Characteristics And Generation Mechanism Of Reactive Oxygen Species In The Rhizosphere Soil And Its Effect On The Degradation Of Polycyclic Aromatic Hydrocarbons

Reactive oxygen species（ROS）,including superoxide radicals(O₂^·-),hydrogen peroxide（H₂O₂）,and hydroxyl radicals（^·OH）,are the intermediates produced by the reduction of O₂.Due to their high reactivity,ROS play critical roles in mediating the transformation of pollutants,element cycling,nutrient release,and biological physiological function regulation.Generally,the production of ROS in different natural environments is mostly related to photochemical processes.Contrastively,the production and role of ROS in soil biogeochemical processes are overlooked due to limited sunlight penetration depth（<0.5 mm）.Recently,the ROS generation under dark conditions also have been explored,which is suggested to be related to the redox reactions at the dynamic interfaces.Rhizosphere,a thin layer of soil at the plant root-soil interface,is a hotspot of intense biogeochemical processes,which may have a strong influence on the production of ROS.An in-depth understanding of rhizosphere ROS production is of great significance for revealing the rhizosphere effects related with ROS.In addition,although studies have been conducted to investigate the production processes and environmental behavior of ROS mediated by single soil components（e.g.,minerals,organic matter and microorganisms）,limited studies have been performed on the characteristics of ROS in whole soil（mixtures of various inorganic minerals and organic matter）and real soil environments affected by climatic conditions,especially the micro-interface mechanism of ROS production and their related environmental effects（e.g.,pollutant transformation）.Therefore,this study systematically studied the production,distribution characteristics,and formation mechanism of ROS in the rhizosphere.The effects of soil properties and external environmental conditions on ROS production were analyzed.Moreover,the role of ROS in the transformation of polycyclic aromatic hydrocarbons（PAHs）in the rhizosphere was further explored.The main contents and results of this study are as follows:（1）Generation and spatiotemporal variation of rhizosphere ROSRhizosphere is a hotspot for the generation of ROS,which shows obvious spatial and temporal distribution characteristics.The accumulation and type of ROS in the rhizosphere were qualitatively identified through chemiluminescence and spin capture methods by using ryegrass and maize as model plants.The microscale spatiotemporal variation of three representative ROS(such as O₂^·-,H₂O₂,and^·OH)in the rhizosphere was quantitatively investigated using molecular probe methods.The production of ROS in the rhizosphere was detected by both ROS-trapping membrane and electron paramagnetic resonance（EPR）spectroscopy,confirming that the rhizosphere is a widespread but previously unrecognized hotspot for ROS generation.The results of chemical molecular probe showed that the contents of three ROS in ryegrass and maize rhizosphere were significantly higher than those in non-rhizosphere soil（p<0.05）,about 2 to 15 times of that in non-rhizosphere soil,and rhizosphere ROS showed obvious spatiotemporal evolution characteristics.Specifically,both O₂^·-and H₂O₂content increased first and then declined throughout the life cycle of ryegrass and maize,while·OH concentration decreased continuously.Spatially,ROS contents remained at a relatively high level at 0-5 mm and then descended with increasing distance.The analysis of physicochemical and biochemical indicators of rhizosphere soil showed that plant growth regulated the release of root exudates and induced changes in soil redox components（Fe（II）,DOC,and water-soluble phenol）and enzyme activities,thus regulating the production of rhizosphere ROS.（2）The influence of soil properties on rhizosphere ROS productionSoil properties significantly affect the production of rhizosphere ROS.The effects of soil properties（e.g.,p H,soil organic carbon,clay minerals,and metal ion contents）on rhizosphere ROS production was investigated by using four typical soils such as black soil,latosol,yellow-brown soil,and red soil.The results showed that there were significant differences（p<0.05）in the content of O₂^·-in the ryegrass rhizosphere of different soils,and the order from the highest to the lowest was black soil（16.64μM/kg）>lateritic soil（15.17μM/kg）>yellow brown soil（13.57μM/kg）>red soil（8.24μM/kg）.In addition,the concentrations of H₂O₂ and^·OH in different rhizosphere soils also had significant differences（p<0.05）,consistent with the trend of O₂^·-.Moreover,the contents of three ROS in maize rhizosphere also had significant differences in different soils（p<0.05）,which was consistent with the trend of ryegrass.Correlation analysis showed that soil physicochemical factors（such as p H,OC,Fe and Mn）were the key factors affecting the generation of ROS in different rhizosphere soils.Among which,p H indirectly affected the generation of ROS by influencing the occurrence of Fenton reaction,while OC,Fe and Mn directly influenced the generation of ROS as electron donors.