Metabolomics of Complex Biological Systems to Uncover Molecular Mechanisms in Rice and Other Organisms
- Author(s): Barding, Gregory Alan
- Advisor(s): Larive, Cynthia K
- Bailey-Serres, Julia
- et al.
The goal of this dissertation is to explore the metabolic reconfiguration of the submergence intolerant rice variety Oryza sativa ssp.japonica cv. M202 and the submergence tolerant cv. M202(Sub1) during and after submergence stress. This work uses multiple analytical techniques, including nuclear magnetic resonance (NMR), gas chromatography - mass spectrometry (GC-MS), and reversed-phase ion-pair ultra-high performance liquid chromatography - mass spectrometry (RPIP-UPLC-MS) to explore the metabolite profiles of the tolerant and intolerant rice varieties in response to abiotic stress.
Untargeted metabolomics was used to query the metabolic impact of submergence on the tolerant and intolerant rice varieties. In results obtained by both NMR spectroscopy and GC-MS, the metabolic profiles of the two varieties diverged as early as 1 d of submergence. Using both techniques, the tolerant variety was shown to significantly conserve carbohydrate resources. A comparison of the NMR and GC-MS results demonstrated that multiple analytical techniques provide the best metabolite coverage. Additionally, a previously unreported NMR resonance was identified as belonging to the methyl protons of the dipeptide alanylglycine (AlaGly). Although the metabolic function of the dipeptide is unclear, AlaGly was the only metabolite to decrease during submergence and not recover during the 1 d recovery period.
RPIP-UPLC-MS analysis provided a more targeted approach to metabolite profiling because of the sample preparation method employed and the selectivity of the RPIP separation for hydrophilic and ionic analytes. Phosphorylated mono- and disaccharides were targeted because of their signaling role in plant metabolism. For example, changes in trehalose-6-phosphate (T6P) levels, a key metabolite responsible for carbon sensing and flowering in plants, were monitored during and after submergence in the tolerant and intolerant rice varieties.
The development of automated and high-throughput chemometric techniques is critical for advances in disease detection, biomarker identification, and toxicological profiling. The coupling of untargeted metabolite profiling by NMR spectroscopy with a new chemometric approach based on z-score analyses, Visual Interpretation of Z-Score Ratios (VIZR), was used to determine differences in spectra from human urine and due to dietary supplementation. This approach can also be extended to biomarker identification in plants and other organisms.