Benzoic Acid Biosynthesis in Plants

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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.