Functional Iron-based Nanoenzyme Mediated Enhancing Soybean Symbiotic Nitrogen Fixation System

Legumes can be identified specifically with Rhizobia to form nodule,the host plant provides nutrients for Rhizobia in this process,while Rhizobia uses the nitrogen fixation activity to convert the nitrogen（N₂）in the atmosphere into ammonia（NH₃）for the plant growth and utilization,both of which are mutually beneficial and symbiotic.Symbiotic nitrogen fixation is regarded as one of the most environmentally friendly and efficient nitrogen fixation methods,accounting for more than 60%of the total global biological nitrogen fixation.Therefore,it is of great significance to improve the efficiency of symbiotic nitrogen fixation by studying the mechanism of symbiotic nitrogen fixation of legumes and the microenvironment of nodule nitrogen fixation,so as to achieve weight loss and increase efficiency and develop green agriculture.In this paper,with the organic combination of nanotechnology and symbiotic nitrogen fixation system,the suppression of nitrogenase by stress factors（ROS）in the nodulation nitrogen fixation microenvironment was improved through the design and synthesis of functional nanoenzymes,so as to realize an increase in nitrogen fixation efficiency.On the contrary,with the controllable mechanism of nanomaterial-mediated Fenton reaction,ROS was continuously induced during the nodulation stage of the symbiotic nitrogen fixation system as a nodulation regulating factor to increase the number of nodules,thus increasing the total nitrogen fixation.The specific research content is as follows:1.Iron-molybdenum quantum dots improve the oxidative stress of root nodules toenhance nitrogenase activityThe Fe Mo QDs@GSH with excellent biocompatibility were synthesized and self-assembled in the PH response by surface modification of glutathione（GSH）.It was found that under neutral or acidic conditions,quantum dots showed aggregated state with peroxidase-like activity,whose fluorescence efficiency would be decreased;while alkaline conditions can induce the dissolution of clusters,weaken peroxidase-like activity and enhance fluorescence.In this respect,the researches on the symbiotic system of root nodules and the autogenous nitrogen fixation system of Azotobacter sphaeroides under different p H conditions were carried out.The results showed that quantum dots had excellent biocompatibility for rhizobia and Azotobacter（Rhizobium and Azotobacter sphaerocephalus）under all p H conditions.Incubation with 250-1000 mg L^-1 Fe Mo QDs@GSH under p H 7.0 conditions,it can significantly enhance the nitrogenase activity of nodules（increased by 26.28%-97.31%）with obvious inhibitory effect on Azotobacter sphaeroides.The incubation of 100 mg L^-1 Fe Mo QDs@GSH at p H 9.0 can effectively enhance the nitrogenase activity of Azotobacter sphaeroides（increased by 22.89%）without significant difference in the symbiotic nitrogen fixation system of nodules.ROS in situ fluorescence analysis of root nodules showed that Fe Mo QDs@GSH significantly reduced ROS levels at p H 7.0.The fluorescence localization of nodule tissue cells confirmed the enrichment of Fe Mo QDs@GSH in the symbiotic zone of cell protoplasts and nodule.Therefore,it’s believe that Fe Mo QDs@GSH at p H 7.0 can significantly reduce the level of ROS in the nodule tissue,thus enhancing the nitrogenase activity of the symbiotic nitrogen fixation system.2.Cobalt ferrite nanozyme for efficient symbiotic nitrogen fixation via regulating reactive oxygen metabolismWe constructed an antioxidant cobalt ferrite（Co Fe₂O₄）nanozyme as a bridge between nanotechnology and biological nitrogen fixation,which was shown to efficiently regulate the reactive oxygen metabolism and protect nitrogenase,thereby significantly enhancing the symbiotic nitrogen fixation efficiency by 260%in soybean（Glycine max）.The Co Fe₂O₄ nanozyme was also revealed to effectively reduce the concentration of ROS in the nodule by 56.6%,creating a superior environment for the proliferation of rhizobia and forming more effective nodules（larger nodules for an increase of 45.6%in the number of parasitic rhizobia）.Furthermore,the Co Fe₂O₄ nanozyme was shown to act as a synergist of leghemoglobin and increase its accumulation by 45.9%,where the high concentration of leghemoglobin in nodular cells can create a relatively hypoxic environment and protect nitrogenase,thus achieving a quantitative leap in nitrogen fixation capacity and simultaneously increasing the soybean photosynthesis by 67.2%.Our study has demonstrated that the Co Fe₂O₄ nanozyme can efficiently regulate the intracellular ROS metabolism and serve as a promising strategy for enhancing symbiotic nitrogen fixation.3.Novel approach to enhance Bradyrhizobium diazoefficiens nodulation through continuous induction of ROS by manganese ferrite nanomaterials in soybeanin this study,we applied manganese ferrite nanoparticles（MF-NPs）with sustainable diatomic catalysis to produce reactive oxygen species（ROS）,thus regulating the nodulation pathway and increasing the number of nodules in soybean（Glycine max）,eventually enhancing symbiotic nitrogen fixation.Symbiosis cultivation of MF-NPs and soybean plants resulted in 50.85%and 61.4%increase in nodule weight and number,respectively,thus inducing a 151.36%nitrogen fixation efficiency increase,finally leading to a 25.70%biomass accumulation increase despite no substantial effect on the nitrogenase activity per unit.Transcriptome sequencing analysis showed that of 31 DEGs related to soybean nodulation were upregulated in late rhizobium inoculation stage（12 d）,indicating that the increase of nodules was derived from nodule-related genes（Nod-R）continuous inductions by MF-NPs.Our results indicated that the nodule number could be effectively increased by extending the nodulation period without threatening the vegetative growth of plants or triggering the autoregulation of nodulation（AON）pathway.This study provides an effective strategy for induction of super-conventional nodulation.