| Plant root exudates play an important role in the process of phytoremediation as an emerging green and in-situ remediation technology using plants to absorb,accumulate,stabilize or volatilize contaminant from soil.They can be modifying the pH and Eh of the rhizosphere,chelating,complexing and depositing with heavy metals,altering the numbers and the activity of rhizoshperic microbes.Then change the chemical existence of heavy metals and increase or decrease their bio-availability.Plant root exudates have many types,in addition to the some certain compounds,whether other compounds play a role in the process of plant tolerate or accumulate the heavy metal,or whether these particular compounds play an main role,it is not clear at present.So,in this study,the mining ecotype of Sedum alfredii,a Cd hyperaccumultor and Pb accumulator native to China,with large biomass,rapid growth and asexual propagation,and the non-mining ecotype of Sedum alfredii,Sedum Sarmentoum Bunge,were cultured in Cd or Pb-containing nutrition solutions.The root exudates released from Sedum alfredii were collected and analyzed by metabolomics method which based on the gas chromatography-mass spectrometer(GC/MS),the components and variation of root exudates from the two ecotypes under different stresses were detected.The potential biomarkers which may be play an important role in the process of Sedum alfredii tolerate or accumulate the heavy metal Cd or Pb were identified,and the metabolic pathway of these compounds were detected.The results have important significations on the improvement of phytoremediation efficiency of the heavy metals contaminated soil by add these compounds or controlled its corresponding metabolic pathway.By optimizing the extraction condition,derivatization condition,separation and scan method of GC and the ion trap MS,the metabolomics analysis method which based on GC/MS was established.The collected root exudates were freeze-dried,and then redissolve with deionized water,freeze-dried again,and then dissolved by cold methanol.The methanol solution was dried by N2,and then successively joined methoxyamine hydrochloride and N-methyl-N-trimethylsilyl trifluoracetamide(MSTFA)to derivatize.Finally,the sample was separated by GC and detected by MS in full scan mode.The accuracy,repeatability and stability of this method were good,the absolute deviation of the retention time of the chromatography peak is less than 0.3 min and the relative standard deviation(RSD)of the peak area is less than 10%.Some different data extraction methods were compared,such as XCMS,MetAlign,MZmine,AMDIS,and so on.AMDIS combined with MET-IDEA was selected as the data extraction method in this study.The qualitative and quantitative information of plant root exudates was obtained by AMDIS analysis when the plant metabolites databases Fiehn and GMD were selected as the AMDIS database and the identified standard of compound was set that the similarity was greater than 70%.Then MET-IDEA was used to process the AMDIS outputs,such as peak alignment and remove the excess peak.The root exudates which released from Sedum alfredii under Cd stress was studied by metabolomics method based on GC/MS.Under hydroponic conditions,the root exudates which released from the two ecotypes of Sedum alfredii under Cd treatment,the hyperaccumulator Sedum alfredii under different Cd exposure concentrations,and the hyperaccumulator Sedum alfredii under different Cd exposure times,were collected,extracted,derivatized and analyzed by GC/MS.69,62,68 compounds were identified respectively which after processed by AMDIS and MET-IDEA.The principal component analysis(PCA)and orthogonal partial least-squares discrimination analysis(OPLS-DA)shows that the samples which have the same condition,such as the ecotype of Sedum alfredii,the Cd exposure concentration and the Cd exposure time,were obviously flock together,the samples which have the different condition were clearly kept separate.18,20,15 compounds which the relative content have significant difference were identified respectively by the loadings plot,the variable importance factor(VIP,larger than 1,are the most relevant for explaining the group)values of the OPLS-DA and analysis of var:iance.And through the change trends of the relative content of these potential biomarkers under different condition to deduced their possible role.Finally,through synthetically analysis of these results,we considered that oxalic acid,benzoic acid，fumaric acid,decanoic acid,1-serine,2-hydroxypentanoic acid,3-hydroxybutanoic acid,threonic acid,oleic acid,dodecanoic acid,erythritol,beta-sitosterol and 1-monooctadecanoylglycerol might be mobilizing Cd,metabolic pathway associated with these compounds have sugar metabolism,amino acid metabolism,lipid metabolism and TCA cycle;phosphoric acid,tetradecanoic acid,glycerol,d-pinitol,octanol,n-octacosane,trehalose and putrescine may be able to stabilize Cd,metabolic pathway associated with these compounds have sugar metabolism,amino acid metabolism,lipid metabolism and energy metabolism.The root exudates which released from Sedum alfredii under Pb stress was studied by metabolomics method based on GC/MS.Under hydroponic conditions,the root exudates which released from the two ecotypes of Sedum alfredii under Pb treatment,the accumulator Sedum alfredii under different Pb exposure concentrations,and the accumulator Sedum alfredii under different Pb exposure times,were collected,extracted,derivatized and analyzed by GC/MS.56,68,72 compounds were identified respectively which after processed by AMDIS and MET-IDEA.The principal component analysis(PCA)and orthogonal partial least-squares discrimination analysis(OPLS-DA)shows that the samples which have the same condition,such as the ecotype of Sedum alfredii,the Pb exposure concentration and the Pb exposure time,were obviously flock together,the samples which have the different condition were clearly kept separate.15,16,18 compounds which the relative content have significant difference were identified respectively by the loadings plot,the VIP values of the OPLS-DA and analysis of variance.And through the change trends of the relative content of these potential biomarkers under different condition to deduced their possible role.Finally,through synthetically analysis of these results,we considered that oxalic acid,hexanoic acid,3-hydroxypropanoic acid,succinic acid,1-alanine,1-proline,galactonic acid,fructose,xylose,glucose,maltose,sucrose,4-methylphenol and 2-methoxyphenol could be attributed to the Pb mobilization,metabolic pathway associated with these compounds have sugar metabolism,amino acid metabolism,nucleotide metabolism and TCA cycle;n-pentadecanoic acid,hexadecanoic acid,9-hexadecenoic acid,oleic acid,azelaic acid,4-hydroxybenzoic acid,octadecanol,glycerol,diethyleneglycol,beta-sitosterol,1-monohexadecanoylglycerol and n-docosane might be closely related to the Pb stabilization,metabolic pathway associated with these compounds have amino acid metabolism and lipid metabolism. |
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