Study On The Signal Transduction Network Of Tobacco Cell Growth Promoted By Engineered Nanomaterials

The increase in grain yield with high efficiency and safety is one of the major challenges in the 21^stcentury.The low bioavailability and high loss rate of traditional pesticides and fertilizers not only cause economic losses,but also lead to pollution of soil and water.Studies have found that engineered nanomaterials（ENMs）can significantly promote the growth of crops.Nano-fertilizers and nano-pesticides not only have high bioavailability,but their slow-release can also benefit plants for a long time,which will reduce cost and increase yields,thereby achieving the goal of environmental protection.ENMs promote plant growth mainly by improving photosynthetic efficiency,increasing nutrient assimilation,and stimulating the production of phytohormone signaling molecules.Auxin（IAA）and cytokinin are important signaling molecules that regulate plant growth and development,especially cell expansion and cell division.However,how ENMs affect these two signaling molecules,and the signaling pathways they stimulate,are currently unclear.In this study,the effects of different types of ENMs on cell growth and signaling transduction were investigated by tobacco（Nicotiana tabacum L.）BY-2 suspension cells.Based on the established optimal ENMs exposure system for promoting cell growth,the underlying signaling molecular mechanisms have been revealed.The main conclusions are as follows:（1）The viability of BY-2 cells exposed to different ENMs（carbon dots（CDs）,nitrogen-doped carbon dots（N-CDs）,nano-silicon oxide（n Si O₂）,nano-selenium（n Se）,nano-titanium dioxide（n Ti O₂）,nano-iron oxide（n Fe₂O₃）,nano-manganese ferrite（nMnFe₂O₄）,nano-cerium oxide（n Ce O₂））was determined by triphenyltetrazolium chloride（TTC）,in order to determine the optimal exposure system for promoting cell growth.The results showed that 1 mg L^-1nMnFe₂O₄ and 0.1 mg L^-1 N-CDs significantly increased cell viability by 30.4%and 11.2%,respectively.Therefore,1 mg L^-1 nMnFe₂O₄ and 0.1 mg L^-1 N-CDs were selected for subsequent investigations.（2）The cell viability,cell morphology,biomass and nutrient uptake under exposure to 1mg L^-1 nMnFe₂O₄ or 0.1 mg L^-1 N-CDs were measured.There was no significant change in cell morphology after the treatment of the two nanomaterials,but the percentage of viable cells increased significantly according to propidium iodide（PI）and fluorescein diacetate（FDA）staining.The dry weight and fresh weight of cells increased by 10.0-48.2%.Both nanomaterials can enhance the nutrient assimilation of cells,nMnFe₂O₄ enhanced the uptake of Fe,Mn,K and Mg,while N-CDs promoted the uptake of K,P,Cu,Mn,Ca and Zn.（3）The cytokinin content was increased by 70.2%after nMnFe₂O₄ treatment for 6 h,while N-CDs significantly increased the content of IAA by 37.2%at 6 h.The net flux of IAA tested by Non-invasive micro-test technique（NMT）showed that the influx of IAA was significantly increased by 3.8-fold after N-CDs treatment for 48 h.The net efflux of H⁺increased by 75.7%after nMnFe₂O₄ treatment for 2 h,while H⁺efflux of cells exposed to N-CDs was not detected until 24 h.Correspondingly,the plasma membrane H⁺-ATPase activity was significantly increased by 23.9%after 4 h of nMnFe₂O₄ treatment.The net influx of K⁺was significantly increased by 2.7-fold after nMnFe₂O₄ treatment for 4 h,while N-CDs treatment had no significant change on the net K⁺influx of cells.（4）The time-dependent investigation of gene expression showed that both nMnFe₂O₄ and N-CDs treatment up-regulated the expression of cell division-related genes CYCB and MAPK.The response of cell division was faster and more significant under N-CDs treatment,and the expression of CYCB was significantly up-regulated by 5.9-fold at 2 h.Both nanomaterials upregulated the expression of auxin synthesis gene AMI1.The down-regulation of IAA9 by nMnFe₂O₄ at 4 h,initiated the expression of ARF1,HAK1,EXP at 2 h by 5.1,2.7,and 3.3-fold,respectively.N-CDs had relatively little effect on the expression of ARF1.Under exposure to N-CDs,the expression of HAK1 was up-regulated by 2.9-fold after 12 h,and that of EXP was up-regulated after 24 h.Accordingly,nMnFe₂O₄ can promote cell expansion by activating the SCF^TIR1/AFB-AUX/IAA signaling pathway,while N-CDs may activate the IAA-mediated ABP1signaling pathway to promote cell division.（5）Metabolomics analysis showed that nMnFe₂O₄ and N-CDs changed cellular metabolic composition in different ways.nMnFe₂O₄ increased the contents of tryptophan,valine,aspartic acid,and histidine,while N-CDs treatment only increased the content of proline and histidine.Both nanomaterials increased benzoic acids,indicating the increase of cell resistance.The metabolic pathways altered by nMnFe₂O₄ and N-CDs mainly involve sphingolipid metabolism,shikimic acid and phenylpropane metabolism,tryptophan metabolism,and arginine biosynthesis.The metabolic changes induced by nMnFe₂O₄ mainly involve cytokinin signal transduction,ATP synthesis,and cell expansion.The metabolic alternations induced by N-CDs treatment mainly involved in promoting cell division and enhancing cell resistance.This study elucidated the cascade of hormonal signals triggered by ENMs from a cellular perspective,contributing to the understanding of the specific mechanisms by which ENMs induce plant growth.The study provided a theoretical basis for the development and application of nano-agricultural technology to protect the environment.