| With the development of industrialization and urbanization,there has been a significant increase in the levels of heavy metals in the environment,which has caused great concern worldwide.Heavy metals are difficult to remove from the environment because they are present in different chemical forms.Among other things,copper contamination causes inhibition of plant growth and affects various physiological processes in plants,causing impaired photosynthesis as well as oxidative damage to cells.Phytoremediation is widely used in the remediation of polluted environments because it is green and cost effective.A large number of aquatic plants have been shown to phytoremediate many types of wastewater.Among them,Lemna minor L.has the characteristics of rapid reproduction,wide distribution and enrichment and uptake of some heavy metals that are not easily removed at low concentrations.However,Lemna minor L.still shows obvious damage symptoms when it encounters heavy metal stress,and several studies have shown that salicylic acid can reduce the toxicity of heavy metals on Lemna minor L.to a certain extent.Therefore,in this study,we analyzed the changes in the growth performance,photosynthesis,tolerance and removal efficacy of Lemna minor L.under copper stress by the application of salicylic acid,conducted screening for the optimal strengthening conditions,and investigated the resistance and strengthening mechanisms of Lemna minor L.under copper stress from the perspective of metabolomics.In the study on the effects of salicylic acid on the growth and photosynthesis of Lemna minor L.under copper stress showed that the application of 0.05 m M,0.5 m M and 1 m M salicylic acid improved the yellowing of Lemna minor L.leaves and reduced the trend of reduction in leaf area,fresh weight and leaf number of Lemna minor L.,and that 0.5 m M salicylic acid was more effective under high copper stress compared to other concentration conditions.The leaf area was higher than that of the control under 5 mg/L,10 mg/L,and 30 mg/L copper stress,and was greater than 0.01cm~2.Fresh weight was about 43%heavier than that of the control under 20 mg/L copper stress.In addition,the application of different concentrations of salicylic acid reduced the reduction of photosynthetic pigments in Lemna minor L.leaves to different degrees,and the reinforcement effect was more pronounced under 0.5 m M salicylic acid.For example,the chlorophyll a and chlorophyll b contents in the leaves of Lemna minor L.were significantly higher than the control under 0-10 mg/L copper stress,the most significant performance was that the chlorophyll a and chlorophyll b contents were respectively 44.14%and 23.42%higher than the control group under10 mg/L copper stress,indicating that 0.5 m M salicylic acid increased the photosynthetic efficiency of the leaves of Lemna minor L.The carotenoid concentration was also higher than the control within 0-10 mg/L copper stress,as shown by the leaf carotenoid concentrations were 19.47%,1.39%and 23.12%higher than the control under 0 mg/L,0.5 mg/L and 10 mg/L copper stress,respectively,indicating that the antioxidant capacity of the leaves was improved.In the study on the effect of salicylic acid on the tolerance and removal efficiency of Lemna minor L.leaves under copper stress.The addition of 0.5 m M salicylic acid significantly reduced the inhibitory effect of copper stress on the growth of Lemna minor L.leaves,and the strengthening effect was most significant at 0.5,10and 20 mg/L copper stress,and the growth inhibition rate was lower than 64.69%,62.59%and 62.70%respectively compared to the control group.The application of0.5 m M salicylic acid improved the tolerance of the leaves to copper stress,mainly in the absence of copper application and 10-30 mg/L copper stress,the application of 0.5m M salicylic acid resulted in significantly lower malondialdehyde concentrations than the control,and reduced the concentration by 18.52%,31.75%,6.54%and 0.04%,respectively.Also electrolyte leakage was significantly reduced compared to the control group and was reduced by 28.65%,46.58%,30.60%,14.17%,13.81%and7.31%under 0-30 mg/L copper stress in the treatment group with 0.5 m M SA applied.Therefore,it can be inferred that salicylic acid activated the antioxidant system in the leaves of Lemna minor L.to reduce oxidative damage.In the treatment group with 0.5m M salicylic acid,the removal of copper from the water was significantly increased by 9.71%,17.07%and 11.95%under 5 mg/L,20 mg/L and 30 mg/L copper stress,respectively,indicating that the uptake of copper in the water by the leaves of Lemna minor L.was enhanced,and the most significant enhancement was achieved under 20mg/L copper stress.In the study of metabolomic analysis of Lemna minor L.under copper stress by the application of salicylic acid:data pre-processing and metabolite content statistics were first performed,and errors in sample collection,preparation and measurement were eliminated by evaluating PCA plots of Lemna minor L.leaf samples and standardized corrections for relative metabolite content.Heat map clustering results indicated that metabolites in each treatment group differed less within groups and more between groups,and the samples could be used to investigate variability between different subgroups.Unsupervised principal component analysis and supervised least squares discriminant analysis further indicated that the four subgroups differed significantly in metabolite composition structure and had significantly different metabolites between treatment groups.Screened by p-values<0.05,and the absolute value of the fold change>2 to Lemna minor L.leaves under control and 0.5 m M salicylic acid conditions.19 differential metabolites were significantly down-regulated and 16 differential metabolites were significantly up-regulated in the positive ion mode,6 differential metabolites were significantly up-regulated and 3 differential metabolites were significantly down-regulated in the negative ion mode beteween the control and 0.5 m M salicylic acid conditions.Between 20 mg/L Cu concentration and 0.5 m M salicylic acid+20 mg/L Cu concentration showed 49 differential metabolites significant up-regulated and 97differential metabolites significant down-regulated in the positive ion mode,and significant up-regulation and down-regulation of 6 differential metabolites in the negative ion mode.Combining ORA enrichment analysis and topological analysis,and considering the top ten overlapping KEGG metabolic pathways of Impact in positive and negative ion mode,it was found that the alanine,aspartate and glutamate metabolic pathways and arginine biosynthesis pathways were overlapping in Lemna minor L.leaves under control and 0.5 m M salicylic acid conditions,and under 20mg/L and 0.5 m M salicylic acid+20 mg/L Cu concentrations.The metabolic pathways of alanine,aspartate and glutamate biosynthesis and arginine biosynthesis were overlapping pathways.The amino acids and organic acids,such as L-glutamic acid,L-glutamine,L-aspartic acid,pyruvic acid,2-oxoglutaric acid,N-acetyl ornithine,2-oxoglutamic acid,N-acetyl-L-glutamic acid,L-alanine and galacturonic succinate,were significantly up-regulated in both pathways after the application of0.5 m M salicylic acid,and these substances could improve the antioxidant activity of the leaves of Lemna minor L.and promote the uptake of copper ions in water,thus strengthening the resistance mechanisms of Lemna minor L.to copper. |
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