| With the large application of fossil fuels such as coal and oil in industrial and agricultural production,transportation and daily life,polycyclic aromatic hydrocarbons(PAHs)have been attracting more attention to all countries in the world.The environmental behavior and biological effects of PAHs are well documented.However,there is little information underlying the reponse mechanism of PAH accumulation in plant leaves.In this study,wheat was used as experimental material of crops,and phenanthrene was employed as the model of PAHs pollutants.The responses to phenanthrene accumulation in wheat leaves were observed.Meanwhile,the methods of phytophysiology and plant biochemistry,plant molecule,proteomics,bioinformatics and other biological techniques were applied to elucidate the physiological and biochemical mechanism of wheat leaf responses under the PAH treatment and to shed light on the growth and development of chloroplasts exposed to PAHs,together with the effects on intracellular organelles.The detailed results are as follows:1.Under the 1.0 and 0.5 mg L'1 phenanthrene treatments for 7 and 9 days respectively,the wheat leaves would be chlorosis at the base.We tested the changes in the concentrations of the precursors in chlorophyll metabolic pathways,such as glutamate,5-aminolevulinic acid,uroporphyrinogen III,Protoporphyrin IX,Mg-protoporphyrin IX and protochlorophyllide.With the accumulation of phenanthrene,the concentrations of glutamate,5-aminolevulinic acid,uroporphyrinogen III,protoporphyrin IX,Mg-protoporphyrin IX and protochlorophyllide increased while the concentrations of porphobilinogen and Chlorophyll b decreased.Also,chlorophyll a content rose initially and then declined.Uroporphyrinogen III synthase and chlorophyllase were activated and porphobilinogen deaminase activity declined in the treatments.Based on the mathematical analysis,the reason for the chlorophyll decrease under 0.5 and 1.0 mg L-1 phenanthrene treatments was that the chlorophyll degradation rate(2.38 and 2.79 mg g-1 min-1)was greater than the chlorophyll synthesis rate(-0.022 and-0.018 mg g-1 min-1)on day 9.We also found that under the phenanthrene treatment,the leaf moisture content increased by 0.91-2.03%;2.Through the observation of transmission electron microscope(TEM),we found that the leaf subcellular structure would turn plasmolysis and distorted and organelles(such as mitochondria and lysosomes)disappeared during the accumulation of phenanthrene.Osmiophilic particles,the biomarker of oxidative stress,occurred and increased with the time and dose of phenanthrene exposed.Then we detected the contents of reactive oxygen species(ROS)and hydrogen peroxide(H2O2)in leaves.Both ROS and H2O2 showed an increase trend and the proportion of H2O2 to ROS increased during the experimental period.Therefore,it is suggeted that H2O2 accumulation leads to cell death under PAH treatment.Furthermore,we detected the changes in nine antioxidants(ascorbic acid,glutathione,polyamine,a-tocopherol,carotenoids,catalase,ascorbate peroxidase,superoxide dismutase and glutathione-S-transferase).Cluster analysis,Pearson correlation analysis,principal component analysis and redundancy analysis were employed.It is indicated that carotenoid and superoxide dismutase are the two most effective antioxidants to scavenge superoxide radicals,and glutathione-S-transferase is a potentially useful antioxidant.Moreover,under the higher concentrations of phenanthrene,ascorbate-glutathione cycle would also turn active.3.Through the observation of TEM,we found that the chloroplasts deformed and began to dissolve under the phenanthrene treatment.The chloroplast would distort from ellipsoid to sphere.As the protein is an important component of the structure and function of living organisms,in order to study this deformation,we used iTRAQ(isobaric tags for relative and absolute quantitation)proteomics to analyze the changes of chloroplast proteins under phenanthrene treatment.A total of 517 proteins were detected by LC-MS/MS in phenanthrene-treated chloroplasts,261 of which were up-regulated.Thylakoid proteins,the structure of chloroplast,were down-regulated in proteomics and the related genes were positively expressed in real-time PCR under phenanthrene treatment,suggesting that the thylakoid destruction is the reason for chloroplast deformation.The proteins related with envelope and stroma were up-regulated,which would be the reason why the chloroplast still kept stable shape after the destruction of the thylakoid.In addition,based on proteomic results,we also concluded that 1)the reduction in the efficiency of the electron transfer chain and the functions of light harvesting complexs was the main change in light reaction;2)the decline of glycolysis was the main cause for respiration reduction;and 3)proteins related with proline,tryptophan and glycine became up-regulated,while those involved in histidine,threonine and arginine turned down-regulated.4.Carotenoid is an active antioxidant under phenanthrene treatments in wheat leaf.Thus,in this study,carotenoid was added as exogenous additive to phenanthrene-treated wheat hydroponic system.It was observed that the addition of exogenous carotenoids could reduce the toxicity of wheat leaf phenanthrene-caused.Compared with the phenanthrene treatment group,the third leaf developed and the chlorosis symptom was not found in carotenoid-added group.Although the relative biomass and relative root length of carotenoid-added groups were lower than those of the control group,both of them were better than the phenanthrene treated groups.At the same time,we also tested chlorophyll a and b contents,antioxidant enzyme,lipid peroxidation and amino acid contents to verify the efficacy of exogenous added carotenoids.The results showed that exogenous addition of carotenoids would make what leaf recover from the phenanthrene accumulation.The content of chlorophyll in wheat leaves increased with the increase in carotenoids added compared with that of phenanthrene treatment group.The activity of SOD and the content of lipid peroxidation in carotenoid-added groups were lower than those in phenanthrene-treated group.In the leaves treated with phenanthrene and 20 mg L-1 carotenoid,amino acid contents would return to normal levels.Therefore,it is confirmed that the exogenous addition of carotenoids could reduce and alleviate the toxic effects of wheat leaves exposed to phenanthrene in the growth and development.In summary,the present study revealed for the first time that the accumulation of phenanthrene would accelerate the degradation of chlorophyll and increased moisture content,then leading to leaf chlorosis in wheat leaves.At the same time,chloroplast,the important carrier of leaf color,would become spherical under phenanthrene treatments.Based on the proteomics and molecular biology methods,it was found that thylakoid gene expression was inhibited,and the amount of related proteins decreased when exposed to phenanthrene.This study also firstly applied mathematical methods to analyze the contribution of antioxidants in wheat leaves and verified that the exogenous addition of carotenoids is an effective way to alleviate the toxicity of PAHs to plants.Our results provide not only novel insights into the mechanisms on response of plant leaves to PAH accumulation,but also the help to optimize strategies for crop safety and phytoremediation of PAH-contaminated soil/water. |
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