Oil body proteins
OBPs stabilize the tightly packed oil bodies in cotyledon cells (Figure 1E). Oleosin accounts for 75-80% of the OBPs associated with B. napus and A. thaliana OBs, while the remaining 20-25% consists of caleosin and steroleosin (Jolivet et al., 2009). Surface-active agents, such as SDS, adsorb to the oil-water interface and results in displacing of OBPs. The polypeptides isolated from camelina OBPs were ~ 15-20 kDa and 26 kDa (Figure 5A) under both non-reducing and reducing conditions and may represent oleosins and caleosins, respectively, while the ~ 37.8 kDa polypeptide may be steroleosins as reported for other crucifers (Jolivelt et al., 2009; Katavic et al., 2006; Tzen, 2012). A washing step at pH 11 was included to remove co-extracted SSPs, particularly napin, and very few low molecular weight proteins were visible in the isolated OBPs. Separation of OBPs by 2DE showed protein spots distributed over a broad pH range of 3-12 (Figures 5B-E). Of the twelve oleosin isoforms encoded by genes in the C. sativa genome (Supplementary Table S1), seven (CsOle1-1-G1, CsOle1-1-G3, CsOle2-1-G2, CsOle3-1-G1, CsOle3-1-G3, CsOle4-1-G1 and CsOle4-1-G2) and few other proteins related to the oleosin family were detected by LC-MS/MS analysis (Figures 5B-E, Supplementary Tables S4 and S5). CsOle1-1-G3 and CsOle2-1-G2 were present in 18 of 20 2DE spots obtained from the pH 3-10 and pH 9-12 OBP fractions. CsOle3-1-G1 was found in only a single 2DE spot. The five isoforms not observed in the OBP fraction were not present in the cruciferin-enriched or napin-enriched fractions. The largest oleosin isoforms identified were CsOle2-1-G1 and CsOle2-1-G2 which comprise 201 amino acid residues, whereas the smallest isoforms were CsOle3-1-G1, CsOle3-1-G2 and CsOle3-1-G3 which comprise 144 amino acid residues (Supplementary Figure S4). The oleosins have a highly conserved central hydrophobic domain (Supplementary Figure S4) that varies in length depending on the isoform (Tzen, 2012).
The genes that are responsible for caleosin and steroleosin expression in camelina are not well-defined; however, proteomics data revealed that caleosin-like proteins encoded by Csa03g006900, Csa05g023090 and Csa07g038560, were present in the OBP fraction (Supplementary Tables S4 and S5). Hydroxysteroid dehydrogenase also was detected. Katavic et al. (2006) reported that hydroxysteroid dehydrogenase is possibly the steroleosin present in B. napus , similar to sesame seed steroleosin. This putative steroleosin in camelina has a molecular weight between ~39 kDa and 42 kDa, which is in agreement with that reported by Tzen, (2012).