Yair Y. Shachar-Hill - US grants

Plant seeds are the world's most important agricultural product and the lowest cost biological source of carbohydrates, oils and proteins. An important goal of plant metabolic engineering is to develop chemical and nutritional production systems in seeds that are amenable to rational genetic engineering. To reach this goal, a quantitative understanding of fluxes through biochemical pathways is needed for seeds. This project will begin to provide such an understanding through analysis and engineering of the accumulation of storage oils in Brassica napus seeds.

Three central questions about oilseed metabolism will be addressed: How do seeds cope with the CO2 generated during oil synthesis? By what pathways does carbon flow from sucrose to oil and storage proteins and how are these pathways influenced by availability of light? What is the source of reductant for fatty acid biosynthesis? These problems are interlinked by shared metabolic intermediates and pathways and the processes they represent must be coordinately regulated during seed development.

This project will lead to a quantitative description of metabolic networks in a major agricultural production system, and to improved strategies for engineering changes in metabolism. The resulting progress in understanding, modeling and engineering seed metabolism and the interdisciplinary training of students and postdocs will help move plant metabolic engineering from the current hit and miss state toward a framework of rational design and analysis. This information will be crucial to expanding the use of plants as green factories that provide renewable and sustainable alternatives to petroleum and also aid nutritional improvements in the seeds that provide most of the world's food.

The arbuscular mycorrhizal symbiosis, a close interaction between plant roots and soil fungi, is essential for the nutrient uptake of approximately 80 % of all known plant species. The arbuscular mycorrhizal fungus with its extraradical mycelium takes up nutrients such as phosphate and nitrogen for the plant, and is supplied in exchange with carbohydrates from the plant. However, our knowledge of the metabolic pathways involved in nitrogen uptake and transfer to the host plant and about the regulation of nutrient exchange between the symbiotic partners is still limited. The goal of this project is to understand the exchange of nutrients in the arbuscular mycorrhizal symbiosis, which is also key to applying arbuscular mycorrhizal fungi as "biofertilizers" in sustainable agriculture. The experimental approach includes stable and radioactive isotope labeling, protein quantification and enzymatic assays, gene expression studies and protein and elemental localization techniques such as immunohistochemistry and energy dispersive X-ray microanalysis. This project will determine how nitrogen is handled in the arbuscular mycorrhizal symbiosis, and how the flux of nitrogen in the symbiosis is regulated. The trainees, including a postdoc, a graduate student and undergraduates will learn an unusually broad range of modern biological techniques and will gain insights into the arbuscular mycorrhizal symbiosis and its importance for the nutrient acquisition of plants.

This award is for the acquisition of a mass spectrometer (a triple quadrupole LC/MS/MS system) and a nitrogen generator for trace level structure-based screening and quantitative profiling of nonvolatile metabolites and reaction products. This system will enable sensitive and high-throughput non-target (metabolomics) and target metabolite analyses. Biology is moving toward a new and interdisciplinary paradigm of systems biology, which seeks to replace the one gene, pathway, or physiological process-at-a-time approaches to understanding complex biological systems with a more efficient and holistic concept. This approach requires the production and datamining of comprehensive and high quality data sets describing dynamic changes in genome, transcriptome, proteome and metabolome. The instruments acquired through this award will allow the investigators to take advantage of the great technological advances that have been made in methods for analysis of metabolites, including the global approach termed metabolomics, largely due to improvements in Mass Spectrometry in recent years.

This mass spectrometer was chosen because it is user friendly, for use by undergraduate and graduate students, as well as postdoctorals and faculty. Individuals trained in mass spectrometer operation, analytical method design, and data interpretation will represent a good gender balance and come from a variety of ethnic, cultural, and educational backgrounds. This is due to recruitment infrastructure on campus and a group of participating faculty who are culturally and ethnically diverse and who seek to train a diverse group of students. Training sessions and workshops will introduce students to the problem-solving power of mass spectrometry, which is one of the most dynamic fields of analytical chemistry. Use of these instruments will accelerate research and improve understanding of gene functions, responses of plants, animals, and microbes to changing environments, and the chemistry governing interactions between organisms.

