Study On Ecotoxicological Mechanism Of Quinolone Antibiotics On Oryza Sativa L.

Due to the overuse and weak metabolism,the environmental behavior and ecotoxicological effects of quinolones have attracted extensive attention.In this paper,the toxic effects of three typical quinolone antibiotics（levofloxacin,enrofloxacin and norfloxacin）on rice（Oryza sativa L.）were studied by transcriptomics,proteomics,metabolomics,microbiomics and resistance genomics,in order to provide a theoretical basis for the rational use and ecological risk assessment of quinolones.The main contents were as follows:（1）Rice seedlings absorbed and enriched quinolone antibiotics by roots and transferred them to shoots.The transport capacity of three quinolone antibiotics in rice seedlings was as follows:levofloxacin>enrofloxacin>norfloxacin.Quinolone antibiotics had a negative effect on the growth of rice seedlings and showed a dose-independence relationship with the exposure concentration.Rice seedlings grew slower and shorter with biomass reduction and albino leaves.Exogenous quinolone antibiotics broke the balance of reactive oxygen species（ROS）production and clearance in rice seedlings,accumulated a large amount of hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）,and increased cell membrane permeability.It indicated that quinolone antibiotics produced oxidative stress on rice seedlings.The stress intensity was positively correlated with the external exposure concentration.The antioxidant enzyme system of rice seedlings was regulated.Low concentration of quinolone antibiotics stimulated superoxide dismutase（SOD）and catalase（CAT）to remove excess ROS and maintain cell homeostasis.However,with the increase of exposure concentration and oxidative stress,the activities of SOD and CAT decreased.Peroxidase（POD）activity decreased gradually with the increase of exposure concentration.Based on the growth inhibition and oxidative stress of rice seedlings,the toxicity of three quinolone antibiotics to rice seedlings was as follows:levofloxacin>enrofloxacin>norfloxacin.（2）Quinolone antibiotics destroyed the structure of mesophyll cells of rice seedlings:cell wall swelled;the number and volume of chloroplasts decreased,the deformation was damaged,the lamellar structure disappeared,a large number of osmiophilic particles were produced,and the mitochondrial cristae structure disappeared.The ability of three quinolones to destroy rice seedling cells was as follows:levofloxacin>enrofloxacin>norfloxacin,.The damage of chloroplast and mitochondrial structure may lead to the decrease of leaf net photosynthetic rate and respiratory rate.The content of chlorophyll and its precursors decreased,indicating that under the stress of quinolone antibiotics,the rate of light energy absorption and light reaction was the rate limiting step of photosynthesis.According to the characteristics of chlorophyll fluorescence and P700 signal,exogenous quinolone antibiotics inhibited the activities of photosystem（PS）I and PS II,reduced the actual photosynthetic efficiency,weakened the ability of photoprotection,and disordered the electron transport chain.The circulating electron flow conducive to photoprotection was activated under norfloxacin stress,but inhibited under enrofloxacin stress.Under the stress of 800μg L^-1enrofloxacin,the transcription level of rice seedlings was affected.GO function enrichment analysis showed that the enrichment degree of chlorophyll biosynthesis was the highest in the differential expression genes（DEGs）.Then,KEGG metabolic pathway enrichment analysis showed that porphyrin and chlorophyll metabolism were the most enriched in up-DEGs.Under the stress of 800μL^-1enrofloxacin,the protein expression of rice seedlings changed.The GO function enrichment analysis showed that the protein abundance of chloroplast,thylakoid,thylakoid membrane and photosystem decreased,the functions of chloroplast binding decreased,the biological processes such as photosynthetic electron transport chain and photosynthetic electron transport chain occurring in PS II were adversely affected,the electron transport activity was inhibited,and the function of electron transporters in circulating electron transport pathway was decreased.It led to the inhibition of biological processes such as light reaction and photosynthesis.KEGG enrichment analysis showed that the metabolic pathways of photosynthesis（ko00195）,oxidative phosphorylation（ko00190）and photosynthesis-antenna protein（ko00196）were down regulated,indicating that the light harvesting capacity and energy production rate of plants decreased.Analysis of protein-protein interaction（PPI）network analysis of the three photosynthesis related metabolic channels for KEGG had shown that proteins were related except PSBS1,and PSBD had a most number of protein-protein associations.It was proved by molecular docking that enrofloxacin could insert into protein related photosynthetic through hydrogen bond and affect their structure.