| With the improvement of China’s industrial,the over-exploitation and smelting of copper mines,and the improper use of copper-containing pesticides and fertilizers have led to increasingly serious heavy metal pollution.Heavy metals have the characteristics of refractory degradation,mutagenicity and cytotoxicity.When heavy metals enter soil and water,they will directly or indirectly affect the soil and water environment through bioaccumulation and biomagnification,and accumulate in animals and plants through the food chain,furthermore cause damage to human organs and even death.Compared with traditional physical and chemical remediation techniques,phytoremediation has the characteristics of low cost,low secondary pollution and large remediation volume.Epipremnum aureum is one of the common aquatic plants,which has the characteristics of high survival rate and large biomass.It is a common plant material for remediating polluted water.The ability of plants to remediate heavy metal-polluted environments mainly depends on their resistance mechanisms to heavy metals.However,phytoremediation is easily affected by heavy metals,resulting in delayed plant growth and low bioavailability,which restricts the transport and absorption of copper by plants.Therefore,in this study,the growth physiological parameter,heavy metal content and copper removal rate of E.aureum under copper stress were measured,and the physiological resistance of E.aureum to copper and the stress effect of copper on E.aureum were analyzed.Metabolomics technology was used to study the response mechanism of E.aureum to copper at the molecular level,and to explore the potential factors that can enhance the absorption and transport efficiency of E.aureum to copper.Based on the results of metabolomic analysis,the response surface methodology was used to optimize the copper repair effect,in order to find the best optimized conditions that can improve the copper removal rate of plant,and verify the optimization results through practical application,so as to provide support for the application of phytoremediation technology in actual copper-contaminated sites.The growth physiological and repair efficiency of plant under copper stress were studied: under the treatment of low concentration of copper（200 mg/L）,the root length of E.aureum increased by 29.06% compared with the control group,indicating that low concentration of copper has an effect on root elongation of E.aureum.Thus,low concentration of copper promoted the root elongation of plant,but the effect of promoting the growth and development of stems and leaves was not obvious.When the copper concentration was 400-500 mg/L,the chlorosis of E.aureum leaves were aggravated and root elongation was inhibited by copper stress.The increase of copper concentration led to the increased degree of lipid peroxidation in E.aureum,which impaired the permeability of cell membrane.At copper concentration of 500 mg/L,MDA increased by 37.65% compared with the control group.In addition,the effect of copper on plant photosynthesis led to the decrease of chlorophyll a and chlorophyll b content.When the copper concentration was 500 mg/L,chlorophyll a and b decreased by 17.17% and 46.15%,respectively,compared with the control group.However,the carotenoid content increased with the increase of copper concentration,which showed that carotenoid increased by 8.17% under 500 mg/L copper treatment compared with the control group.The content of carotene scavenged a large number of free radicals produced by oxidative stress and slowed down the toxic effect of copper on E.aureum.The content of proline and hydroxyproline in E.aureum leaves increased with the increase of copper concentration.When the copper concentration was 500mg/L,the proline content of E.aureum leaves increased by 48.35% compared with the control group,and the hydroxyproline content in the same copper concentration was 3 times that of the control group.E.aureum regulated the osmotic imbalance caused by copper and promoted the thickening of the cell wall by increasing the content of proline and hydroxyproline,thereby resisting the toxicity of copper stress to plants.When the copper concentration was 500 mg/L,the copper content in the roots was 11 times that in the stems and leaves.E.aureum inhibited the transport of copper to the stem and leaves to resist copper stress,thereby improving the tolerance of E.aureum to copper.Metabolomics was used to study the response mechanism of E.aureum to copper at the molecular level: There were 586 significantly differential metabolites in E.aureum leaves under copper stress,of which 256 significantly up-regulated differential metabolites and 330 significantly down-regulated differential metabolites,indicating that the effect of copper stress on E.aureum resulted in the down-regulation of most differential metabolites in leaf cells.Ten key metabolic pathways were screened by enrichment analysis and topology analysis,among which the differential metabolite expression in the arginine and proline metabolism pathway,the indole alkaloid biosynthesis pathway and the brassinosteroid biosynthesis pathway were up-regulated,indicating that E.aureum may respond to the toxicity of copper through the above three pathways.Under copper stress,E.aureum improved the resistance of plants to copper by promoting the conversion of proline to hydroxyproline.Hydroxyproline promoted the synthesis of an important structural protein in the plant cell wall,the hydroxyproline-rich glycoprotein.This structural protein promoted the thickening of the plant cell wall,thereby inhibiting the entry of copper into plant cells to damage organelles.E.aureum protected against copper-induced oxidative stress by promoting the synthesis of indole alkaloids and scavenging intracellular reactive oxygen species.E.aureum promoted plant growth and development by promoting the biosynthesis of brassinosteroid,thereby improving the absorption and transport efficiency of E.aureum on copper.In addition,brassinosteroid promoted antioxidant enzyme activity to defend against oxidative damage caused by copper.Optimization of the repair efficiency of E.aureum under copper stress by response surface method was studied: based on the results of metabolomics,salicylic acid（SA）,nitroprusside SNP（NO donor）and brassinosteroid phytohormone 2,4-EBL were exogenous enhancer to optimize the repair efficiency.The complete randomized design showed that the copper removal rate reached the maximum when the concentration of SA was 50 μM,the concentration of SNP was 50 μM and the concentration of 2,4-EBL was 1 μM.According to the complete randomized design results,the response surface optimization test was carried out with SA concentration（25,50 and 100 μM）,SNP concentration（25,50 and 100 μM）and 2,4-EBL concentration（0.5,1 and 2 μM）as the influencing factors,and copper removal rate as the response value.The results showed that when the concentration of SA was 36.50μM,the concentration of SNP was 100 μM and the concentration of 2,4-EBL was 2μM,the copper removal rate reached the theoretical optimum value of 85.84%.The theoretical optimal conditions obtained by the response surface were verified,and the copper removal rate was 84.39%,which was 30.19% higher than that before optimization,indicating that the optimal conditions obtained by the response surface could be applied in actual repair. |
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