The relationship between tryptophan and pyridine nucleotide metabolism in plants

从单细胞生命到哺乳动物和植物，必不可少的细胞代谢物吡啶核苷酸在调节所有生物体的能量代谢中起着重要作用。然而，在植物中，如何制造吡啶核苷酸的问题仍然是一个未解决的研究领域，即使在这个基因组学时代，也存在一些历史矛盾。了解吡啶核苷酸的生物合成不仅对了解其在植物生物学中的功能具有重要意义，而且对提高食用植物的营养品质也至关重要。此外，由于最近的研究已经阐明了吡啶核苷酸在细胞存活和细胞通讯中的多种作用，因此在分子和遗传水平上了解吡啶核苷酸代谢的细节对于设计植物在不断变化的全球环境中逆境生存的策略至关重要。通过这个项目的研讨会，早期的职业科学家将被介绍到代谢途径发现的现代方法。吡啶核苷酸在所有生物体中都是由简单的代谢前体合成的。虽然反应的具体步骤因生物而异，但一个共同的特征是由氨基酸（色氨酸或天冬氨酸）生成喹啉酸。早期的研究表明，单子叶植物具有利用色氨酸或天冬氨酸的遗传能力，但最近的基因组分析表明，它们缺乏从色氨酸合成吡啶核苷酸的能力。然而，犬尿氨酸代谢途径中的关键中间体可以在玉米中找到，这些中间体的来源将通过拟议的研究来解决。基于稳定同位素方法和高分辨率质谱的先进代谢分析将用于阐明吡啶核苷酸的代谢起源。在玉米和其他植物中，犬尿氨酸代谢途径转化为吡啶核苷酸将被测量，以确定犬尿氨酸代谢途径在什么时候发育以及在什么组织中是主要的。将研究正常野生型植物和突变体在色氨酸和吲哚生物合成方面的比较代谢转化。结合稳定同位素标记前体的小分子抑制剂将用于吡啶核苷酸代谢网络的靶向高分辨率质谱代谢组学分析。植物对天冬氨酸和犬尿氨酸途径的相对利用将通过测量早期前体的通量来确定。一旦通过同位素分析确定了途径，测定指导的分馏将被用来克服明显的进化分歧，这使得遗传同源性分析变得困难。实验指导分离和蛋白质组学将用于获得犬尿氨酸途径中两个关键蛋白的氨基酸序列。为研究生，博士后科学家和其他对稳定同位素方法用于代谢测量和途径发现感兴趣的人举办的植物代谢组学夏季在线研讨会将扩大这些方法的欣赏，这些方法使用高分辨率质谱法进行稳定同位素分析。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.