（3）Enrofloxacin activated the activity of nitrogen assimilation enzyme in rice seedlings and increased the content of nitrogen assimilation intermediates.The contents of nitrate nitrogen（NO₃^--N）,nitrite nitrogen,amino nitrogen,and protein,and the activities of nitrogen assimilating enzymes such as nitrate reductase,nitrite reductase,glutamine synthase,glutamate synthase,and glutamate dehydrogenase increased with the increase of exposure concentration.The increase of NO₃^--N content might be related to the degradability of enrofloxacin as a nitrogen source.Ammonium nitrogen content first increased and then decreased with the increase of exposure concentration,which may be related with the increase of assimilation enzyme activity in the downstream under the stress.Excessive NO accumulation caused nitrosation causing adverse effects on plants.Low concentration(2μg L^-1)of enrofloxacin had no significant effect on metabolites in rice seedlings,while high concentration(800μg L^-1)of enrofloxacin significantly changed the types and contents of metabolites,resulting in changes in metabolic pathways.There were five significant disturbed metabolic pathways in the shoots of rice seedlings（P<0.05）.They were:1)glycine,serine,and threonine metabolism;2)aminoacyl-t RNA biosynthesis;3)alanine,aspartate,and glutamate metabolism;4)butyric acid metabolism;5)glyoxylic acid and dicarboxylic acid metabolism.In addition,there were two significant disturbed metabolic pathways in roots（P<0.05）,tyrosine metabolism and citric acid（TCA）cycle.According to the correlation analysis,the change of metabolite could affect the activity of nitrogen assimilation enzyme,and then affect the nitrogen content in plants.（4）Under the stress of 800μg L^-1enrofloxacin,the number of amplitude sequence variables（ASV）of microorganisms in the phyllosphere and roots of rice seedlings decreased.The richness and diversity of phyllosphere microbial community changed slightly,while the richness and diversity of root microbial community decreased.The phyllosphere microbial community structure changed slightly at the phylum and genus.In roots,the abundance of Bacteroidetes,Actinomycetes,Firmicutes and Planctomycetota increased（P<0.05）,and the abundance of Abditibacteriota decreased（P<0.05）.The dominant genera in root microbial community were transferred.The abundance of Planctomycetota with anaerobic ammonium oxidation and metabolism ability,Gordonia and Sphingobium with organic compound degradation ability and pathogenic bacteria Chryseobacterium increased,while the abundance of biological nitrogen fixing bacteria Azohydromonas and Azospirillum decreased.According to PICRUSt2,the function of microbial community was predicted.Many KEGG pathways such as lipid metabolism,amino acid metabolism,carbohydrate metabolism,lipopolysaccharide biosynthesis,exogenous degradation and metabolism,pyrimidine metabolism,translation,m RNA monitoring pathway,ribosome,bacterial secretion system and signal pathway were affected to varying degrees.The reduction of pyrimidine metabolism,translation,and m RNA monitoring pathway might be related to enrofloxacin’s bacterial activation mechanism.Enrofloxacin also changed the composition and abundance of microbial resistance genes in the roots of rice seedlings.The abundance of quinolone antibiotic resistance genes（ARGs）increased,while the total abundance of ARGs and mobile genetic elements（MGEs）decreased.Correlation analysis showed that MGEs promoted the transmission of most ARGs,but negatively correlated with the abundance of total ARGs,which may be caused by the enhancement of bacterial lineage negatively correlated with ARGs and the reduction of potential ARG hosts.There was a good correlation between ARGs,MGEs and root dominant microorganisms in rice seedlings.The pathogen Chryseobacterium was positively correlated with a few ARGs,indicating that the survival of bacteria may only need to express one or several ARGs under single antibiotic stress.