Study On The Stress Response Of Cole And Maize Using NMR-based Metabonomics

NMR-based plant metabolomics mainly use NMR(Nuclear Magnetic Resonance)and multivariate data analysis to detect and analyze the plant fluids and tissues systematically,which contains tracking detection,quantitative analysis and qualitative classification to the dynamic changes of metabolites over time from a complete organism(rather than individual single-cell).The given biological informations,which generate by external interference acted on plant physiological processes,could help to determine the sites of action,identify relevant biomarkers,reveal the biological significance and discover the movement law of plant life in the molecular level.This work studies two crops,cole(Brassica napus L.)and maize(Zea mays L.var.ceratina Kulesh),disturbed by external interference,and analyzes the corresponding metabolic changes coming from physiological processes.Extraction strategy on Brassica napus L.leaves is also optimized.Three aspects are covered:1.Metabolites in plants are various,which polarity,type and concentration level are different.Therefore they couldn't be extracted completely with one solvent.To investigate the molecular composition of plant and its metabolic change comprehensively,four solvents are selected to extract the Brassica napus L.leaves.NMR combined with pattern recognition method is applied to evaluate the different extraction strategies.The result indicates that there are 36 metabolites in Brassica napus L.leaves,including sugars,amino acids,organic acid,flavonoids and so on.Compared with Na2HPO4/NaH2PO4 buffer solvent,50%methanol contains low level of lactate,isoleucine,while high level of sucrose,frumaric acid,malic acid and trans-/cis-phenylpropanoid.50%chloroformic methanol extracts more fatty chain than 50%methanol,but less sugars,amino acids and organic acids.Moreover,acetone is an excellent solvent for selective extraction of fatty compounds.2.Spraying pesticides usually causes crop plant to make certain stress response,which is sure to lead the metabolic changes of the plant.A series of NMR techniques,such as 1H NMR,COSY,TOCSY,HMBC,HSQC and JRES,combined with the principal component analysis(PCA)and orthogonal partial least squares-discriminant analysis(OPLS-DA)are performed to study the stress response of Brassica napus L.for spraying pesticide HWS02.The result shows that the concentration of ?/?-glucose,valine,GABA,choline,linolenic acid,fumaric acid,malic acid,threonine and isoleucine changes after the pesticide HWS02 is sprayed to cole.These metabolites are mostly related to tricarboxylic acid(TCA)cycle,which means that the pesticide HWS02 could affect the cole through the TCA cycle.This not only provides the vital information on the stress response of the cole to the pesticide HWS02,but also offers some theoretical support for the reasonable application and development of pesticides.3.Usually,the pesticides of herbicide class are selective.For example,the pesticide HWS02 can selectively get rid of barnyard grass,but it is relatively safe for maize.This part investigates the metabolic changes of maize after the pesticide HWS02 is sprayed,and compares the stress response difference of the two crops in the disturbing of HWS02.Principal component analysis(PCA)and partial least squares-discriiminant method(PLS-DA)are performed to study the metabolic changes in different growth stages for both treated and control groups.The result shows that,when pesticide HWS02 is sprayed,the concentration of sucrose decreases firstly,but increases subsequently,while the metabolite U2 is on the contrary.And there is no obvious change to other metabolites.Those indicate that pesticide HWS02 could bring more metabolic changes in the growth of cole than maize,which means that pesticide HWS02 is relative safer for maize.