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Associate Professor of Plant Biology Department of Plant Biology Plant Biology Department
Selected Recent Publications; Publication List; Current Funding; Teaching; Lab Members |
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Research
NMR spectroscopy for probing plant metabolism and transport
Much of our work involves the use of Nuclear Magnetic Resonance (NMR) spectroscopy to tackle problems in plant metabolism and transport and I have a longstanding interest in the development and application of NMR methods for this purpose. NMR can be used with or without isotopic labeling to identify, quantify and localize metabolites, to define the intracellular environment, and to determine metabolic and transport fluxes. Its advantages include its non-destructive nature, the ease of sample preparation, the simultaneous detection of multiple metabolites, and the positional isotopic information that spectra give directly.
In recent years progress in several fields has made NMR even more useful. One area of progress is the advent of molecular biological methods that make both the analysis and the manipulation of gene expression increasingly straightforward. Another is the development of equipment and techniques that greatly increase sensitivity and discrimination in NMR spectra, both in vivo and of extracts. A third area is the development of theoretical tools for determining fluxes in metabolic networks from the isotopic labeling patterns in metabolic products. The synergy between these developments and NMR is exemplified below in the areas of metabolism in the arbuscular mycorrhizal symbiosis and plant metabolic engineering.
Links: Max T. Rogers NMR facility at MSU , George Ratcliffe, collaborator on Plant NMR
Metabolism and transport in the Arbuscular Mycorrhizal Symbiosis
Working model of carbon fluxes in the AM symbiosis
"The mycorrhiza rather than the root is the chief organ of nutrient uptake" Smith and Read 1997
The arbuscular mycorrhizal (AM) symbiosis is the relationship between plants and a group of beneficial fungi. These fungi have been growing in and around the roots of most land plants worldwide for hundreds of millions of years and they greatly increase the ability of plants to take up nutrients (particularly phosphorous) from the soil. In return for assistance in nutrient uptake, AM fungi receive their carbon from the host plant. We are studying the uptake, transport, exchange and metabolism of carbon and nitrogen compounds by the host roots and fungus. We use NMR spectroscopy and mass spectrometry together with stable isotope labeling to follow the biochemical processes of the symbiosis. In this part of the work we collaborate with Philip Pfeffer and coworkers at the USDA labs in Philadelphia. In collaboration with Peter Lammers at New Mexico State University we are using molecular biological tools to analyze the gene expression involved in these biochemical processes and their regulation.
Our results are revealing the nature of the metabolic pathways, storage compounds, pathways and regulation that are involved in what is arguably the world's most important symbiosis.
Metabolic Engineering in Plants
Our knowledge of the regulation and compartmentation of metabolism in plants is far from complete. This contributes dramatically to the failure rate of metabolic engineering efforts, which are usually unsuccessful in their practical aims for unforeseen (and often unexamined) reasons. To learn from failures and to increase the chances of future success one needs to analyze the metabolic network involved and the effects upon it of genetic manipulations. This involves the assignment of gene function and the analysis of flux through the metabolic network. In two projects involving plant metabolic engineering we are engaged in this effort:
Metabolic flux analysis of developing seeds
In collaboration with John Ohlrogge we are mapping the fluxes through central metabolism in developing oilseed rape (canola) seeds that lead to the production of oil protein and carbohydrates. We are using a combination of steady-state stable isotopic labeling, mass balancing, GC-MS, NMR and computer-aided flux modeling to address questions about the routes and rates of carbon flow, and the sources and sinks of cofactors and CO2.
Flux map for central carbon metabolism of B. napus embryos during storage oil and protein accumulation. Only net fluxes are shown. Biomass constraints (red arrows) were derived considering the three main biomass components seed oil, protein and starch using the stoichiometry of their biosynthesis. Additional flux measurements (blue arrows) are derived by 13C- and 15N-labeling experiments and flux parameter fitting. Dependent fluxes are shown with black arrows. Abbreviations: Ac-CoA, acetyl coenzyme-A; Cit, citrate; E4P,erythrose-4-phosphate; HP, hexose-phosphate; KG, keto-glutarate; ME, malic enzyme; ACL, ATP:citrate lyase; OAA, oxaloacetate; PEP, phosphoenol pyruvate; PGA, 3-phospho-glycerate; PP, pentose-phosphate; Pyr, pyruvate; TP, triose-phosphate.
In collaboration with Andrew Hanson (U. FL, Gainesville) we are studying the function of non-photosynthetic one-carbon genes. This involves the integration of biochemical, molecular biological, metabolic modeling and analytical expertise. NMR has thus far contributed to testing model-generated hypotheses about glycine betaine metabolism, to identifying the function of a putative pipecolinate oxidase and to identifying the product of p-aminobenzoate metabolism in plants. We are currently studying the function of 5-Formyl-THF cycloligase in arabdopsis. The network of non-photosynthetic one carbon metabolism in plants showing the location of genes whose function is being studied and the sources and sinks for labeled one carbon units
Selected Recent Publications
Last RL, Jones AD, Shachar-Hill Y (2007) Towards the plant metabolome and beyond. Nature Reviews Molecular Cell Biology 8: 167 - 174
Ratcliffe RG and Shachar-Hill Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant Journal 45: 490-511
Govindarajulu M, Pfeffer PE, Jin H, Abubaker J, Douds DD, Allen JW, Bücking H,Lammers PJ, Shachar-Hill Y (2005) Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature 435: 819-823
Schwender J, Goffman F, Ohlrogge JB, Shachar-Hill Y (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432: 779-782
PLB 801,Graduate Seminar
PLB 803 Integrative Topics in Plant Biology
PLB 802/BMB 961 Special Engineering and Quantifying Metabolic Networks
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Eva Collakova, Postdoctoral Research Associate |
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Joanna Cross, Postdoctoral Research Associate |
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Jackson Gehan, Graduate Student |
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Igor Libourel, Postdoctoral Research Associate |
