Glutathione Metabolism and Proteome Analysis of Ectomycorrhizal Fungi in Response to Heavy Metal Stress

Abstract

Ectomycorrhizal (ECM) fungi hold a potential role in bioremediation of heavy metal polluted areas because of its metal accumulation and detoxification properties. However, the incomplete information about the molecular response of these fungi restricts its potential applications. The present study focuses on scrutinizing the ECM fungi for its potential role in heavy metal detoxification and understanding the molecular mechanisms involved in its tolerance. ECM fungi Laccaria bicolor and Hebeloma cylindrosporum when exposed to increasing concentrations of cadmium (Cd) and arsenic (As), accumulated the metal(loid) intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS) were highly regulated by Cd and As stress. Both Cd and As coordinately upregulated the expression of both γ-GCS and GS genes, thus resulting in increased γ-GCS and GS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (γ-GCS and GS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating Cd and As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating Cd and As induced responses and its detoxification in ECM fungi L. bicolor and H. cylindrosporum. Further, the proteomic analysis of ECM fungi in response to Cd stress provides deep insight into the mechanisms of Cd toxicity and the response of ECM fungi in mitigating it. The comparative proteomic analysis of L. bicolor reported 997 differentially expressed proteins under Cd stress. The KEGG annotation of these differentially expressed proteins revealed that Cd induced toxicity in ECM fungi by altering various metabolisms like carbohydrate metabolism, nucleotide metabolism, energy metabolism, lipid metabolism and genetic information processing like DNA replication, DNA repair, transcription, translation, protein folding and chromosome metabolism. In defense, the ECM fungi confront the Cd induced toxicity by up-regulating the enzymes involved in oxidative response including glutathione metabolism, inducing signaling pathway and increase the amino acid and protein biosynthesis. These observations provide deep understanding of the Cd toxicity mechanisms and highlight the biomarkers for Cd toxicity in L. bicolor. This study also provides the reference dataset on fungal proteome changes under Cd stress.