The Biological Effect And Mechanism Of Carbon Nanomaterials On Rhizobium-legume Symbiosis In Lotus Japonicus

The problem of nitrogen fertilizer in agricultural production has long been the major strategic issues,waiting to be solved by scientists all over the world.Due to the increasing demand for nitrogen to increase grain yield and the serious environmental pollution caused by excessive use of nitrogen fertilizers,the study of rhizobium-legume symbiosis system has become a research hotspot in the biological field.Enhancing the ability of symbiotic nitrogen fixation can improve the use efficiency of nitrogen fertilizers in agricultural production,which is important for the sustainable development of agriculture.Currently,nanomaterials have been extensively studied and applied in the biological field due to the rapid development of nanotechnology,and some studies have reported that carbon nanomaterials(CNMs)can affect the growth and biochemical reactions in plants,leading to the emergence of nano-biological effects.Nevertheless,there have been few reports about the study of CNMs in rhizobium-legume symbiosis system.Therefore,this study was aimed to investigate the effects of CNMs on the symbiotic nitrogen fixation of the legume,which could enrich our knowledge about the biological effects of CNMs.Moreover,it is expected that the application potentials of CNMs could help to develop new strategies to promote nitrogen fixation in rhizobium-legume symbiosis system,which will provide important implications for the wide use of CNMs in plant biotechnology.Through chemical characterization,data comparison analysis,electron microscope observation and gene expression analysis,we here investigated the effects of three important kinds of CNMs(o-SWCNTs,o-MWCNTs and GO)on the development and physiology of symbiotic nitrogen fixation in Lotus japonicus and discussed the molecular mechanism in depth.1.Through examining the growth curve and colony-forming units of rhizobia,we studied the effects of o-SWCNTs,o-MWCNTs and GO on the growth of rhizobia.The results showed that o-MWCNTs have no obvious effects on the growth activity of rhizobia.In the treatments with different concentrations of o-MWCNTs,only slight or no significant influence was observed on the bacterial growth.But the bacterial growth was obviously inhibited by 50 ?g/m L GO or 500 ?g/m L o-SWCNTs,suggesting that the toxicity of GO or o-SWCNTs to rhizobia is higher than that of o-MWCNTs.2.Under the autogenic condition without the inoculation of rhizobia,we studied the effects of o-SWCNTs,o-MWCNTs and GO on the growth and development of Lotus japonicus.The results showed that plant seedlings treated with o-MWCNTs exhibited a significantly higher elongation of stem,but the growth of roots under the same treatment was promoted;on the other hand,the growth of root was obviously inhibited by o-SWCNTs or GO treatment,resulting in decreased root length.Further investigation showed that o-MWCNTs at a series of gradient concentrations still had a promoting effect on the stem of the plants cultured under different conditions,suggesting that o-MWCNTs can promote the growth and development of Lotus japonicas to a certain extent.3.Under the symbiotic condition without the inoculation of rhizobia,we focused on studying the effect of CNMs on the rhizobium-legume symbiosis system.The preliminary statistical analysis revealed that the amount of nodules on the roots treated with o-MWCNTs was increased compared with the roots treated with o-SWCNTs or GO at 14 dpi.Further investigation showed that from 14 dpi to 28 dpi,plants treated by o-MWCNTs started to possess obviously more nodules(about 40% more than that of the control).At the same time,nitrogen fixation was enhanced in the root nodules exposed to o-MWCNTs at 28 dpi.Based on the synthetic effect of the above two aspects,o-MWCNTs finally stimulates the development of plant and results in a dramatic increase in vegetative biomass.These results indicate that o-MWCNTs have positive effects on nodulation and biological nitrogen fixation.4.Qualitative analysis of o-MWCNTs in plant cells(mainly root and nodule cells)was performed by Raman spectroscopy and additionally by transmission electron microscopy(TEM).The specific D band and G band of carbon nanotubes were observed in the detection of Raman spectroscopy,indicating the presence of o-MWCNTs in root and nodule cells.Then,TEM analysis was performed to validate this speculation.These results suggest that after contact with the plants,o-MWCNTs can penetrate the cell wall,pierce through the cell membrane,and are finally transferred into the cytoplasm.5.Based on the model signaling pathway of nodulation in Lotus japonicus,the expression of twelve genes essential for nodulation was detected by quantitative real-time PCR method in the roots treated by o-MWCNTs.The results showed that in different periods,the expression of some genes was up-regulated after o-MWCNTs treatment,particularly under the symbiosis condition.The expression of NIN was significantly elevated compared with that of the control,which is consistent with the increase of nodules in MWCNTs-treated roots.In addition,it was found that the expression of relevant nitrogenase genes was down-regulated obviously,which indirectly demonstrates the feedback inhibition caused by the enhancement of nitrogenase by o-MWCNTs.Our results indicate that o-MWCNTs can directly or indirectly regulate the rhizobium-legume symbiosis system at the molecular level in Lotus japonicus.