Hydroxy fatty acids (HFAs) are major chemical feedstocks for industries manufacturing cosmetics, paints, polymers and resins. Currently, Ricinus communis (Castor) is the only agronomic source of HFA but cultivation of castor is banned in US due to the presence of toxin in its seed. Physaria fendleri (Lesquerella) is a native plant from southwest US, non-toxic, and accumulates about 60% hydroxy fatty acids in its seed oil/triacylglycerol (TAG). However, P. fendleri is not a crop and requires breeding or engineering to be a high yielding oilseed crop. Therefore, it is important to understand the triacylglycerol assembly pathway involved in accumulation of HFA in P. fendleri seeds. Castor uses linear Kennedy pathway with efficient acyl-editing to accumulate triacylglycerol with HFAs whereas other oilseed plants use membrane lipid phosphatidylcholine (PC) as an intermediate to make TAG without HFAs. Most plants keep HFAs out of their membrane lipids so, P. fendleri was thought to use linear Kennedy pathway as castor to avoid unusual fatty acids in membrane lipids. But seed transcriptomics data suggested that expression of TAG assembly genes in P. fendleri are similar to other brassica species which use PC-derived DAG (diacylglycerol) pathway, thus making TAG assembly pathway in P. fendleri unclear. Metabolic labeling experiments using radioactive substrates in developing seeds demonstrated that P. fendleri uses a novel pathway known as TAG remodeling to make final TAG molecule containing two HFAs. In the TAG remodeling pathway, PC is used as an intermediate to first make 1HFA-TAG, subsequently a non-hydroxy fatty acid is removed and replaced with a second hydroxy fatty acid to make the final 2HFA-TAG species. A triacylglycerol lipase and acyl-CoA:diacylglycerol acyltransferase (DGAT) might work in tandem for TAG remodeling mechanism. Protein-protein interactions studies using Yeast two-hybrid and Bimolecular fluorescence complementation assays were able to identify the candidate genes for TAG remodeling. Green fluorescent protein (GFP) fused version of these candidate genes were used for their subcellular localization in tobacco leaves. In addition to identifying possible genes, Arabidopsis thaliana HFA line overexpressing those candidate enzymes from P. fendleri were used to test if TAG remodeling can be reconstituted in a different species.