Cytological Study Of Plant Salt Tolerance Regulated By Engineered Nanomaterials

Land salinization has received great attention,threatening the soil environment and human food production and security.Plants possess a series of responses to resist salt stress,but with little success,and engineered nanomaterials（ENMs）have great potential in enhancing plant salt tolerance.ENMs can enhance photosynthesis and anti-oxidative capacity.However,the mechanism of ENMs enhancing plant salt tolerance is still unclear,especially the ENMs-mediated salt tolerance signaling network,initial responses and molecular mechanisms need to be studied.In this study,tobacco（Nicotiana tabacum L.）BY-2 cell suspensions,cultured in MS（Murashige&Skoog）medium,were used as experimental materials and exposed to nano-cerium oxide（nCeO₂）,nitrogen-doped carbon dots（N-CDs）,nano-titanium dioxide（nTiO₂）,nano-selenium（nSe）,nano manganese ferrite（nMnFe₂O₄）,nano-silicon dioxide（nSiO₂）,carbon dots（N-CDs）and nano-iron oxides（n Fe₂O₃）,respectively.The cell viability of control group（CK,1/2 MS medium）,salt-treated group（S,1/2 MS medium+100 mM NaCl）and ENMs-treated group exposed to salt stress（S+ENMs,1/2 MS medium+100 mM NaCl+ENMs）was determined to identify the optimal ENMs types and exposure dose.On this basis,two ENMs,nCeO₂ and N-CDs,were selected to reveal the initial response（early-stage signal）and adaptive changes of plants exposed to ENMs,enhancing the salt tolerance of BY-2 cells.In addition,nCeO₂,N-CDs,nTiO₂ and nSe were selected to study the metabolic regulation mechanisms of different ENMs enhancing the salt tolerance of plants.The main findings are as follows:（1）Salt-stressed BY-2 cells were exposed to different types and concentrations of ENMs,results showed that 0.1 mgL^-1 nCeO₂ was the optimal ENMs for improving the salt tolerance of plants（the cell viability increased significantly,6 h-72 h,7.6%-19.4%）,0.5 mgL^-1 N-CDs（24 h,8.8%）,0.1 mgL^-1 nTiO₂（6 h,8.1%）,0.5 mgL^-1 nSe（12 h,13.6%）and 0.5 mgL^-1 CDs（4 h,3.4%）also enhanced the tolerance to salt stress,while n Mn Fe₂O₄,nSiO₂ and n Fe₂O₃ had no significant effect on plant salt tolerance.（2）In salt-stressed BY-2 cells exposed to 0.1 mgL^-1 nCeO₂,the cell biomass increased significantly（4 h,19.3%;72 h,26.5%）compared with that of S treatment,the net influx of Ca²⁺（4 h,293.6%）,Ca²⁺content（4 h,73.8%）and Na⁺net efflux（6 h,191.2%）significantly increased in the early stage of ENMs exposure,and the Na⁺content（12 h,7.8%）significantly decreased;The expression of gene IPUT1 encoding glycosyl-inositol phosphateceramide（GIPC）which senses Na⁺and regulates Ca²⁺,was significantly enhanced（4 h,5.2-fold）,and the expression of genes related to salt over sensitive（SOS）-SOS3,SOS2 and SOS1 was also significantly up-regulated（6 h,4.4-5.3 fold）.Therefore,nCeO₂ could enhance the salt tolerance of BY-2 cells through the GIPC（Na⁺sensing）-SOS pathway（Na⁺efflux）.In salt-stressed BY-2 cells exposed to 0.5 mgL^-1 N-CDs,the biomass remained unchanged,the net efflux of Na⁺and the net influx of Ca²⁺did not change significantly,but the net influx of K⁺increased significantly（24 h,297.8%）,the Na⁺content was significantly decreased（48 h,24.5%）.The expression of IPUT1,SOS3,SOS2 and SOS1 was significantly up-regulated compared with those of the S treatment group,but the up-regulation appeared during the late stage of exposure.The expression of Na⁺/H⁺transporter-related gene NHX1 was significantly up-regulated（6 h,7.8-fold）,thus,N-CDs could enhance the salt tolerance of BY-2 cells via the K⁺signaling-Na⁺compartmentation.（3）The metabolite composition of the S+ENMs（nCeO₂,N-CDs,nTiO₂,or nSe）treatment group was significantly changed compared with the S treatment group.In S+nCeO₂ treatment,the contents of fatty acids and conjugates were significantly increased,thereby strengthening cell membranes and the Na⁺sensing process of GIPCs.InS+N-CDs treatment,the contents of amino acids,carbohydrates and TCA acids were significantly increased,thereby maintaining cell osmotic balance and energy metabolism.In S+nTiO₂ treatment,fatty acids and conjugates,TCA acid,amines,monosaccharides,pyrimidines and purines and other small molecular substances significantly increased,these substances help to maintain the physiological state of cells under salt stress.In S+nSe treatment,the contents of amino acids and indole substances increased significantly,in addition,the increase in the content of regulatory substances such as proline and salicylic acid enhanced the salt tolerance of BY-2 cells.The tricarboxylic acid cycle and nucleotide metabolism may play an important role in the regulation of Na⁺efflux by ENMs.