Cruciferins
2DE of the cruciferin-enriched fraction yielded 29 proteins in the neutral pH region in the range of 17-55 kDa (Figures 4A, B). Based on the normalized total spectral (NTS) values (Table 2), these spots consisted primarily of cruciferin isoforms (89.8-100%), with vicillins, napins and non-storage proteins present in minor amounts. Eleven of the twelve cruciferin isoforms encoded by genes in the C. sativagenome (Supplementary Table S1) were identified in the isolated protein (Supplementary Table S2). CsCruA-1-G2 was the most prevalent isoform and was present in all of the 29 2DE spots, followed by CsCruC-1-G2 (27 spots), CsCruC-1-G3 (27 spots), CsCruB-1-G3 (25 spots) and CsCruB-1-G1 (22 spots). The only isoform that was not present in the purified cruciferin fraction was CsCruD-1-G3; however, CsCruD-1-G3 was found in the purified oil body fraction (Supplementary Tables S4 & S5). All identified cruciferins were in the molecular weight range of 50-65 kDa, indicative of intact monomers/protomers. Cruciferin protomers consist of α- (~30-40 kDa) and β- (~20 kDa) polypeptide chains attached via a S-S linkage (Wanasundara, 2011). Camelina cruciferins ranged from 453 (CsCruD-1-G1 and CsCruD-1-G2) to 586 (CsCruB-1-G1) amino acid residues with variations depending on the respective gene (Supplementary Figure S1). The cruciferin C isoforms possess the longest α-chain (3-4 kDa longer), the highest isoleucine and tyrosine levels and the lowest alanine, phenylalanine and serine levels compared to the cruciferin A, B and D isoforms. Seeds from camelina lines devoid of cruciferin C isoforms (three cruciferin C genes edited by CRISPR/Cas9 technology) showed differences in amino acid and fatty acid compositions (Lyzenga et al., 2019).