http://www.greatlakesbioenergy.org/ Project Goals: Design of Improved Plant Oil Structure for Fuel Use. The high viscosity of plant oils causes problems with standard diesel engines. Therefore, most biodiesel is produced by conversion of triacylglycerol to methyl or ethyl esters. Acetyl-glycerides are abundant in some plant species and represent a form of triacylglycerol with acetyl rather than acyl groups at sn-3. This structure results in oil with predicted lower viscosity that should directly replace diesel fuel #4 without need for transesterification. The goal of this study is to identify genes involved in biosynthesis of this valuable type of plant oil. Unlike microarrays, EST sequencing provides a method of transcript analysis that allows quantitative comparisons between genes and between different plant species. In order to identify genes involved in plant oil biosynthesis and the transcription factors and other regulatory systems that control oil accumulation, Michigan State, together with JGI has sequenced over 10 million ESTs from a variety of oilseeds and other oil rich tissues. Why do we need millions of ESTs? Key enzymes of lipid metabolism (e.g. acyltrasferases, phospholipases, thioesterases) are very low abundance and can be difficult to detect by conventional EST sequencing. Deep EST sequencing using 454 pyrosequencing provides a large increase in EST sequence information and allows us to accurately quantify low level expression. By sequencing libraries from multiple species we obtain information on what similarities and differences distinguish oil synthesis in seeds producing unusual fatty acids and in seeds compared to other tissues such as mesocarp that produce high oil levels. Replicate analysis of samples (including cDNA synthesis and PCR) gave a 0.99 correlation coefficient between # reads per gene. Therefore, 454 sequencing is technically and biologically reproducible and provides an accurate measure of gene expression. We have observed that core enzymes of fatty acid biosynthesis are, in general, expressed in consistent stoichiometric ratios in a number of different oilseeds and tissues. Therefore, those genes that fall outside the usual stoichiometry offer insight into unique metabolism. For example, we observe very low expression of the FatB thioesterase that controls saturated fatty acid production in castor, which agrees with the fact that castor is an oilseed with extremely low saturated fatty acid content. Endosperm tissue from Euonymus alatus (Burning Bush) accumulates high levels of 3-acetyl-1,2- diacyl-sn-glycerols (ac-TAGs) as the major storage lipids. Ac-TAGs are unusual triacylglycerols (TAGs) with an sn-3 acetate group instead of a long-chain fatty acid and have added value applications in direct use as biodiesel and lubricant oil feedstocks. In addition to producing ac-TAGs, Euonymus fruit also synthesize normal, long-chain TAGs (lc- TAGs) in their aril tissue. The close developmental coordination and spatial proximity of two tissues with the ability to produce different TAGs presents a unique opportunity to understand the accumulation of unusual TAGs in plants. By sequencing ESTs from these tissues we have identified candidate genes involved in ac-TAG biosynthesis. One such candidate, subsequently named EaDAcT (Euonymus alatus diacylglycerol acetyl-transferase) was highly expressed in the endosperm and absent from the aril. Expression of EaDAcT in yeast resulted in the accumulation of ac-TAGs, but not lc-TAGs. In vitro assays with microsomes from yeast expressing EaDAcT demonstrated that the enzyme possesses acetyl-CoA diacylglycerol acetyltransferase activity, but not