Physiological homeostasis alteration and cellular structure damage of Chlorella vulgaris exposed to silver nanoparticles with various microstructural morphologies.

The toxicity of silver nanoparticles (AgNPs) with a single morphology to aquatic organisms has been well demonstrated in the past decade, but few studies have been carried out to evaluate the differences in toxicity among AgNPs with various microstructural morphologies. In this work, C. vulgaris was used as the tested organism to examine the differences in toxic effects among AgNSs, AgNCs, and AgPLs at concentrations of 0.5, 1.0, 2.0, and 5.0 mg/L. The results showed that the cell density and chlorophyll a content of C. vulgaris decreased when the dose of AgNPs was increased, while the inhibiting effects that were caused by AgPLs were stronger than those that were caused by AgNCs and AgNSs. Under short-term exposure to AgPLs, the ROS content was significantly higher than those under exposure to AgNCs and AgNSs, while the MDA content fluctuated without obvious regularity. The dose of AgPLs affected the antioxidative enzyme activity and lipid peroxidation more obviously than those of AgNSs and AgNCs. The superoxide dismutase and catalase contents in the former case were distinctly higher than those in the latter cases. Consequently, the cell apoptosis rate under exposure to AgPLs reached 83%, which was higher than those under exposure to AgNSs (50%) and AgNCs (71%). This work shows that the level of toxicity to C. vulgaris was in the order of AgPLs > AgNCs > AgNSs. The obtained results demonstrate that the microstructural morphologies of AgNPs determined their potential toxicity.

Toxicity of BPNSs against Chlorella vulgaris: Oxidative damage, physical damage and self-protection mechanism.

Black phosphorus nanosheets (BPNSs) has extensive application prospect in the fields of optoelectronics and biomedicine, due to its unique physicochemical properties. Therefore, a systematic toxic study is necessary to assess its environmental safety. Herein, BPNSs was prepared by liquid exfoliation procedure, the primary producer Chlorella vulgaris (C. vulgaris) was used as a test subject. After the exposure for 120 h at 15, 45 and 75 mg/L BPNSs, the cell viabilities were 45.05%, 18.86% and 4.60% for each treatment group, respectively. The extent of lipid peroxidation and peroxidative damage in C. vulgaris was confirmed by measuring reactive oxygen species (ROS) levels, superoxide dismutase (SOD) and catalase (CAT) activities, followed by determination of malondialdehyde (MDA) content. Morphological analysis results (i.e., SEM and TEM) showed that BPNSs adhered to the cell surface and enter the cell to severely damage cell structure. Furthermore, BPNSs were shown to accelerate apoptosis in C. vulgaris by flow cytometry analysis. Finally, GC-MS was used to explore the metabolic regulatory mechanism of C. vulgaris in response to BPNSs stress. The results of this study can provide theoretical support for subsequent studies on the potential enrichment risk of BPNSs in the water environmental food chain.

Exploration of defense and tolerance mechanisms in dominant species of mining area - Trifolium pratense L. upon exposure to silver.

This present study investigated detoxification mechanisms of leguminous forage Trifolium pratense L. (red clover) seedlings upon exposure to Ag ions (Ag+) on an atomic level. Depressed plant growth (maximum inhibition rate: 46.57%) and significantly altered antioxidase/antioxidant substances levels (maximum inhibition rate: 65.45%/55.41%) revealed that the physiological metabolism was disturbed. Notable lesions were observed in both leaf and root cells at 588 µM Ag+ treatment. All differentially expressed genes (DEGs) were remarkably mapped to biological metabolism related pathways. Red clover seedlings were speculated to initially transform and immobilize Ag+ in the culture medium, then transporting and fixing them inside the cell, mainly as unreduced Ag+ bound to oxygen-, nitrogen-, sulfur-, chloride-containing biological molecules. A portion of Ag+ was reduced to Ag0 and aggregated to form crystalline argentiferous nanoparticles. Effective reducing agents such as alcohols, carboxylic acid, and etc, which are capable of coordinating heavy metals to reduce and stabilize them, were assumed to play a role in Ag+ reduction. The research results are of great value to understand the defense and tolerance mechanisms of red clover to Ag+ and explore the main existing forms of Ag+ in vivo and in vitro, which could indicate contamination condition in regional ecological environment such as mining area and its potential effects.

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.