Physiological, biochemical, and transcriptional regulation in a leguminous forage Trifolium pratense L. responding to silver ions

Trifolium pratense L. (red clover) is an important leguminous crop with great potential for Ag-contaminated environment remediation. Whereas, the molecular mechanisms of Ag tolerance in red clover are largely unknown. Red clover seedlings were used for physiological and transcriptomic investigation under 0, 20, 50, and 100 mg/L Ag+ stress in our research to reveal potential molecular resistance mechanism. Research showed that red clover possessed fairly strong Ag absorbance capacity, the Ag level reached 0.14 and 2.35 mg/g.FW in the leaves and roots under 100 mg/L AgNO3 stress condition. Root fresh weight, root dry weight, root water content, and photosynthetic pigments contents were significantly decreased with elevating AgNO3 concentration. Obvious withered plant tissue, microstructure disorder, and disrupted organelles were observed. In vitro evaluations (e.g., PI and DCFH-DA staining) represented that AgNO3 at high concentration (100 mg/L) exhibited obvious inhibition on cell viability, which was due possibly to the induction of reactive oxygen species (ROS) accumulation. A total of 44643 differentially expressed genes (DEGs) were identified under Ag stress, covering 27155 upregulated and 17488 downregulated genes. 12 stress-responsive DEGs was authenticated utilizing real-time quantitative PCR (qRT-PCR). Gene ontology (GO) analysis revealed that the DEGs were mostly related to metal ion binding (molecular function), nucleus (cellular component), and defense response (biological process). Involved DEGs in sequence-specific DNA binding transcription factor activity, response to various hormones (e.g., abscisic acid, IAA/Auxin, salicylic acid, and etc), calcium signal transduction, and protein ubiquitination were concluded to play crucial roles in Ag tolerance of red clover. On the other hand, Kyoto Encyclopedia of Genes and Genomes (KEGG) database annotated several stress responsive pathways such as plant-pathogen interaction, phenylpropanoid biosynthesis, ubiquitin mediated proteolysis, hormone signal transduction, and autophagy. Several down-regulated genes (e.g., RSF2, RCD1, DOX1, and etc) were identified indicating possible metabolic disturbance. Besides, protein-protein interaction network (PPI) identified several pivotal genes such as ribosomal proteins, TIR, and ZAT.