Fatty acids produced by plants form the core of membranes and serve many other essential functions including providing surface protection from biotic and abiotic stress and as precursors for signaling molecules. In addition, oils produced by seeds are a major source of calories in diets and an important source of lubricants, detergents and chemical feedstocks. A long term goal of my laboratory is to understand how plants control the activity of fatty acid synthesis and lipid metabolism pathways and how their products are channeled into diverse roles and locations within or outside the plant cell. My lab is currently focusing on understanding the following aspects of plant lipid metabolism. 1) We are characterizing the biosynthesis of cutin and suberin. These structures are polymers of fatty acids that occur outside the cell and provide a barrier to water and gas exchange and protection against pathogens (more info 1). 2) In order to guide rational efforts toward metabolic engineering of seeds we are developing flux models of central metabolism in oilseeds. Recently we discovered a new metabolic route for carbohydrate conversion to oil in seeds . 3) We have characterized genes for several enzymes of lipid metabolism and are studying the regulation of these genes through microarrays and other genomic approaches. We also transform plants with genes involved in lipid metabolism in sense and antisense constructs both to study their physiological role and to obtain useful alterations in plant oils.
Understanding the Chemistry and Biosynthesis of Lipid Polymers of Arabidopsis
All aerial parts of vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells. The cuticle is composed of a cutin polymer matrix with waxes embedded in the matrix and also deposited on its surface (Figs 1, 2 and 3). Suberin has similar functions for roots, periderm and wounded tissues. Cutin and suberin are the most abundant lipid polymers in nature. They are assembled as polyesters from fatty acid monomers but many aspects of their structure and biosynthesis are poorly understood.
Finding Candidate Genes For Cutin Biosynthesis: Microarrays Of Arabidopsis Epidermis Arabidopsis has a very fragile cuticle but epidermal tissue can be harvested by peeling from stems (Fig 5). RNA was isolated from top or bottom of 10 cm long stems and probes prepared and hybridized to Affymetrix 22K array. Whole stem sections were used as reference. Analysis of gene expression in epidermis and its relation to lipid synthesis is described in: Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F. (2005) Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol. 139:1649-65
New Insights into Oilseeds from Metabolic Flux Analysis
The life of all cells depends on the coordinated, efficient and regulated flux of biochemical reactions. We want to better understand carbon fluxes and their subcellular distribution during oilseed metabolism so that we can more effectively use metabolic engineering to design improved crops. We are establishing experimental systems and modeling methods that provide quantitative measurements of fluxes involved in the networks of central carbon metabolism. Developing oilseed embryos are labeled with a variety of 13C-labeled precursors and the distribution of 13C in individual atoms of the products of metabolism is analyzed by GC/MS and NMR. We use steady-state labeling because this method offers the ability to make quantitative determinations of flux ratios at branch points of metabolism. This methodology (sometimes called Metabolic Flux Ratio Analysis or MetaFor) can be used to investigate fluxes in vivo in systems that have not been perturbed by cell disruption, mutations or transgenes. We also have investigated the "carbon use efficiency" of developing seeds by measuring the proportion of carbon taken up by embryos that is recovered in storage end products. Results from our labeling and biochemical analysis have so far led to the following conclusions about oilseed metabolism: Most carbon enters the plastid from the cytosol as PEP or higher intermediates, rather than pyruvate, malate or acetate.The oxidative pentose phosphate pathway does not provide the major source of NADPH for fatty acid synthesisLight contributes substantially to increasing oil synthesis in green seeds.RuBisCo plays a major role in the carbon economy of green oilseedsTissue [CO2] concentrations during seed development are extremely high.
