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

RUI：植物邻氨基苯甲酸分支点的分子进化和调控

基本信息

批准号：
2214883
负责人：
Cynthia Holland
金额：
$ 37.54万
依托单位：
Williams College
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-15 至 2025-04-30
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2214883&HistoricalAwards=false
关键词：
RUI Molecular evolution regulation anthranilate

项目摘要

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.

为了防御食草动物，葡萄、玉米、柑橘和草莓等植物会释放一种名为邻氨基苯甲酸甲酯的挥发物，它可以作为天然的鸟类威慑剂并吸引寄生昆虫。虽然至少有十个植物科产生邻氨基苯甲酸甲酯，但所有植物都会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，色氨酸 (Trp) 对植物生长和发育至关重要。这项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调节邻氨基苯甲酸代谢。由于邻氨基苯甲酸甲酯在商业上用作食品和饮料行业的葡萄调味剂以及田间和农作物的防禽喷雾剂，因此从这些实验中获得的知识可能会对生物技术和农业产生影响。除了在主要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不同本科生外，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实验室套件，以进行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由初级代谢物（如氨基酸）或初级代谢途径中的中间体合成的专门代谢物。尽管氨基酸生物合成在植物防御中发挥着重要作用，但色氨酸生物合成途径的早期步骤仍有待在微生物之外进行生化表征。 Trp 途径首先形成邻氨基苯甲酸，然后使用转移酶 (PAT1) 将其与磷酸核糖结合。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，而柑橘科植物则产生邻氨基苯甲酸甲酯作为有毒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至色氨酸与邻氨基苯甲酸甲酯生物合成的分子机制，并追踪途径调节的演变。该项目将利用丰富的可用植物基因组序列数据，并使用综合方法来研究使用邻氨基苯甲酸的酶。目标 1 将使用诱变、活性测定和 X 射线晶体学来确定 PAT1 调节的分子决定因素和进化。目标 2 将使用结构引导的系统发育方法结合离体代谢组学测定来追踪邻氨基苯甲酸 O- 和 N- 甲基转移酶的趋同进化。目标 3 将使用系统级方法研究植物中的表型、代谢和基因表达变化，进一步加深我们对邻氨基苯甲酸调节的理解。这项研究将为基本氨基酸生物合成途径的调控机制提供新的线索，并帮助我们理解趋同专门代谢的进化。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准。