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一种名为甲酰苯甲酸的挥发性物质，这种物质可以作为天然的鸟类威慑物，并吸引寄生虫。虽然至少有10个植物科产生甲酰苯甲酸，但所有植物都合成甲酰苯甲酸前体，作为生物合成氨基酸色氨酸（Trp）的中间体，而色氨酸是植物生长发育所必需的。本研究的目的是了解植物如何调节氨基酸合成和防御代谢之间的界面上的邻氨基甲酸酯代谢。由于甲酰苯甲酸甲酯在食品和饮料工业中被商业上用作葡萄调味剂，并作为田地和作物的防鸟喷雾剂，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。除了在一所主要的本科院校培训蛋白质生物化学、结构生物学、系统生物学和植物遗传学方面的本科生外，该项目还将用于为马萨诸塞州和佛蒙特州农村县的中小学生开发一个实验室工具包，以调查植物化学物质在抵御食草动物方面的重要性。植物化学防御通常是由初级代谢物合成的专门代谢物，如氨基酸，或初级代谢途径中的中间体。尽管氨基酸生物合成在植物防御中发挥着重要作用，但Trp生物合成途径的早期步骤仍然需要在微生物之外进行生化表征。色氨酸途径首先形成邻氨基苯甲酸酯，然后利用转移酶（PAT1）将其偶联到磷酸核糖糖上。一些植物通过甲基化将邻氨基苯甲酸从色氨酸途径中吸走，形成挥发性的邻氨基苯甲酸甲酯，而柑橘家族产生n -邻氨基苯甲酸甲酯作为有毒的吖啶酮生物碱的前体。本研究的目的是确定控制邻氨基苯甲酸分配到色氨酸和甲基邻氨基苯甲酸生物合成的分子机制，并追踪途径调控的进化。该项目将利用丰富的现有植物基因组序列数据，并使用综合方法来研究利用邻氨基苯甲酸的酶。目的1将利用诱变、活性测定和x射线晶体学确定PAT1调控的分子决定因素和进化。目的2将使用结构引导的系统发育方法结合体外代谢组学分析来追踪邻氨基苯甲酸O-和n -甲基转移酶的趋同进化。目的3将利用系统水平的方法来研究植物的表型、代谢和基因表达变化，进一步了解邻氨基苯甲酸调节。这项研究将为基础氨基酸生物合成途径的调控机制提供新的线索，并有助于我们对趋同特化代谢进化的理解。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。

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.