New Analytical Methods Of CE-MS Based Metabolomics And Their Applications In Tobacco Leaf Studies

Plant metabolomics is a science, which aims at detection of all metabolites within a plant under specific environmental or genetic conditions. Compared with components in other species, metabolome in plant is more diverse and complex. However, investigations of metabolome both from comprehensive and fine accurate perspectives make it a big challenge for analysis methods. Mass spectrometry is very useful analysis tool in metabolomics. However, one analytical platform or single omics technique has its own limitation to understand the biology functions in plant from a holistic outlook. To this end, it needs to develop integrated metabolomics methods based on multi-platform and multi-omics technologies. In this thesis, we carried out investigations of novel analytical methods based on capillary electrophoresis-mass spectrometry (CE-MS) and their applications in metabolomics studies of model plant tobacco to improve the coverage of metabolites.(1) A novel analytical method was established for determining intermediates involved in sulfur pathway with high throughput and high sensitivity. The strategy of the method was achieved by continuously multiple and large volume injections and on line sample preconcentration based on CE-MS. The developed method was successfully applied in tobacco and cell samples. It realized to optimize various experimental conditions at the same time and reduce the time of experimental optimization. Compared with normal method, the method throughput was enhanced 5 times, and its sensitivity was increased 14.4-33.1 times. These results indicated that the developed method was very suitable for study the sulfur pathway.(2) In order to further enlarge the coverage of polar metabolome, extraction solvent for polar metabolites was optimized and finalized by the design of experiments. A non-targeted tobacco metabolomics method was developed based on CE-MS. A total of 154 polar metabolites was identified in tobacco leaf including amino acids, organic acids, nucleosides, vitamins and coenzymes etc. The developed method possessed advantages of simple sample preparation, good repeatability and wide coverage of plant polar metabolome, indicating its extensive applicability in polar metabolome research for tobacco.(3) A multi-platform metabolomics approach was established with the combination of gas chromatography-mass spectrometry (GC-MS) and CE-MS. The two platforms showed satisfactory complementarity by covering a wide range of measurable polar metabolites. The established method was successfully used in field-grown mature tobacco leaves in three typical tobacco-growing provinces in China. Approximately 240 polar metabolites were determined in tobacco leaves. A metabolic correlation network analysis showed that carbon-nitrogen contribution and transformation were significantly related to regional characteristics. In order to make the metabolic regulations clear, the significantly related pathway analysis was performed. The results showed that nitrogen assimilation and tricarboxylic acid (TCA) cycle were more activated in Yunnan and Guizhou than in Henan, but the substrates of TCA cycle and the regulation of nitrogen contribution were different from Yunnan and Guizhou. The ability of photosynthesis and nitrogen metabolism was higher in Yunnan than in Henan and Guizhou. Compared with Yunnan and Guizhou, pentose phosphate pathway had a close correlation with shikimic acid metabolism in Henan.(4) To overcome the limitation of one platform and single omics technique for comprehensive understanding the biology functions, an integrated multi-platform and multi-omics method was developed to investigate tobacco leaf variations in response to topping. The multi-platform metabolic profiling in tobacco leaf was generated by pseudo-targeted and non-targeted GC-MS methods, targeted and non-targeted liquid chromatography-mass spectrometry/ultraviolet (LC-MS/UV) methods and non-targeted CE-MS method. A total of 367 metabolites was identified which involved in primary and secondary metabolism. Metabolomics data showed that the topping treatment had a much greater impact on upper leaves than middle and lower leaves and the metabolic regulations were different between early stage and mature of topping. In order to further investigate these metabolic mechanisms, integrated metabolomics and transcriptomics was used. Compared with control group, topping induced lipid mobilization and enhancement of photosynthesis and carbon fixation in treated group at the early phase. Furthermore, topping changed nutrient distribution patterns and the degree of leaf senescence. At the mature stage, secondary metabolites were accumulated at the upper leaves. These results indicated that integrated metabolomics based on multi-platform and multi-omics systemically elucidated the metabolic mechanisms at the molecular level.