Effects Of Pristine And Aged ZnO Nanoparticles On Medicago Truncatula And Soil Microbial Community

With the rapid development of nanotechnology,the application and demand of nanomaterials in various fields is increasing.In recent years,nanomaterials?mostly metal nanomaterials?have been gradually applied in agriculture as fertilizers,pesticides and other agricultural preparations.However,the effect of nanomaterials on plant growth and its microbial community has become a hot topic in the field of nano-toxicology.It is considered that the chemical behavior of nanomaterials will change after they enter the soil,this study intends to select the pristine zinc oxide nanoparticles?pZnO NPs?and its aging product sulfidized ZnO NPs?sZnO NPs?as the test materials,the legume model plant Medicago truncatula is used as the test plant.The soil exposure experiment was used to study the response of Medicago truncatula under pZnO NPs and sZnO NPs stress at physiological,subcellular,and molecular levels.Combining transcriptomics to discover differential genes and metabolic pathways,and analyzing the response of soil microbial communities via high-throughput sequencing technology,so as to reveal the potential mechanism of pZnO NPs and sZnO NPs toxicity on Medicago truncatula and soil microbial community.The main findings are as follows:1. The three zinc treatments significantly inhibited the shoots elongation and biomass accumulation?including fresh biomass and dry biomass of shoots?at high concentration level,but low concentration level had no significant effects.The ZnSO₄ and pZnO NPs inhibited the dry biomass of root and numbers of nodules of Medicago truncatula at both concentrations levels,while sZnO NPs only showed the same negative effect at high concentration.The content of Znin plant tissues exposed from all forms of Zntreatments increased significantly compared with the control,and content of Znin root is significantly higher than shoot.2. ZnSO₄,pZnO NPs and sZnO NPs damaged the leaf subcellular structure of Medicago truncatula.The expansion and deformed chloroplasts structure with different degrees was observed at pZnO NPs and sZnO NPs treatments,and the number of starch grains in the chloroplasts was increased significantly at 500 mg·kg^-1 pZnO NPs and sZnO NPs(100,500 mg·kg^-1)treatments.ZnSO₄ caused chloroplast expanding only at high concentration level.In addition,sZnO NPs significantly reduced the content of chlorophyll a and chlorophyll b in leaves of Medicago truncatula at both concentrations levels,but pZnO NPs had no effect on chlorophyll biosynthesis.3. Both pZnO NPs and sZnO NPs treatments significantly increased the soil dissolved organic carbon content.There are significant differences in the composition and structure of soil bacterial microbial community under three zinc treatments.There are also significant differences between the high and low concentration of the same treatment.Compared with the soil bacterial community,the response of the soil fungal community composition and structural diversity was not sensitivity.Compared with control,only ZnSO₄ treatments showed significant differences in fungal structural diversity,while pZnO NPs and sZnO NPs treatments had less differences.The effects of the three zinc treatments on the relative abundance of soil bacterial community were consistent,all of which caused the relative abundance of soil Proteobacteria to increase by about 10%compared to control,while the relative abundance of Cyanobacteria was a significant decline.As for the abundance of soil fungal community,exposure of ZnSO₄ at both concentrations levels resulting in an increase in the relative abundance of Ascomycota by about 30%,while the relative abundance of Zygomycota and Basidiomycota decreased significantly.The relative abundance of Basidiomycota was significantly higher than control in the pZnO NPs treatments at both concentrations levels,while the sZnO NPs have no effect on the abundance of soil fungal community.4. The transcription profiles of pZnO NPs and sZnO NPs treatments had changed significantly compared to control and ZnSO₄ treatment,and there is obvious difference between these two transcription profiles.GO enrichment analysis of shoots showed that pZnO NPs significantly affected genes related to oxidation-reduction process?oxidoreductase activity,oxidative stress,etc.?.Most of the DEGs in the sZnO NPs treatment were highly enriched in GO terms such as“response to stimulus”,“membrane part”.KEGG enrichment analysis found that DEGs in the pZnO NPs and sZnO NPs treatments were highly enriched in pathways related to plant hormone signal transductio,terpenoid biosynthesis and unsaturated fatty acid synthesis.For root analysis,pZnO NPs significantly affected the expression of genes related to response to stimulus and membrane part.DEGs in sZnO NPs treatment were mainly enriched in GO terms such as“catalytic activity”.KEGG enrichment analysis showed that both pZnO NPs and sZnO NPs interfered with sugar metabolism,phenylpropane biosynthesis and amino acid metabolism of Medicago truncatula roots.In summary,exposure of pZnO NPs and its aging product sZnO NPs at test concentration had a negative effect on the growth of Medicago truncatula.The three treatments all affected the subcellular structure of alfalfa leaves,but the stress of pZnO NPs and sZnO NPs on the chloroplast subcellular structure was slightly more severe than that of ZnSO₄.However,the negative effects caused by pZnO NPs and sZnO NPs are significantly different from ZnSO₄.At the level of gene transcription expression,the transcription profiles of the three zinc treatments are significantly different,and some biological processes involved in the DEGs induced by the pZnO NPs and sZnO NPs treatments showed their own differences.In addition,the composition and structural diversity of soil bacterial and fungal communities also differed significantly between the nanoparticle treatments?pZnO NPs and sZnO NPs?and the zinc ion treatment?ZnSO₄?.In summary,from the response processes and differences of“alfalfa-soil microorganisms”at different levels of physiological growth,subcellular structure and molecular,it can be seen that the toxicity mechanism of pZnO NPs and sZnO NPs on alfalfa and soil microbial community is significantly different from zinc ions?ZnSO₄?.At the same time,the aging product sZnO NPs showed a different toxic effect than pZnO NPs,indicating that the environmental transformation process of nanoparticles in soil has an effect on regulating the biological effects of ZnO NPs.