The relationship between tryptophan and pyridine nucleotide metabolism in plants

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The essential cellular metabolites known as pyridine nucleotides play an important role in regulating energy metabolism in all organisms, from single cell life to mammals and plants. However, in plants, the question of how pyridine nucleotides are made remains an unsettled area of research with several historical contradictions even in this genomics era. Understanding pyridine nucleotide biosynthesis is not only important for a general understanding of their functions in plant biology but is critical for improvement of the nutritional quality of food plants. Also, because recent research has elucidated multiple roles of pyridine nucleotides in cell survival and cell communication, understanding the details of pyridine nucleotide metabolism at the molecular and genetic level is expected to be critically important for designing strategies for plant survival under stress in a changing global environment. Early career scientists will be introduced via this project’s workshop to modern methods for metabolic pathway discovery.Pyridine nucleotides are synthesized from simple metabolic precursors in all organisms. While the specific steps of reactions differ among organisms, a common feature is the generation of quinolinic acid from an amino acid, either tryptophan or aspartate. Earlier studies suggested that monocotyledonous plants had the genetic capacity to utilize either tryptophan or aspartate, but more recent genomic analyses suggest that they lack the capacity to synthesize pyridine nucleotides from tryptophan. Key intermediates in the kynurenine metabolic pathway, however, can be found in maize and the origins of these intermediates will be resolved by the proposed research. Advanced metabolic analysis based on stable isotope methods and high-resolution mass spectrometry will be used to elucidate the metabolic origins of pyridine nucleotides. The kynurenine metabolic pathway to pyridine nucleotides in maize and other plants will be measured to determine when in development and in what tissues the kynurenine pathway is primary. Comparative metabolic transformations in normal wildtype plants and mutants in tryptophan and indole biosynthesis will be studied. Small molecule inhibitors coupled with stable isotope labeled precursors will be used for targeted high-resolution mass spectrometry based metabolomic analysis of the pyridine nucleotide metabolic network. Relative plant utilization of both the aspartate and kynurenine pathways will be determined by measurement of the flux from early precursors. Once pathways are determined by isotopic analysis, assay guided fractionation will be employed to overcome the apparent evolutionary divergence that has made genetic homology analysis difficult. Assay guided fractionation and proteomics will be used to obtain amino acid sequence for two key proteins in the kynurenine pathway. A summer and online workshop in plant metabolomics for graduate students, postdoctoral scientists and others interested in stable isotope methodology for metabolic measurement and pathway discovery will broaden the appreciation of these approaches using modern applications of high resolution mass spectrometry to stable isotope analysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.