SummarySeed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn-3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several nonfood and food applications.

Two Brassicaceae species, Physaria fendleri and Camelina sativa, are genetically very closely related to each other and to Arabidopsis thaliana. Physaria fendleri seeds contain over 50% hydroxy fatty acids (HFAs), while Camelina sativa and Arabidopsis do not accumulate HFAs. To better understand how plants evolved new biochemical pathways with the capacity to accumulate high levels of unusual fatty acids, transcript expression and protein sequences of developing seeds of Physaria fendleri, wild-type Camelina sativa, and Camelina sativa expressing a castor bean (Ricinus communis) hydroxylase were analyzed. A number of potential evolutionary adaptations within lipid metabolism that probably enhance HFA production and accumulation in Physaria fendleri, and, in their absence, limit accumulation in transgenic tissues were revealed. These adaptations occurred in at least 20 genes within several lipid pathways from the onset of fatty acid synthesis and its regulation to the assembly of triacylglycerols. Lipid genes of Physaria fendleri appear to have co-evolved through modulation of transcriptional abundances and alterations within protein sequences. Only a handful of genes showed evidence for sequence adaptation through gene duplication. Collectively, these evolutionary changes probably occurred to minimize deleterious effects of high HFA amounts and/or to enhance accumulation for physiological advantage. These results shed light on the evolution of pathways for novel fatty acid production in seeds, help explain some of the current limitations to accumulation of HFAs in transgenic plants, and may provide improved strategies for future engineering of their production.

Over 450 structurally distinct fatty acids are synthesized by plants. We have developed PlantFAdb.org, an internet-based database that allows users to search and display fatty acid composition data for over 9000 plants. PlantFAdb includes more than 17 000 data tables from >3000 publications and hundreds of unpublished analyses. This unique feature allows users to easily explore chemotaxonomic relationships between fatty acid structures and plant species by displaying these relationships on dynamic phylogenetic trees. Users can navigate between order, family, genus and species by clicking on nodes in the tree. The weight percentage of a selected fatty acid is indicated on phylogenetic trees and clicking in the graph leads to underlying data tables and publications. The display of chemotaxonomy allows users to quickly explore the diversity of plant species that produce each fatty acid and that can provide insights into the evolution of biosynthetic pathways. Fatty acid compositions and other parameters from each plant species have also been compiled from multiple publications on a single page in graphical form. Links provide simple and intuitive navigation between fatty acid structures, plant species, data tables and the publications that underlie the datasets. In addition to providing an introduction to this resource, this report illustrates examples of insights that can be derived from PlantFAdb. Based on the number of plant families and orders that have not yet been surveyed we estimate that a large number of novel fatty acid structures are still to be discovered in plants.