RUI: Molecular evolution and regulation of the anthranilate branch point in plants

为了保护自己免受食草动物的侵害，葡萄、玉米、柑橘和草莓等植物会释放出一种名为邻氨基苯甲酸甲酯的挥发物，这种挥发物可以作为天然的鸟类威慑物，吸引寄生昆虫。虽然至少有十个植物科产生邻氨基苯甲酸甲酯，但所有植物都合成前体邻氨基苯甲酸，作为氨基酸色氨酸（Trp）生物合成的中间体，这对植物生长和发育至关重要。本研究的目的是了解植物如何在氨基酸合成和防御代谢之间的界面上调节邻氨基苯甲酸酯代谢。由于邻氨基苯甲酸甲酯在商业上被用作食品和饮料工业中的葡萄调味剂，以及农田和作物的防禽喷雾剂，因此从这些实验中获得的知识可能对生物技术和农业产生影响。除了在主要的本科院校培养蛋白质生物化学，结构生物学，系统生物学和植物遗传学方面的不同本科生外，该项目将用于为马萨诸塞州和佛蒙特州的农村县的小学生和中学生开发一个实验室工具包，以研究植物化学物质在防御食草动物方面的重要性。植物化学防御通常是合成的专门代谢物，从初级代谢物，如氨基酸，或初级代谢途径中的中间产物。尽管氨基酸生物合成在植物防御中起着重要作用，但Trp生物合成途径的早期步骤仍然需要在微生物外进行生化表征。Trp途径开始于形成邻氨基苯甲酸，然后使用转移酶（PAT 1）将邻氨基苯甲酸与磷酸核糖基糖缀合。一些植物通过甲基化邻氨基苯甲酸酯，形成挥发性的邻氨基苯甲酸O-甲酯，而柑橘类植物产生N-甲基邻氨基苯甲酸酯作为有毒吖啶酮生物碱的前体。本研究的目标是确定邻氨基苯甲酸分配的分子机制，色氨酸与邻氨基苯甲酸甲酯的生物合成和跟踪的演变途径的调节。该项目将利用丰富的植物基因组序列数据，并使用综合方法来研究邻氨基苯甲酸酯使用酶。目的1将确定分子决定因素和演变的PAT 1调节使用诱变，活性测定，和X-射线晶体学。目标2将跟踪邻氨基苯甲酸O-和N-甲基转移酶的趋同进化，使用结构指导的系统发育方法，结合离体代谢组学分析。目的3将进一步了解邻氨基苯甲酸调制使用系统水平的方法来调查植物的表型，代谢和基因表达的变化。这项研究将照亮一个基本的氨基酸生物合成途径的调控机制的新的光，以及在我们的会聚专门代谢的演变的理解援助。这个奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

To defend themselves against herbivores, plants such as grape, maize, citrus, and strawberry emit a volatile called methyl anthranilate, which acts as a natural bird deterrent and attracts parasitic insects. While at least ten plant families produce methyl anthranilate, all plants synthesize the precursor, anthranilate, as an intermediate in the biosynthesis of the amino acid tryptophan (Trp), which is essential for plant growth and development. The goal of this research is to understand how plants regulate anthranilate metabolism at the interface between amino acid synthesis and defense metabolism. Since methyl anthranilate is used commercially as a grape flavoring agent in the food and beverage industry and as an anti-avian spray for fields and crops, knowledge gained from these experiments may have implications in biotechnology and agriculture. In addition to training diverse undergraduates in protein biochemistry, structural biology, systems biology, and plant genetics at a primarily undergraduate institution, this project will be used to develop a laboratory kit for elementary and middle school students in rural counties in Massachusetts and Vermont to investigate the importance of plant chemicals in defense against herbivores.Plant chemical defenses are typically specialized metabolites that are synthesized from primary metabolites, like amino acids, or intermediates in primary metabolic pathways. Despite the fundamental role of amino acid biosynthesis in plant defense, the early steps of the Trp biosynthetic pathway remain to be biochemically characterized outside of microorganisms. The Trp pathway starts by forming anthranilate, which is then conjugated to a phosphoribosyl sugar using a transferase (PAT1). Some plants siphon anthranilate away from the Trp pathway by methylating it, forming the volatile O-methyl anthranilate, while the Citrus family generates N-methyl anthranilate as a precursor to toxic acridone alkaloids. The goals of this research are to identify the molecular mechanisms governing anthranilate allocation to Trp versus methyl anthranilate biosynthesis and to trace the evolution of pathway regulation. This project will take advantage of the wealth of available plant genome sequence data and use integrative approaches to investigate anthranilate-using enzymes. Aim 1 will identify the molecular determinants and evolution of PAT1 regulation using mutagenesis, activity assays, and X-ray crystallography. Aim 2 will trace the convergent evolution of anthranilate O- and N-methyltransferases using a structure-guided phylogenetic approach coupled with ex vivo metabolomics assays. Aim 3 will further our understanding of anthranilate modulation using a systems-level approach to investigate phenotypic, metabolic, and gene expression changes in planta. This study will shine new light on the regulatory mechanisms of a fundamental amino acid biosynthetic pathway, as well as aid in our understanding of the evolution of convergent specialized metabolism.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.