Study On Exogenous H₂S Alleviating Toxicity Of CuO NPs In Tomato Seedlings

Copper Oxide Nanoparticles(CuO NPs)are important semiconductor metal oxide.Its large-scale use will inevitably enter the environment.Excessive CuO NPs accumulated in the environment will enter the organism along with the food chain/food web,which will cause harm to human health.Hydrogen sulfide(H2S)is the third gas signal molecule after CO and NO.Exogenous H2S plays an important role in plant heavy metal stress.However,the mechanism of H2S regulating plant response to CuO NPs stress is still unclear.Therefore,in this study,tomato,a model plant,was used as the test organism,CuO NPs as the pollutant,and exogenous H2S as the signal regulator.The mechanism of hydrogen sulfide alleviating the toxicity of CuO NPs in tomato seedlings was studied from the aspects of physiological trend of tomato seedling growth,distribution of copper content,changes of cell wall components,and antioxidant system.The main research contents and results are as follows(1)The growth index of tomato seedling(root length,plant height,leaf area,fresh weight,dry weight and root cell length)were observed and measured,and the physiological indexes(photosynthetic pigment content,MDA content and root cell activity)were measured and analyzed.It was found that the inhibition effect of tomato growth increased with the increase of CuO NPs concentration.The high concentration(100 mg/L)CuO NPs significantly inhibited the growth of tomato seedlings and decreased the accumulation of biomass.The NaHS of physiological concentration could enhance the activity of tomato root cells and photosynthesis intensity under the stress of CuO NPs,which further promoted the growth of tomato seedling plants and biomass accumulation under the stress of CuO NPs,thus alleviating its toxic effect.(2)By measuring the Cu distribution in tomato seedling tissue,Cu distribution in subcellular components,cell wall polysaccharide and cell wall polysaccharide,it was found that Cu was mainly distributed in tomato seedling root system and mainly concentrated in cell wall(80.6%-95.2%).According to the results of Cu content in cell wall,pectin was the main part of Cu accumulation,alkali soluble pectin(NSP),it plays an important role in Cu accumulation.H2S reduced the Cu content in the NSP under CuO NPs stress by 24.28%and 33.32%,respectively.the decrease was the most significant,followed by the CSP,Cu content was decreased by 7.77%and 27.51%,respectively..Besides pectin,a certain amount of Cu was accumulated in hemicellulose,while H2S significantly reduced Cu content(19.66%-27.51%)in hemicellulose 1(HC1).The effect of H2S on Cu content in cellulose is not significant,which may be due to the less Cu content accumulated in cellulose than pectin and hemicellulose.The results of ion chromatography(ICS)analysis showed that the content of pectin,hemicellulose and cellulose was significantly increased by the addition of exogenous H2S.The results of ICS analysis showed that the basic monosaccharide content,such as glucose,galactose and arabinose,which constituted the cell wall cellulose and pectin,increased significantly under the action of H2S.In addition,the accumulation of lignin was significantly decreased and the synthesis of callose was increased with H2S under high concentration of CuO NPs.In conclusion,we speculate that exogenous H2S may promote the cell wall structure of tomato to be more compact and complete,thus preventing the external CuO NPs from entering tomato body.(3)The results showed that the stress of CuO NPs made tomato seedlings accumulate excessive reactive oxygen species(ROS)(73.66-123.74%),inhibit the activity of antioxidant enzymes and the synthesis of antioxidants.The results showed that exogenous H2S could significantly activate the activities of antioxidant enzymes such as SOD,APX,POD and GR in vivo and in vitro.The improvement of these enzymes was beneficial to the removal of ROS,and thus alleviated oxidative stress.The increase of these enzymes depends on the up regulation of the expression level of the related genes.Therefore,H2S can regulate the expression level of antioxidant gene and activate antioxidant enzymes in tomato seedlings,thus play its own role in scavenging ROS.In addition,the vitro sulfhydrylation analysis showed that the antioxidant enzymes existed sulfuration.The sulfhydrylation sites of the protein SlCAT1 were C234,the sulfhydrylation sites of SlcAPX1 were C168,and the thiol sites of SlPOD5 were located in C46 and C61,which were both on the surface of protein and the active domain of enzyme,which could be easily modified by S-thiomercaptosylation.Further research found that NaHS induced S-thiothiolation on CAT,cAPX and POD protein in a dose-dependent manner,that is,with the increase of NaHS concentration of H2S donor,the level of thiol of recombinant protein was enhanced.