Biosynthesis of Isoflavonoid Phytoalexins in Pea

本提案的目的是进一步了解（+）pisatin的生物合成，（+）pisatin是豌豆产生的类翼石芹类植物抗毒素。虽然（-）异构体更为常见，但在自然界中，翼龙类以两种立体异构体中的任何一种存在。最近的研究结果表明（+）pisatin和（-）pterocarpan的生物合成共用一个具有（-）手性的中间体。（+） pisatin的合成也很不寻常，因为它的一个不对称碳上的氧来自H2O，而（-）pterocarpan的类似碳上的氧来自O2。这些观察结果表明，与（-）翼龙烷相比，（+）翼龙烷与（-）翼龙烷中建立手性和氧结合的机制根本不同。编码产生（-）手性中间体和推定的鱼素生物合成终端步骤的酶的基因已被克隆。这些基因和它们编码的酶，将通过结合传统的示踪研究和生化分析，以及生产缺乏这些酶的转基因豌豆组织，来解决（+）pisatin手性的起源。这样的实验应该最终确定这些酶是否参与了鱼素的生物合成，如果是的话，将随后用于确定这一途径中的新步骤。此外，这项工作将定义一个实验系统，其中这种植物抗毒素的生物学功能可以描绘。许多植物合成抗生素，称为植物抗菌素，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抵御疾病的一种机制。这项拨款提案的目的是确定由豌豆制成的植物抗菌素pisatin的生产过程中涉及的生化步骤。我们将使用标准的生化程序，除了在可疑步骤中进行特定的突变来表征该途径。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。克隆的基因可用于在植物中设计新的生物合成途径，目的是增强植物的抗病机制。该研究项目由代谢生物化学项目、分子与细胞生物科学部和综合植物生物学项目、综合生物学和神经科学部联合资助。＊＊＊

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. ***