Biosynthesis of Isoflavonoid Phytoalexins in Pea

9319733 VanEtten这项建议的目的是加深对(+)pisatin生物合成的了解，(+)pisatin是由豌豆产生的类翼果苷植物抗毒素。蕨类在自然界中以两种立体异构体中的任何一种存在，尽管(-)异构体更为常见。最近的结果表明，(+)pisatin和(-)ptercarpans的生物合成共享一个具有(-)手性的中间体。(+)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(-)紫檀烷类似碳上的氧来自O2。这些观察表明，与(-)果皮相比，根本不同的机制建立了(+)pisatin中的手性和氧的结合。编码产生(-)手性中间体和pisatin生物合成终末步骤的酶的基因已经被克隆。这些基因及其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手性的来源。这样的实验应该最终确定这些酶是否参与了pisatin的生物合成，如果是的话，随后将被用来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，在其中可以描述这种植物抗毒素的生物学功能。许多植物合成抗生素，称为植物保卫素，以应对微生物的感染。这些抗菌化合物的产生被认为是植物抵御疾病的一种机制。这项赠款提案的目的是确定生产由豌豆制成的植物抗菌素pisatin的生化步骤。除了在可疑步骤中进行特定突变外，我们还将使用标准的生化程序来表征该途径。从这项研究中获得的信息最终可能被用于克隆与植物保卫素生物合成有关的基因。克隆的基因可以用来设计植物中新的生物合成途径，目的是增强植物的抗病机制。该研究项目由新陈代谢生物化学项目、分子和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共同资助。***

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. ***