Effects Of Graphene Oxide On Wheat Nitrate Uptake And Assimilation And Nitrogen Nutrient Transformation In Soil

Graphene has broad application prospects in various fields due to its unique physical and chemical properties.With the continuous increase in production and usage,graphene will inevitably enter the environment,thereby posing risks to the ecological environment and human health.So far,many studies have reported the environmental ecotoxicological effects of graphene.Plants are important producers in the ecosystem,the effects of graphene materials on their physiology and mechanisms have been reported in multiple ways,but the reports on the influence of graphene on the uptake and transformation of nitrogen,which is an essential element for plant growth,are still lacking.In addition,soil is the most likely receptor for graphene,and the influence of graphene on the transformation of the most important nitrogen nutrient in the soil also urgently needs further study.In this study,wheat and soil were selected to be materials in this experiment.First,the effects of graphene oxide（GO）on the uptake and assimilation of nitrate nitrogen by wheat and its intrinsic mechanisms were studied,then the effects and mechanisms of GO on nitrogen transformation in soil were revealed.This study is expected to provide basic information on the impact of graphene on plant nitrogen use efficiency and soil nitrogen cycle.The research mainly achieved the following research results:（1）Nitrate concentrations were significantly decreased in the roots of wheat plants treated with GO at 200-800 mg·L^-1.Non-invasive microelectrode measurement demonstrated that GO could significantly inhibit the net NO₃^-influx in the meristematic,elongation,and mature zones of wheat roots.Further analysis indicated that GO could be trapped in the root vacuoles,and that the maximal root length and the number of lateral roots were significantly reduced.Additionally,root tip whitening,creases,oxidative stress,and weakened respiration were observed.These observations indicate that GO is highly unfavorable for vigorous root growth and inhibits increase in root uptake area.At the molecular level,GO exposure caused DNA damage and inhibited the expression of most nitrate transporters（NRTs）in wheat roots,with the most significantly downregulated genes being NRT1.3,NRT1.5,NRT2.1,NRT2.3,and NRT2.4.We concluded that GO exposure decreased the root uptake area and root activity,and decreased the expression of NRTs,which may have consequently suppressed the NO₃^-uptake rate,leading to adverse nitrate accumulation in stressed plants.（2）GO displayed concentration-dependent inhibitory effects on the fresh weight,length and number of lateral roots in shoot.Moreover,the above indicators have no significant difference between s GO and m GO treatments.Further analysis found that high concentration of GO treatments significantly reduced the nitrate nitrogen,assimilated nitrogen and total nitrogen concentrations in the roots.Among them,the reductions of nitrate nitrogen,assimilated nitrogen and total nitrogen in roots under 400 mg·L^-1s GO was 0.9,1.3 and 1times higher,respectively,than that of m GO treatment.Analysis of root oxidative status and in-situ NO₃^-uptake revealed that s GO caused greater damage to the root tip and lower NO₃^-net influx rate,compared with m GO.In addition,the expression of nitrate transporter（NRTs）genes in wheat roots including NRT1.5,NRT2.1,NRT2.2,NRT2.3,and NRT2.4 under s GO treatment was also lower than m GO treatments.Although both s GO and m GO with concentrations higher than 400 mg·L^-1significantly reduced the nitrogen concentration in shoots,there was no statistically significant difference between the two kinds of GO treatments.Overall,compared with m GO treatment,s GO induced oxidative stress,and inhibitions on nitrate uptake and accumulation of wheat roots were more significant.（3）The accumulation of wheat fresh weight under 400 mg·L^-1GO treatment was significantly inhibited,as compared with the control.This level of GO also resulted in the observation of 4895 and 10987 differentially expressed genes in the shoots and roots,respectively.Further analysis showed that GO caused a significant down-regulation of only glutamine synthetase（GS）-encoding genes in the shoots,and most of the nitrogen metabolic enzymes in the roots,including GS,nitrate reductase（NR）,glutamate synthase（GOGAT）and glutamate dehydrogenase（GDH）,showed significant downward-regulation trends in some of their encoding genes.The activities of above enzymes in wheat roots were also reduced under GO treatment.Among them,the activity of NR which is the rate-limiting enzyme of nitrate assimilation,were approximately 75%lower with 400 mg·L^-1GO supply than the control,and the activity of GS,the key enzyme for NH₄⁺assimilation,was reduced by 76%.Correspondingly,GO appears to exert a negative regulation on multiple nitrogen assimilation products including nitrous nitrogen,ammonium nitrogen,glutamine（Gln）,glutamate（Glu）and soluble protein.In summary,this study shows that GO has adverse effects on the nitrogen assimilation of plants,and NR and GS are the most affected sites.（4）In the soils fertilized with urea,GO significantly reduced the soil microbial biomass and caused a decline in microbial diversity.Among them,the abundance of nitrogen transformation related bacteria as Firmicutes,Nitrospirae,Proteobacteria,Planctomycetes and Cyanobacteria were significantly decreased with GO incubation.Moreover,the response of above bacteria to multilayer GO was more significant than that of single-layer GO.Among the enzymes related to nitrogen transformation,nitrate reductase is most sensitive even at low concentrations of GO,followed by ammonia monooxygenase and urease,which reduced by13%-31%,5%-26%and 9%-19%respectively,as compared with control.Compared with the control,addition of high concentrations of GO significantly increased the retention of soil urea by 32%-59%,and the contents of ammonium nitrogen and nitrate nitrogen were22%-28%and 55%-69%lower,respectively.Taken together,GO could reduce microbial activity and thereby hampering the hydrolysis of urea,and decrease the transformation of amide nitrogen to ammonium and nitrate,which ultimately delay the nitrogen cycle in soils.