（3）The influence of environmental conditions on rhizosphere ROS productionEnvironmental conditions have important effects on the generation of rhizosphere ROS.The influences of environmental factors on rhizosphere ROS production were explored by setting different environmental conditions（such as temperature,humidity and oxygen availability）.The results showed that temperature,humidity and oxygen availability had significant effects on rhizosphere ROS（p<0.05）production.Specifically,three ROS(including O₂^·-,H₂O₂,and^·OH)contents increased first and then decreased with the increase of temperature and humidity,reaching the maximum at 25℃and 45%,respectively,indicating that the moderate increase of temperature and humidity was beneficial to the generation of ROS in the rhizosphere.The ROS content gradually increased with the increase of oxygen availability,reaching the maximum when the oxygen availability is 8.97 m² 10⁶s^-1.The analysis of rhizosphere soil physicochemical and biochemical indicators indicated that temperature and humidity regulated the release of root exudates to change the contents of water-soluble phenol and DOC,thus indirectly regulating the production of ROS,while oxygen directly mediated ROS production as a precursor of ROS production.（4）The generation mechanism of rhizosphere ROSThe generation mechanism of rhizosphere ROS is regulated by coupling biotic-abiotic processes.The key factors,potential pathways and mechanisms of ROS generation in the rhizosphere were probed by using statistical methods.Redundancy analysis（RDA）revealed that the formation of rhizosphere ROS was mainly regulated by biological factors（such as bacteria,Chloroflexi,Bacteroidota,Actinobacteria,and Actinobacteriota,and fungi,Basidiomycota and Ascomycota）and abiotic factors（such as root exudates,water-soluble phenols and Fe（II））.Structural equation model（SEM）further revealed that root exudates,secreted by the plant along with its growth,could influence the O2^·-generation in two pathways.First,root exudates could provide substrates for microbial growth,facilitating the biotic generation of O₂^·-through extracellular release.Second,water-soluble phenols,as the main component of root exudates with redox-metastable properties,could facilitate the electrons transfer to oxygen and promote the abiotic production of O₂^·-.The formed O₂^·-was easily transformed into H₂O₂ via dismutation and hydrolysis,which was further decomposed to produce^·OH through Fe（II）-mediated Fenton reactions.During this process,fungi could regulate Fe（II）/Fe（III）cycling,and thus indirectly affected the production of^·OH.Overall,the combined processes,including Fe（II）and water-soluble phenol-mediated electron transfer,microbial community-driven extracellular O₂^·-release,and Fe（II）/Fe（III）cycling,may be responsible for ROS production.（5）The degradation of PAHs by rhizosphere ROSRhizosphere ROS promote the transformation of PAHs,which is affected by the molecular structural characteristics of PAHs.The effects of PAHs on maize growth,rhizosphere ROS production,and the potential degradation of PAHs mediated by ROS were explored by using PAHs with different molecular structures,such as naphthalene（NAP）,phenanthrene（PHE）,and anthracene（ANT）as model compounds.The results showed that PAHs had no significant effect on the growth of maize.Whereas three PAHs compounds could significantly increase the content of rhizosphere ROS(such as O₂^·-,H₂O₂,and^·OH),and the promotion effect was molecular specific:ANT>PHE>NAP.The analysis of physicochemical and biochemical indexes of rhizosphere soil indicated that PAHs might regulate ROS production by regulating the changes of root exudates and water-soluble phenols.By comparing the differences in the transformation of different PAHs,it was found that the transformation rate of PAHs was in the order of ANT>PHE>NAP,which was consistent with the trend of ROS,indicating that the degradation of PAHs might be related to rhizosphere ROS.The quenching experiments further confirmed that rhizosphere ROS promoted the transformation of PAHs.Overall,the rhizosphere is a widespread but previously unappreciated hotspot for ROS production,which possesses obvious spatial and temporal distribution characteristics.Meanwhile,soil physicochemical properties and environmental conditions have important effects on rhizosphere ROS production,which is regulated by the coupling effects of biological（e.g.microbial community structure and enzyme activity）and abiotic（Fe（II）/Fe（III）cycling,root exudates,and water-soluble phenols）factors.Moreover,PAHs can increase rhizosphere ROS content,which plays an important role in PAHs degradation.This study elucidated the mechanism of rhizosphere ROS production,providing insight into the rhizosphere effects associated with ROS and a new perspective on phytoremediation of pollutants.