hg concentration in roots resulted in a substantial deposition of Hg in the root cell wall due to the upregulation of MsPCS1 and MsGSH1 genes in roots. It implies that GSH induces PC accumulation in roots enabling excess Hg bound to the cell wall, thereby limiting the transport of Hg to the aerial part of alfalfa. In silico analysis further suggests a conserved motif linked to the phytochelatin synthase domain (CL0125). In addition, GSH induced the GSH concentration and GR activity in protecting alfalfa plants from Hg-induced oxidative damage. These findings can be useful to formulate GSH-based fertilizer or to develop Hg-tolerant alfalfa plants. ABSTRACT: Mercury (Hg) is toxic to plants, but the effect of glutathione in Hg alleviation was never studied in alfalfa, an important forage crop. In this study, Hg toxicity showed morphological retardation, chlorophyll reduction, and PSII inefficiency, which was restored due to GSH supplementation in alfalfa plants treated with Hg. Results showed a significant increase of Hg, but Fe and S concentrations substantially decreased in root and shoot accompanied by the downregulation of Fe (MsIRT1) and S (MsSultr1;2 and MsSultr1;3) transporters in roots of Hg-toxic alfalfa. However, GSH caused a significant decrease of Hg in the shoot, while the root Hg level substantially increased, accompanied by the restoration of Fe and S status, relative to Hg-stressed alfalfa. The subcellular analysis showed a substantial deposition of Hg in the root cell wall accompanied by the increased GSH and PC and the upregulation of MsPCS1 and MsGSH1 genes in roots. It suggests the involvement of GSH in triggering PC accumulation, causing excess Hg bound to the cell wall of the root, thereby reducing Hg translocation in alfalfa. Bioinformatics analysis showed that the MsPCS1 protein demonstrated one common conserved motif linked to the phytochelatin synthase domain (CL0125) with MtPCS1 and AtMCS1 homologs. These in silico analysis further confirmed the detoxification role of MsPCS1 induced by GSH in Hg-toxic alfalfa. Additionally, GSH induces GSH and GR activity to counteract oxidative injuries provoked by Hg-induced H(2)O(2) and lipid peroxidation. These findings may provide valuable knowledge to popularize GSH-derived fertilizer or to develop Hg-free alfalfa or other forage plants.