Camelina sativa produces oil that is rich in polyunsaturated linoleic (18:2 Δ9,12) and linolenic (18:3 Δ9,12,15) acids. These fatty acids are obtained by the successive desaturation of oleic (18:1 Δ9) acid, which is catalyzed in the endoplasmic reticulum by two different microsomal desaturases: oleate Δ 12 desaturase (CsFAD2) and linoleate Δ 15 desaturase (CsFAD3). The objective of the present study is to investigate the contribution of these two desaturases to the composition and properties of C. sativa seed oil, and look over the effect that temperature exerts on their activity. Three different copies of both these genes were identified, which when analysed contained three histidine rich motifs (HXCGHX, HRXHH and HVXHH) and six highly conserved transmembrane domains. Comparing their sequences, the CsFAD2 copies accommodated four conservative changes (Glu-36-Asp, Arg-48-His, Val-97-Ala and Ala-177-Pro) and two semi-conservative ones (Val-63-Ile and Leu-249-Met), whereas only one semi-conservative change (Ala-327-Ser) was detected in CsFAD3 but with two extra amino acids (His-147 and Gly-148). The CsFAD2 and CsFAD3 cDNAs were heterologously expressed in Saccharomyces cerevisiae to confirm that they were active enzymes and their dependence on temperature was investigated by growing the recombinant yeast cells at low (22 °C) and optimal (30 °C) temperatures. The conversion of 18:1 Δ9 to 18:2 Δ9,12 by CsFAD2 was slightly better at 30 °C than at 22 °C, and it was also able to desaturate palmitoleic acid (16:1 Δ9) to hexadecadienoic acid (16:2 Δ9,12). By contrast, the conversion driven by CsFAD3 was enhanced 5-fold at 22 °C compared to 30 °C, although it was not able to desaturate 16:2 Δ9,12. The distribution of these microsomal desaturases was also studied in C. sativa, which were expressed most strongly in expanding leaves and developing seeds.
Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into glycerolipids. Acyl-ACP thioesterases are classified into two families, FatA and FatB, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. Camelina sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of Camelina sativa oil was discussed in the light of these results.
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