Global Analysis of Gene Transcription during Seed Development
Seeds represent the major source of food for the worlds population and represent the major value of most agricultural products. We have used cDNA microarrays to examine changes in gene expression during Arabidopsis seed development and to compare wild-type and mutant (wrinkled1) seeds that have an 80% reduction in oil. Between 5 to 13 days after flowering (DAF), a period prior to and including the major accumulation of storage oils and proteins, approximately 35 % of the genes represented on the array changed at least 2 fold, but a larger fraction (65%) showed little or no change in expression. Genes whose expression changed most tended to be expressed more in seeds than in other tissues. Genes related to biosynthesis of storage components showed several distinct temporal expression patterns. For example, a number of genes encoding core fatty acid synthesis enzymes displayed a bell-shaped pattern of expression between 5 and 13 DAF. In contrast, the expression of storage proteins, oleosins and other known ABA regulated genes increased later and remained high. Genes for photosynthetic proteins followed a pattern very similar to that of fatty acid synthesis proteins implicating a role in CO2 refixation and supply of cofactors for oil synthesis. Expression profiles of key carbon transporters and glycolytic enzymes reflected shifts in flux from cytosolic toward plastid metabolism. Despite major changes in metabolism between wrinkled1 and wild-type seeds, less than 1% of genes differed more than 2-fold, and most of these were involved in central lipid and carbohydrate metabolism.
Allen, D.K., LaClair, R.W., Ohlrogge, J.B., and Shachar-Hill, Y. (2012). Isotope labeling of RuBisCO subunits provides in vivo information on subcellular biosynthesis and equilibration of amino acids. Plant Cell and Environment In press.
Tjellstrom, H., Yang, Z., Allen, D.K., and Ohlrogge, J.B. (2011). Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids. Plant Physiol.
Chapman, K.D., and Ohlrogge, J.B. (2012). Compartmentation of triacylglycerol accumulation in plants. J Biol Chem 287, 2288-2294.
Troncoso-Ponce, M.A., Kilaru, A., Cao, X., Durrett, T.P., Fan, J., Jensen, J.K., Thrower, N.A., Pauly, M., Wilkerson, C., and Ohlrogge, J.B. (2011). Comparative deep transcriptional profiling of four developing oilseeds. Plant J 68, 1014-1027.
JB, O., and K, C. (2011). The seeds of green energy - expanding the contribution of plant oils as biofuels. The Biochemist 33, 34-38.
Bourgis, F., Kilaru, A., Cao, X., Ngando-Ebongue, G.F., Drira, N., Ohlrogge, J.B., and Arondel, V. (2011). Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Natl Acad Sci U S A 108, 12527-12532.
Yang, W., Pollard, M., Li-Beisson, Y., Beisson, F., Feig, M., and Ohlrogge, J. (2010). A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol. Proc Natl Acad Sci U S A 107, 12040-12045.
Durrett, T.P., McClosky, D.D., Tumaney, A.W., Elzinga, D.A., Ohlrogge, J., and Pollard, M. (2010). A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds. Proc Natl Acad Sci U S A 107, 9464-9469.
Zhang, M., Fan, J., Taylor, D.C., and Ohlrogge, J.B. (2009). DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development. Plant Cell 21, 3885-3901.
Yang, Z., and Ohlrogge, J.B. (2009). Turnover of fatty acids during natural senescence of Arabidopsis, Brachypodium, and switchgrass and in Arabidopsis beta-oxidation mutants. Plant Physiol 150, 1981-1989.
Ohlrogge, J., Allen, D., Berguson, B., Dellapenna, D., Shachar-Hill, Y., and Stymne, S. (2009). Energy. Driving on biomass. Science 324, 1019-1020.
Molina, I., Li-Beisson, Y., Beisson, F., Ohlrogge, J.B., and Pollard, M. (2009). Identification of an Arabidopsis feruloyl-coenzyme a transferase required for suberin synthesis. Plant Physiol 151, 1317-1328.
Li-Beisson, Y., Pollard, M., Sauveplane, V., Pinot, F., Ohlrogge, J., and Beisson, F. (2009). Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester. Proc Natl Acad Sci U S A 106, 22008-22013.
Bates, P.D., Durrett, T.P., Ohlrogge, J.B., and Pollard, M. (2009). Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos. Plant Physiology 150, 55-72.