Phenolic compounds in Vitis vinifera contribute important flavor, functionality, and health qualities to both table and wine grapes. The plant phenolic metabolic pathway has been well characterized, however many important questions remain regarding the influence of environmental conditions on pathway regulation. The synthesis and/or degradation of anthocyanins is reported to be sensitive to vineyard temperature, with lower color found in warm climate fruit, but the cause for less color is not understood. As a diagnostic for this pathway's regulation, in vitro and in situ techniques to incorporate the stable-isotopic tracer, L-phenyl- 13C6-alanine (Phe13), were developed as well as the accompanying high throughput analytical LC-DAD-MS/MS method to quantify and track the label into phenylalanine metabolites. Variability between vines, clusters and berries was assessed to foster a robust sampling plan for further studies. This methodology provided a framework for examining the regulation of 27 phenolic metabolites at different stages of maturity or under different environmental conditions. The technique was then used to track the development of labeled and unlabeled phenolics in vineyard Cabernet Sauvignon grapes over two years. Phenolic profiles during ripening were consistent with previous research, however, individual anthocyanins accumulated with different profiles during ripening; malvidin species continually climbed in concentration while other anthocyanins tended to plateau or drop near the end of the growing season. The isotopic label was predominantly incorporated into anthocyanins, presumably because of their dominant accumulation during ripening. Notably, the incorporation of label continued long after levels of Phe13 had dropped to below 1 nmol/berry, preventing an accurate assessment of the hypothesized turnover of anthocyanins. While our tracer did not perform exactly as we had expected, the exciting results of this study indicated the presence of a previously unreported pool of phenylpropanoid pathway substrate. To probe the degradation of grape anthocyanins in hot climates, grape berries were labeled in vitro and subjected to heated and unheated treatments. To analyze these samples, a complete, untargeted metabolomic approach was taken using microfluidic chip liquid chromatography paired with high mass accuracy mass spectrometry. The data was then analyzed using MetExtract software algorithms, which identified any isotopomer with 6 C-13's. From the study, 53 molecular features were found to be significantly different between heated and control samples. Of these, 3 features were found to be greater in quantity in the 45C than in 25C grape treatments. These yet-to-be identified compounds represent the most likely suspects for products of anthocyanin degradation. Overall, the methods provided in this work present a basis for examining the regulation of phenolic metabolism at different stages of maturity or under different environmental conditions while the results from this work shed light on previously unknown qualities of phenylpropanoid metabolism.
|Advisor:||Waterhouse, Andrew L.|
|Commitee:||Adams, Douglas O., Negre-Zakharov, Florence|
|School:||University of California, Davis|
|Department:||Agricultural and Environmental Chemistry|
|School Location:||United States -- California|
|Source:||DAI-B 74/10(E), Dissertation Abstracts International|
|Subjects:||Food Science, Plant sciences, Analytical chemistry|
|Keywords:||Anthocyanin, Metabolism, Metabolomics, Phenylalanine, Phenylpropanoid, Stable-isotope|
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