Fatty acids are produced by plants form the core of membranes and serve many other essential functions such as 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. Our group has focused on the following aspects of plant lipid metabolism. 1) We engineer plants using genes involved in lipid metabolism to obtain useful alterations in their seed oils. 2) In order to guide metabolic engineering of oils we use radioisotopes and stable isotopes to understand biosynthesis of lipids in oilseeds. These studies led to the discovery of a new metabolic route for carbohydrate conversion to oil in seeds. 3) 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. 4) We have characterized genes for several enzymes of lipid metabolism and are studying these genes and enzymes through biochemical and genomic approaches.
Metabolic Engineering of Oilseeds
Our lab has engineered the oilseed crop Camelina to produce high levels of a special oil with reduced viscosity, freezing point and calorific value.
This oil (acetyl-triacylglycerol or acetyl-TAG) is unusual because it has a 2-carbon acetyl group, rather than 16-20 carbon fatty acids on the sn-3 carbon of glycerol (Fig. 1).
The properties of acetyl-TAG compared to conventional seed oils can enable its use in several food and non-food ‘value-added’ applications, including as emulsifiers, food coatings and plasticizers. These special oils may also have advantages as biodegradable lubricants and as ‘drop-in’ biodiesel for some engines.
To achieve high levels of acetyl-TAG in the oil, Camelina was engineered to express an enzyme (diacylglycerol acetyltransferase (EaDAcT)) discovered from Euonymus alatus (burning bush (Fig 1)). The transgenic Camelina was grown in MSU fields to provide gallons of oil for testing (fig 2).
Discovery of a New Pathway for Triacylglycerol Biosynthesis in Plants
The surface of the fruits Bayberry (Myrica pensylvanica) is covered with an extremely thick and unusual layer of crystalline wax. Bayberry fruits have the highest production of surface wax in nature, reaching up to 25% of the total fruit mass. The composition of Bayberry wax is also striking, consisting completely of saturated triacylglycerol (TAG) and diacylglycerol (DAG), with 85% palmitate and 14% myristate as the dominant acyl chains. To understand the production and secretion of Bayberry wax we characterized the transcriptome of the wax secreting tissue and we monitored wax biosynthesis through [14C]-acetate and [14C]- glycerol radiolabeling. We found that the most abundantly expressed genes at all stages of Bayberry wax synthesis are genes associated with surface lipid production. Transcripts for enzymes involved in seed TAG synthesis were 10-100 fold less abundant. There was also accumulation of monoacylglycerol (MAG), with the acylchain on the sn-2 position of glycerol (sn-2-MAG), which was previously shown to be an early intermediate in the biosynthesis of the surface lipid polyester cutin. Radiolabeling also identified sn-2 MAG as the first labeled lipid product, and the kinetics of MAG and DAG labeling supported their precursor-product relationship to TAG. Thus, we believe that enzymes involved in surface lipid biosynthesis have evolved to produce the massive accumulation of extracellular TAG and DAG. Such a pathway for triacylglycerol synthesis in plants has not been previously described.
Video of wax secretion
Video of wax secretion
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.
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 use experimental systems that provide quantitative measurements of fluxes involved in the networks of lipid and central carbon metabolism. Developing oilseed embryos are labeled with a variety of 14C and13C-labeled precursors. We use kinetic and steady-state labeling to make quantitative determinations of flux ratios at branch points of metabolism. This methodology is 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. Some conclusions from our labeling and biochemical analysis of 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 synthesis
- Light contributes substantially to increasing oil synthesis in green seeds.
- RuBisCo plays a major role in the carbon economy of green oilseeds
- Tissue [CO2] concentrations during seed development are extremely high.