Study On Chemical Defense Substances In Maize Seedling Stage

Maize is an important food crop in China and its metabolism is strong at seedling stage,but it is highly harmful by pests and diseases.In this dissertation,phytochemical extraction and separation technology is used to systematically separate the secondary metabolites of the widely cultivated cultivar maize(Danyu 336)in the Northeast at the seedling stage.The structures of these secondary metabolites are determined by nuclear magnetic resonance spectrum(1D and 2D NMR),single-crystal X-ray diffraction,chemical synthesis and chiral resolution.The Ultra Performance Liquid Chromatography-tandem mass spectrometry(UPLC-MS/MS)is used to investigate the distribution and concentration changes of these metabolites in the seedling stage and combine with biological activety screen to reveal the defense function of main secondary metabolites against diseases and pests.(1)A new natural product ZL-1(MAHBOA)is isolated from maize(Danyu 336)at the seedling stage and its planar structure is determined by NMR.The amount of ZL-1 is enriched by chemical synthesis,and the structure of ZL-1 is determined by single crystal diffraction that the compound is determined to be a chiral isomer.The chiral chromatographic separation method is used to resolve the chiral isomers,and the stereochemical structure of MAHBOA-1 and MAHBOA-2 are determined by ECD calculation that it is found that the ratio of MAHBOA-1 to MAHBOA-2 in the plant is 1: 3.The quantitative analysis of UPLC-MS/MS show that the content of the compound in the above-ground part of maize is higher than that in the underground part and the content reach the highest in 7th day at the seedling stage.During this period,the content of MAHBOA-1 in the above-ground and underground parts are 1.21 ± 0.24 and 0.22 ± 0.08 ?g·g-1 DW,and the contents of MAHBOA-2 are 3.22 ± 0.65 and 0.59 ± 0.21 ?g·g-1 DW,respectively.In addition,the compound is also stably present in seedlings of other gramineous food crops.Based on the analysis of biosynthetic pathways of similar substances,it is speculated that the biosynthetic precursor of MAHBOA is indole-3-acetic acid(IAA).IAA and MAHBOA are modified by deuteration substitution and the deuteratiom products d2-IAA and d2-MAHBOA are obtained.The maize seedlings are fed with d2-IAA,and d2-MAHBOA is detected in the above and below ground parts,which indicates that IAA is the biosynthetic precursor of MAHBOA.Further analysis of the content of MAHBOA in the maize mutant bx2,there is no change in the contents of MAHBOA-1 and MAHBOA-2 in the mutant compared with the control maize,and the results show that the MAHBOA and benzoxazinoids have different synthetic pathways.Biological activity screen show that MAHBOA has certain inhibitory activity on the growth of Bacillus subtilis.Furthermore,MAHBOA also show strong antifeedant activity against Helicoverpa armigera.The antifeedant concentration of the 1: 3 chiral isomer mixture is 13.25 ± 0.79 ?g/cm2 and the antifeedant concentrations of MAHBOA-1 and MAHBOA-2 are 16.49 ± 2.56 and 12.67 ± 0.69 ?g/cm2.These results indicate that MAHBOA-2 has the strongest antifeedant activity.Compounds MAHBOA-1 and MAHBOA-2 are fed to Spodoptera litura,Mythimnaseparata walker and Helicoverpa armigera.The results of metabolic analysis show that all three insects can convert MAHBOA-2 to MAHBOA-1,and Spodoptera litura has stronger transformation ability than Mythimnaseparata walker,and Helicoverpa armigera with the lowest metabolic transformation ability.All three insects cannot convert MAHBOA-1 to MAHBOA-2 which indicates that the insects can reduce the toxic effect of the compound by transforming the R configuration to the S configuration.(2)18 compounds are isolated from maize seedlings(Danyu 336)using chromatographic separation technology.The structures of these compounds are identified by NMR and MS,including 6 benzoxazinoids,3 Terpenoids and 9 phenolic acids.A UPLC-MS/MS method for the determination of 12 compounds is established with enabling rapid and accurate quantitative analysis of these compounds within 17 minutes.Quantitative analysis of some compounds in maize by this method,it shows that the content of compound MBOA(ZL-2)continues to decrease from day 3 to 28,while the content of compounds M2BOA(ZL-3),HMBOA(ZL-4),BOA(ZL-6)and HBOA(ZL-7)are higher when the maize grows to the 7th day.Qualitative and quantitative analysis of compounds in the hydroponic solution and soil of maize(Jingke 968 and Shenyu 26)show that the root exudates of maize are mainly MBOA,M2 BOA and HMBOA,and the secretion of maize is the highest in 5-7 days in hydroponic solution.Moreover,the content of MBOA is higher in root exudates than in all soil types.(3)The seed coating agent provides favorable conditions for the preservation of maize germplasm,but the pesticide components in the seed coating agent will be transported and metabolized by plant seedlings.In this paper,three carbamate compounds are transported or metabolized by maize seedlings including carbofuran(ZL-20),3-hydroxy-carbofuran(ZL-21)and 3-keto-7-phenolcarbofuran(ZL-22).The quantitative analysis of UPLC-MS/MS showed that the content of carbofuran in the seedlings is the highest,which may be the substance transported by plants from seed coat agent.The content of 3-hydroxy-carbofuran and 3-keto-7-phenolcarbofuran is relatively low,which may be the metabolites of carbofuran.In conclusion,the secondary metabolism in maize seedlings is highly active,which can not only synthesize and release a large number of defensive benzoxazinoids,terpenoids and phenols but also have a strong ability to transport and metabolize toxic substances.The discovery of MAHBOA-1 and MAHBOA-2 with IAA as precursors shows that maize can use the growth substances in seedling stage to transform into defense substances,which provides a foundation for the study of plant growth and defense mechanism.