Intellectual Merit: Metabolites are the building blocks and energy sources of life. Metabolomics, the large-scale study of metabolites, is revolutionizing our understanding of plant metabolism and novel gene function, but its full scientific promise has not been realized due to multiple technical challenges. These challenges include the accurate chemical identification of metabolites and a sophisticated understanding of the spatial/temporal distribution of metabolism. To address these issues, a synergistic international team of plant metabolomics and metabolism experts will use an integrated approach to push key technical developments. The project will use these enabling technologies to gain a better understanding of carbon sequestration and allocation in relationship to energy and a low carbon society. The major biological objective is to discover and elucidate key genes involved in metabolism of cell wall components (especially lignin and phenylpropanoids) and energy dense lipids. The major technical objective is to develop advanced analytical technologies and use them for the systematic and data directed metabolome annotation of two model plant species, Arabidopsis thaliana and Medicago truncatula (a close relative of alfalfa, Medicago sativa).
Broader Impacts: This project will enable the engineering of plants for bioenergy by advancing our understanding of carbon allocation and partitioning and, thus, contribute to a low carbon society. Novel and sophisticated metabolomics technologies will be developed, resulting in increased metabolomics coverage. Valuable spectral libraries will be generated and distributed to the metabolomics community. Collaborative relationships between US and Japanese scientists and institutions will be strengthened through a foreign exchange program. The project will provide multi-disciplinary training for four USA postdoctoral researchers in plant metabolomics, molecular biology, integrated systems biology, and cutting-edge analytical biochemistry. Hands-on metabolomics workshops will be conducted each year to provide advanced educational opportunities for the international metabolomics community. This project will support three summer Research Experiences for Undergraduates (REU) students each year.
Intellectual Merit: Metabolites are the building blocks and energy sources of life and the large-scale study of metabolites is known as metabolomics. Metabolomics is revolutionizing our understanding of plant metabolism and the discovery of novel gene functions. Although the growing utility of metabolomics is well documented, its full scientific promise has not yet been realized due to multiple technical challenges. These challenges include the accurate chemical identification of metabolites and a sophisticated understanding of the spatial/temporal distribution of metabolism. To address these issues, a synergistic, international team of plant metabolomics and metabolism experts was assembled, and this team used an integrated approach that united clear biological drivers to both push key technical developments and then pull from these enabling technologies a better understanding of carbon sequestration and allocation in relationship to energy and a low carbon society. The specific biological objectives of this project were to discover and characterize key genes that are involved in cell wall metabolism (especially lignin and phenylpropanoids) and energy dense lipids. Studies on the lignin pathway in Medicgao trunctaula by the Dixon laboratory yielded a set of homozygous mutant plants for most steps in the lignin pathway and several regulatory transcription factors. These are an important community resource for continued studies related to lignin. Metabolomics analyses of the homozygous lignin mutants were performed and revealed substantial changes in phenylpropanoid and triterpene saponins levels. These results provided new insights into the cross talk between lignin and other metabolic pathways and represent critical information necessary for engineering plants with enhanced bioenergy/bioprocessing capacity. The Nikolau lab has identified and characterized genes that encode subunits of the Type II fatty acid synthase (FAS) system and appear to be at the heart of mitochondrial FAS. The Nikolau lab has further characterized a series of T-DNA lines that carry mutations in the three ELO genes that appear to catalyze the condensation reactions which result in the elongation of the acyl-chain length in the ER-located fatty acid elongase system. The Nikolau and Saito groups have also integrated metabolomics databases developed at each institution, i.e. PMR and MeKO. The major technical objectives of this project were to develop advanced analytical technologies and use these for the systematic and biologically directed annotation of two model plant species metabolomes, namely Arabidopsis thaliana and Medicago truncatula, which is a close relative of alfalfa (Medicago sativa). Much success was achieved related to these specific objectives. The Sumner lab produced a powerful, novel software package called Plant Metabolite Annotation Toolbox (PlantMAT) that enables the automated prediction of metabolite identify based upon multiple and orthogonal experimental data, custom and public databases, and literature content. The Sumner lab also assembled a sophisticated analytical ensemble consisting of a Waters Acquity IClass ultrahigh pressure liquid chromatograph (UHPLC), Bruker maXis quadrupole time of flight mass spectrometer (QToF-MS), Bruker/Spark Holland solid phase extractor (SPE), a Gilson 215 liquid sample handler robot, and a Bruker Avance III 600 MHz nuclear magnetic resonance (NMR) spectrometer with 1.7 mm TCI cryoprobe. This ensemble has increased the efficiency for higher-throughput metabolite identifications. The identities for approximately 120 specialized metabolites were predicted by the Sumner lab based upon UHPLC-MS/MS experimental data. From these predictions, over 40 novel metabolites were identified by NMR, and our ‘production’ pipeline continues. In addition, over 100 metabolites have been identified by GCMS spectral matching to authentic standards. Mass spectral libraries have also been developed and are available to the public at www.noble.org/apps/Scientific/WebDownLoadManager/DownloadArea.aspx. The Nikolau lab has also developed ultra-high resolution, FT-ICR-Mass Spectrometry combined with stable-isotope labeling for metabolite annotation. This platform was used to identify two previously undetected phospholipids and for the identification of other fatty acid biosynthetic intermediates. Broader Impacts: The overall outcomes of this project have been significant advances in our understanding of carbon allocation and partitioning in plant specialized and lipid metabolism that has led to greater opportunities for engineering renewable bioenergy resources resulting in a low carbon society. Novel and sophisticated metabolomics technologies were developed resulting in increased metabolomics coverage. Strong, collaborative relationships were built between US and Japanese scientists. These synergistic interactions accelerated progress and provided a much deeper knowledge base from which to build success. This project and the NSF-JST Metabolomics for a Low Carbon Society program were also a spring-board for establishing a new Plant, Algae, and Microbial Research Coordination Network (PAMM-NET; NSF IOS Award#1340058). The goals of PAMM-NET are to promote effective communication, enhance opportunities for collaboration, build community consensus, identify key challenges in metabolomics, and to facilitate coordinated community empirical efforts to meet these challenges. The project has provided multi-disciplinary training for four USA postdoctoral researchers, two graduate students and three undergraduate interns in plant metabolomics, molecular biology, integrated systems biology, and cutting-edge analytical biochemistry. Metabolomics workshops were conducted each year to provide advanced educational opportunities for the international metabolomics community.
