Biosynthesis of Isoflavonoid Phytoalexins in Pea

9319733 VanEtten该建议的目的是进一步了解豌豆产生的（+）pisatin（pterocarpanoid phytoalexin）的生物合成。 紫檀烷在自然界中以两种立体异构体之一存在，尽管（-）异构体更常见。 最近的研究结果表明，（+）pisatin和（-）pterocarpans的生物合成共享一个中间体，具有（-）手性。 （+）pisatin的合成也是不寻常的，因为它的一个不对称碳上的氧来自H2O，而（-）pterocarpans的类似碳上的氧来自O2。 这些观察结果意味着，与（-）紫檀烷相比，（+）pisatin中氧的手性和掺入机制根本不同。 已经克隆了编码产生（-）手性中间体的酶和在pisatin生物合成中推定的末端步骤的基因。 这些基因和它们编码的酶，将被用来解决（+）pisatin手性的起源，结合传统的示踪研究和生化分析与生产转基因豌豆组织缺乏这些酶。 这样的实验应该最终确定酶是否参与pisatin的生物合成，如果是这样的话，将随后用于确定在这一途径中的新步骤。 此外，这项工作将定义一个实验系统，在该系统中可以描绘这种植物抗毒素的生物学功能。 许多植物合成抗生素，称为植物抗毒素，以应对微生物感染。 这些抗微生物化合物的产生被认为是植物防御疾病的一种机制。 这项资助计划的目的是确定豌豆生产植物抗毒素pisatin的生化步骤。 我们将使用标准的生化程序，除了在可疑的步骤中进行特定的突变，以表征途径。 从这项研究中获得的信息可能最终用于克隆参与植物抗毒素生物合成的基因。 克隆的基因可用于设计植物中新的生物合成途径，以增强植物的抗病机制。 该研究项目由分子细胞生物科学部代谢生物化学项目和综合生物学和神经科学部综合植物生物学项目联合资助。 ***

9319733 VanEtten The objective of this proposal is to further the understanding of the biosynthesis of (+) pisatin, the pterocarpanoid phytoalexin produced by pea. Pterocarpans exist as either of two stereoisomers in nature although the (-) isomer is more common. Recent results suggest that the biosynthesis of (+) pisatin and (-) pterocarpans share an intermediate that has a (-) chirality. Synthesis of (+) pisatin is also unusual in that the oxygen on one of its asymmetric carbons derives from H2O, whereas the oxygen on the analogous carbon of (-) pterocarpans is derived from O2. These observations imply that fundamentally different mechanisms establish the chirality and incorporation of oxygen in (+) pisatin compared to the (-) pterocarpans. Genes that encode the enzymes which produce the (-) chirality intermediate and the putative terminal step in pisatin biosynthesis have been cloned. These genes, and the enzymes they encode, will be used to resolve the origin of chirality in (+) pisatin by combining traditional tracer studies and biochemical analyses with the production of transgenic pea tissue lacking each of these enzymes. Such experiments should establish conclusively whether the enzymes are involved in pisatin biosynthesis and, if so, will subsequently be used to identify the novel steps in this pathway. Furthermore, this work will define an experimental system in which the biological function of this phytoalexin can be delineated. %%% Many plants synthesize antibiotics, called phytoalexins, in response to microbial infection. Production of these antimicrobial compounds is believed to be one mechanism used by plants to defend against disease. The objective of this grant proposal is to identify the biochemical steps involved in the production of the phytoalexin pisatin made by garden pea. We will use standard biochemical procedures in addition to making specific mutations in suspected steps to characterize the pathway. The information gained f rom this research may eventually be used to clone genes involved in the biosynthesis of phytoalexins. The cloned genes could be used to engineer new biosynthetic pathways in plants with the goal of augmenting the plant's disease resistance mechanisms. This research program is funded jointly by the Metabolic Biochemistry Program, Division of Molecular & Cellular Biosciences, and the Integrative Plant Biology Program, Division of Integrative Biology and Neuroscience. ***