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).