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).