title: Functional characterization of flax fatty acid desaturase FAD2 and FAD3 isoforms expressed in yeast reveals a broad diversity in activity.
abstract: With 45 % or more oil content that contains more than 55 % alpha linolenic (LIN) acid, linseed (Linum usitatissimum L.) is one of the richest plant sources of this essential fatty acid. Fatty acid desaturases 2 (FAD2) and 3 (FAD3) are the main enzymes responsible for the Δ12 and Δ15 desaturation in planta. In linseed, the oilseed morphotype of flax, two paralogous copies, and several alleles exist for each gene. Here, we cloned three alleles of FAD2A, four of FAD2B, six of FAD3A, and seven of FAD3B into a pYES vector and transformed all 20 constructs and an empty construct in yeast. The transformants were induced in the presence of oleic (OLE) acid substrate for FAD2 constructs and linoleic (LIO) acid for FAD3. Conversion rates of OLE acid into LIO acid and LIO acid into LIN acid were measured by gas chromatography. Conversion rate of FAD2 exceeded that of FAD3 enzymes with FAD2B having a conversion rate approximately 10 % higher than FAD2A. All FAD2 isoforms were active, but significant differences existed between isoforms of both FAD2 enzymes. Two FAD3A and three FAD3B isoforms were not functional. Some nonfunctional enzymes resulted from the presence of nonsense mutations causing premature stop codons, but FAD3B-C and FAD3B-F seem to be associated with single amino acid changes. The activity of FAD3A-C was more than fivefold greater than the most common isoform FAD3A-A, while FAD3A-F was fourfold greater. Such isoforms could be incorporated into breeding lines to possibly further increase the proportion of LIN acid in linseed.
authors: Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB, R3T 2M9, Canada.
authors: Natasa
authors: N
authors: Radovanovic
authors: Dinushika
authors: D
authors: Thambugala
authors: Scott
authors: S
authors: Duguid
authors: Evelyn
authors: E
authors: Loewen
authors: Sylvie
authors: S
authors: Cloutier
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000378:metabolism; D019597:Flax / Q000201:enzymology / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D014644:Genetic Variation; D019787:Linoleic Acid / Q000378:metabolism; D008043:Linseed Oil / Q000737:chemistry / Q000378:metabolism*; D019301:Oleic Acid / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
chemical_list: D020033:Protein Isoforms; D019301:Oleic Acid; D008043:Linseed Oil; D019787:Linoleic Acid; D044943:Fatty Acid Desaturases; C040448:delta-12 fatty acid desaturase