Identification of multiple lipid genes with modifications in expression and sequence associated with the evolution of hydroxy fatty acid accumulation in Physaria fendleri

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. Significance Statement Comparison of protein sequences and transcript expression from developing seeds of Physaria fendleri that accumulate high amounts of hydroxy FAs, to Camelina sativa, a closely related plant that does not accumulate hydroxy FAs revealed multiple genes, many of which have not been previously considered, that likely co-evolved to enable hydroxy FA accumulation in Physaria. These genes provide new strategies to improve the ability of transgenic commodity crops to accumulate these and other value-added oils.