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

The study of plant biology has advanced our understanding in areas not only related to agriculture and food quality, but also in the areas of human biology. Recent advances in technologies in the area of biochemistry, cell biology and genetics have enabled plant scientists to address biological questions previously unattainable. This award provides support for the acquisition of a laser capture microdissection (LCM) system and ancillary equipment for advanced histologic analysis of plant materials at Michigan State University. LCM is an advanced method for analyzing single cells in living multicellular organisms. Using this technique, cells from a target microscopic region can be selected, isolated, and analyzed. In short, this yields precise cellular snapshots of cell diversity and the mechanisms controlling processes as varied as development, stress, metabolism, pathogenesis, and senescence. As a world leader in the experimental plant sciences, MSU is extremely well placed to use and disseminate this powerful technology, and the addition of LCM will leverage existing technological strengths at MSU to catalyze breakthroughs in plant biology. In addition, through hands-on training and education, post-graduate, graduate, undergraduate and even high school students will receive specialized training using LCM, as well as downstream genomic and proteomic techniques at MSU.

Plant biomass such as wood, grass straw, and agricultural residues represent an abundant, cheap, and renewable feedstock for the production of liquid transportation fuels and chemicals. One way to make fuel out of biomass is through a process called fast pyrolysis, where the solid plant material is rapidly heated in the absence of air to decompose it to a mixture of gas, solid char, and a liquid called bio-oil, which can be upgraded to liquid transportation fuel. Plant biomass is a mixture of three biologically- produced polymers - cellulose, hemicellulose, and lignin - arranged into a complex three-dimensional network. The origin of pyrolysis products from this complex mixture is not well understood, since all these materials interact with one another during biomass pyrolysis. This lack of understanding has hampered efforts to maximize bio-oil production from a given plant species. This project seeks to map the origin of biomass pyrolysis products to specific biomass components within real biomass materials. A key innovation is the use of plant cell technology to label the cellulose and lignin components so that their fate can be tracked during fast pyrolysis. The educational activities associated with this project include a summer residential program for high school students using topics developed from the research. Students will see how plants capture and store carbon and energy, use chemical probes to help discover biological processes, and discuss the opportunities and challenges of obtaining fuels and chemicals form renewable resources.

The overall goal of the research is to identify and map reaction pathways for fast pyrolysis of lignocellulosic biomass to specific cellulosic and lignin molecular constituents. The research plan has two objectives to trace the origins of the fast pyrolysis products. The first objective is to develop a methodology to enable mapping of the reaction pathways through carbon-13 labeling. Towards this end, cells of the model plant Arabidopsis thaliana will be heterotrophically cultured on a mixture of unlabeled and 13-C labeled glucose and phenylalanine, precursors for cellulose and lignin biosynthesis respectively, to separately tag the carbohydrate and lignin fractions in the Arabidopsis cell wall. Arabidopsis stem cuttings will then be pyrolyzed, with and without catalyst, using 13C-labeled Arabidopsis cells as probes at different temperatures. The results will be compared to the products of simple mixtures of unlabeled cells with labeled small-molecule building blocks. By tracking the fate of the labeled sites, it will be possible to construct a reaction network that describes both their incorporation in the plant matter, and the chemical events that form the pyrolysis products. The second objective is to use the methodology developed under objective 1 on woody plants within genera Populus and Paniceae that are relevant to bioenergy production. The research outcomes will lead to improved, mechanism-based reaction and kinetic models for predicting product yields from reaction pathways for fast pyrolysis from real lignocellulosic biomass substrates, not just model compounds. This information can then be used to identify reactor design and operation strategies to stabilize and optimize the yield of bio-oil and its downstream conversion into liquid transportation fuel.