Benzoic Acid Biosynthesis in Plants

该奖项是根据2009年《美国复苏和再投资法案》(公法111-15)提供资金的。知识价值：许多植物化合物，包括天然植物生长调节剂、防御性化合物、病原菌诱导的植物保卫素、传粉者引诱剂和风味化合物，含有从苯甲酸衍生的苯甲酰基部分，在自然生态系统中植物生存和繁殖成功中发挥关键作用。尽管苯甲酸的结构简单，分布广泛，对植物生活史具有重要意义，但导致其形成的生化途径在很大程度上仍不清楚。这项拟议研究的目标是发现导致苯甲酸生物合成的未知生化途径，对所涉及的酶进行生化表征，并确定它们在细胞中的定位。我们将采用遗传学、代谢组学、功能基因组学和代谢通量分析等一种独特的综合方法来剖析植物中苯甲酸的生物合成途径。矮牵牛花释放大量的苯类化合物，是解释导致苯甲酸形成的代谢途径的理想模型系统，将在本研究中使用。从矮牵牛花中自然释放出苯甲酸甲酯和其他各种挥发性苯甲酸衍生物，从而能够通过苯类网络快速、定量地分析通量。参与苯甲酸生物合成的候选基因将使用RNAi方法进行沉默。花瓣特异性启动子的使用将确保苯甲酸的合成只在花瓣中受到影响，从而将对植物生存、生长和繁殖的有害影响降至最低。具有苯甲酸代谢改变的转基因生物将接受代谢概况、同位素标记实验和计算机辅助途径建模，以评估苯甲酸网络内的碳流，并检测代谢扰动期间的通量重新分配。由沉默导致苯甲酸代谢变化的基因编码的酶将被生化表征。这些酶的亚细胞定位将通过实验确定，以评估隔室在调节苯甲酸通量方面的潜在作用。这项研究将导致参与苯甲酸生物合成的基因和酶的基础性发现，并填补我们对苯甲酸代谢知识的重要空白。所得结果将为全面了解苯甲酸网络的结构和调控性质，并为未来合理的植物代谢工程奠定基础，以改善植物的风味和香气质量，增强植物对环境逆境的抵抗力，增加传粉者的吸引力和增加生物活性化合物的数量。更广泛的影响：该项目为下一代植物生物化学家提供了强大的多学科培训部分。本科生和研究生以及博士后科学家(包括女性和少数族裔)将获得植物酶学、分子生物学、活体同位素标记、遗传学和综合建模方面的经验。除了在实验室培训学生外，国际植物代谢研究所还将组织植物次生代谢生物化学和代谢工程研究生研究研讨会，该研讨会将把来自美国的研究生和博士后与来自世界各地的其他人聚集在一起，并将在2011年植物代谢工程戈登研究会议之前举行。这次研讨会将使年轻科学家对植物新陈代谢有更深入的了解，更积极地参与科学和教育，并帮助他们与未来的国外同事建立专业关系，特别是因为植物生物化学在某些国家(如德国、日本)很有帮助。这项研究中使用的结果和方法也将通过开发侧重于植物专业新陈代谢的课程模块，整合到普渡大学的本科生和研究生教育中。国际和平研究所将为学员提供指导，帮助他们在未来的科学工作中取得成功。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-15). Intellectual Merit: Numerous plant compounds, including natural plant growth regulators, defensive compounds, pathogen-induced phytoalexins, pollinator attractants and flavor compounds, contain benzoyl moieties derived from benzoic acid and play crucial roles in plant survival and reproductive success in natural ecosystems. Despite the simple structure of benzoic acid, widespread distribution and importance for the plant life cycle, the biochemical pathways leading to its formation remain largely unknown. The goal of the proposed research is to discover unknown biochemical pathways leading to benzoic acid biosynthesis, biochemically characterize the enzymes involved and determine their localization in the cell. A unique integrative approach including genetics, metabolomics, functional genomics and metabolic flux analysis will be employed to dissect the pathways to benzoic acid biosynthesis in plants. Petunia flowers, emitting large amounts of benzenoid compounds, represent an ideal model system for the elucidation of the metabolic pathways leading to benzoic acid formation and will be used in this research. The natural emission of methylbenzoate as well as a variety of other volatile benzoic acid derivatives from petunia flowers enables a rapid, quantitative analysis of flux through the benzenoid network. Candidate genes involved in benzoic acid biosynthesis will be silenced using RNAi methodology. The use of a petal-specific promoter will ensure that benzoic acid synthesis will be affected in petals only, thus minimizing unwanted effects on plant viability, growth, and reproduction. Transgenics with altered benzoic acid metabolism will be subjected to metabolic profiling, isotopic labeling experiments and computer-assisted pathway modeling to assess the carbon flow within benzoic acid network and detect flux redistribution during metabolic perturbation. Enzymes encoded by genes whose silencing leads to changes in benzoic acid metabolism will be biochemically characterized. Subcellular localization of these enzymes will be determined experimentally to evaluate the potential role of compartmentalization in regulating flux to benzoic acid. This research will result in the fundamental discovery of genes and enzymes involved in benzoic acid biosynthesis and fill important gaps in our knowledge of benzoic acid metabolism. Obtained results will provide a comprehensive understanding of structural and regulatory properties of the benzoic acid network and lay a foundation for future rational metabolic engineering of plants to improve flavor and aroma quality, boost plant defenses against environmental adversities, increase pollinator attraction and heighten amounts of biologically active compounds. Broader Impacts: This project has a strong multidisciplinary training component for the next generation of plant biochemists. The undergraduate and graduate students, as well as post-doctoral scientists (including women and minorities) will gain experience in plant enzymology, molecular biology, in vivo isotopic labeling, genetics, and integrative modeling. In addition to training students in the laboratory, the PI will organize a Graduate Research Seminar on biochemistry and metabolic engineering of plant secondary metabolism that will bring graduate students and postdocs from the US together with others from around the world and will precede the Gordon Research Conference on Plant Metabolic Engineering in 2011. This seminar will provide young scientists with a deeper understanding of plant metabolism and more actively involve them in science and education, as well as help them build professional relationships with future colleagues from abroad, particularly helpful since plant biochemistry is quite strong in certain foreign countries (e.g., Germany, Japan). Results and approaches used in this research will also be integrated into undergraduate and graduate education at Purdue University by developing course modules focused on plant specialized metabolism. The PI will provide mentorship to trainees for success in their future scientific endeavors.