Molecular Mechanism Underlying Multi-walled Carbon Nanotubes And Magnesium Hydride-alleviated Copper Toxicity In Alfalfa

Although Copper（Cu）plays essential roles in plants,excess Cu can cause phytotoxicity via deactivating important proteins/enzymes related to key metabolism.To cope with agricultural production and food security problems caused by Cu stress,nanotechnology might be an important driver for the sustainable agriculture,including the phytoremediation of metal contaminated soils.However,whether or how multi-walled carbon nanotubes（MWCNTs）influence excess Cu-caused phytotoxicity is still elusive.In addition,the development of hydrogen agriculture has opened up a new direction to deal with abiotic stresses in plants.Existing studies have shown that hydrogen（H2）has an important role in a variety of abiotic stresses response.With the increasing application of the new hydrogen storage material magnesium hydride（MgH2）,it remains unclear whether MgH2 can play a role in enhancing copper tolerance in plants.In response to solve the above problems,a series of experiments were carried out using alfalfa as research material.The results are as follows:Low concentration of MWCNTs（5 mg/L）can effectively relieve copper stress in alfalfa.Electron microscopy was used to observe that MWCNTs could penetrate the cell wall of alfalfa root tissue into the cell,regulate the expression of heavy metal transporting ATPase5（HMA5）genes,and reduce the absorption of copper ions.At the same time,MWCNTs treatment reduced the level of lipid peroxidation in the root of alfalfa and reduced the content of endogenous thiobarbituric acid reactant（TBARS）and hydrogen peroxide（H2O2）.MWCNTs control related antioxidant enzyme activities and corresponding transcripts,and restore redox balance.MWCNTs increase the levels of non-protein thiols（NPTs）,chelate excess Cu2+,and significantly increase the expression of genes related to cell wall biosynthesis and cell wall polysaccharide modification in the alfalfa root system,and promote cell wall capture,thereby maintaining more in the cell wall and vacuole Cu2+.In addition,MWCNTs can partially restore the absorption and distribution of nutrients disturbed by copper poisoning.The new high-efficiency magnesium-based hydrogen storage material MgH2 has stable hydrogen release ability.Both 0.01 g/L MgH2 solution and nitric oxide（NO）donor nitroprus（SNP）can significantly reduce the absorption of Cu2+ by alfalfa root tissue and promote the synthesis of endogenous NO to enhance the antioxidant capacity of alfalfa,and reduces the levels of ROS and TBARS to effectively alleviate the inhibitory effect of alfalfa root growth under Cu stress.At the same time,the above changes can be significantly affected by the NO scavenger PTIO.In addition,the nitrate reductase（NR）inhibitor tungstate can reverse the alleviating effect of MgH2 on Cu toxicity in alfalfa,suggesting that NR is the key enzyme to MgH2 regulating endogenous NO synthesis.The above results show that:MWCNTs and new high-efficiency magnesium-based hydrogen storage material MgH2 can alleviate the growth inhibition of alfalfa under Cu poisoning.Our results thus provide a theoretical basis for further exploration of plant growth regulators.