Toxicity And Defense Mechanism Of Chlamydomonas Reinhardtii Under Silver Nanoparticles Stress

Nano-metallic materials as new materials have been widely used in various fields such as catalysis,biomedicine,electronics industry,and environmental protection.However,the large-scale production and application of nano-metallic materials have led to the continuous release into the aquatic environment,and caused serious ecological risks with the accumulation of food chain.The potential environmental risks and biological toxicity of nano-metallic materials has become a hot topic in domestic and foreign research.This article selected typical nano-metallic material(silver nanoparticles)and aquatic organism(Chlamydomonas reinhardtii)as the research objects.The toxicity and defense mechanism of Chlamydomonas reinhardtii(C.reinhardtiii)under silver nanoparticles(Ag-NPs)stress were studied by using flow cytometry(FCM),fourier transform infrared spectroscopy(FTIR),transmission electron microscopy(TEM),fluorescence quantitative PCR and other techniques.The purpose was to evaluate the potential impact of nano-metallic materials on aquatic ecosystems and the resistance of aquatic organisms to nano-metallic materials.The main results are as follows:(1)Ag-NPs had inhibitory effects on the growth and reproduction of C.reinhardtiii,and showed a certain dose-response relationship and time-response relationship as well as the 96-h EC₅₀ was 32.19 mg/L.The single cell size of algae cells became smaller and the complexity of cell changed under Ag-NPs stress.In addition,the photosynthetic pigments content and chlorophyll autofluorescence both decreased with the concentration of Ag-NPs increased,which impaired photosynthesis.When the Ag-NPs concentration was higher than 10 mg/L,the chloroplast of C.reinhardtii was destroyed,the thylakoid lamellae was blurred,and the arrangement was disordered.(2)Ag-NPs induced C.reinhardtiii to produce excessive reactive oxygen species(ROS),and the proportion of ROS-producing cells increased.After 72 h exposure to 50 mg/L Ag-NPs,the proportion of ROS-producing cells was 35.20±1.13%.Excessive ROS led to lipid peroxidation reaction,the content of malondialdehyde(MDA)gradually increased,the cell membrane permeability enhanced,which accelerated the internalization of Ag-NPs.The ultrastructure and cell surface functional groups of C.reinhardtiii changed under high concentrations of Ag-NPs stress,causing cell wall lysis,protoplast contraction,plasmolysis,organelle damage,and even induced cell apoptosis.(3)The exopolysaccharide content of C.reinhardtii gradually increased with the prolonged time of Ag-NPs stress.When 10 mg/L Ag-NPs was stressed for 72 h,the exopolysaccharide content was the highest,which was 1.45 times that of the control group.The extracellular protein content increased significantly under Ag-NPs stress.In order to eliminate excess ROS,the activities of superoxide dismutase(SOD)and peroxidase(POD)of antioxidant enzyme defense pathway in cells were significantly increased.At the same time,the algal cells relieved oxidative stress by consuming reduced glutathione(GSH),and the GSH content decreased gradually with increasing stress concentration.GSH was oxidized to oxidized glutathione(GSSG),and the content of GSSG and total glutathione(T-GSH)gradually increased.Moreover,the phytochelatin(PC)content increased with the increase of Ag-NPs concentration,which was involved in the defense pathway of heavy metal chelation.(4)When C.reinhardtii was stressed by Ag-NPs,the expression levels of MSOD,QTOX2,CAT1,GPX2,APX and VTE3 functional genes involved in the defense process were all up-regulated to varying degrees in a short time(3 and 6 h).Among them,the up-regulation of MSOD gene was the most obvious.The maximum relative expression level appeared at 50mg/L Ag-NPs stress for 3 h,and the maximum was 163.80±26.04 times that of algae cells without Ag-NPs stress.The algae cells showed certain adaptability and the expression level was low under long-term(72 h)stress.Ag-NPs stress caused the up-regulation of defense genes in a short time,and then regulated the synthesis of antioxidants to resist oxidative stress.