Mol Biotechnol DOI 10.1007/s12033-014-9737-1

RESEARCH

Functional Characterization of Flax Fatty Acid Desaturase FAD2 and FAD3 Isoforms Expressed in Yeast Reveals a Broad Diversity in Activity Natasa Radovanovic • Dinushika Thambugala • Scott Duguid • Evelyn Loewen • Sylvie Cloutier

Ó Her Majesty the Queen in Right of Canada as represented by: the Minister of Agriculture 2014

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 D12 and D15 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

Electronic supplementary material The online version of this article (doi:10.1007/s12033-014-9737-1) contains supplementary material, which is available to authorized users. N. Radovanovic  S. Cloutier (&) Cereal Research Centre, Agriculture and Agri-Food Canada, 195 Dafoe Road, Winnipeg, MB R3T 2M9, Canada e-mail: [email protected] D. Thambugala  S. Cloutier Department of Plant Science, University of Manitoba, 66 Dafoe Road, Winnipeg, MB R3T 2N2, Canada S. Duguid  E. Loewen Morden Research Station, Agriculture and Agri-Food Canada, 101 Route 100, Unit 100, Morden, MB R6M 1Y5, Canada

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. Keywords Fatty acid desaturase  Linseed  Yeast expression  FAD isoform activity

Introduction Flax (Linum usitatissimum L.) is a dual purpose crop that can be grown for its seed oil (linseed) or for its celluloserich stem fiber (fiber flax). The main fatty acids of linseed oil are palmitic (PAL, C16:0), stearic (STE, C18:0), oleic (OLE, C18:1cisD9), linoleic (LIO, C18:2cisD9,12), and linolenic acid (LIN, C18:3cisD9,12,15), accumulated as triacylglycerols (TAGs) and serving as storage lipids. Because humans cannot synthesize them, LIO and LIN constitute essential omega-6 and omega-3 fatty acids that must be obtained in their diet [1]. Higher plants such as soybean, corn, canola, sunflower, safflower, and flax are a major source of these essential polyunsaturated fatty acids (PUFAs), shown to have positive effects on human health [1, 2] by, for example, playing important roles in the prevention of coronary heart diseases, hypertension, and type 2 diabetes [3]. Typical low omega-3 fatty acid intake in the Western diet combined with high amounts of omega-6 results in a high omega-6/omega-3 ratio, which contributes to the pathogenesis of many diseases, including cancer,

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cardiovascular, inflammatory, and autoimmune diseases [4]. An additional health benefit to a higher LIN intake is exemplified by the ability of the human body to further desaturate and elongate LIN into eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) creating very long PUFAs (VL-PUFAs) that have an important role in brain development and function and have very strong antiinflammatory potential [5–7]. Therefore, oils with a high LIN content have therapeutic and nutraceutical applications, creating new markets for oils with specific fatty acid composition. Current linseed varieties display a wide range of fatty acid composition, as high as 70–73 % LIN in ‘‘high-LIN’’ or Nulin varieties and as low as 2–3 % LIN in solin varieties [8]. Conventional linseed varieties generally contain 50–59 % LIN, an amount considerably higher than other commonly used vegetable oils such as canola (11 %), soybean (8 %), and corn (1 %). Chia (Salvia hispanica), with 52–69 % LIN, and Camelina (Camelina sativa), with 30–38 % LIN, are also excellent sources of plant-based omega-3 fatty acids although their oil content is substantially less than linseed [9–12]. The oil content of chia ranges from 26 to 38 % [9, 12] and that of Camelina from 23 to 33 % [13]. Linseed oil content ranged from 31 to 46 % in a germplasm collection [14], but most current linseed varieties exceed 45 % [15–17]. In most plant tissues, over 75 % of the fatty acids are unsaturated [18]. They are synthesized in planta through a series of elongation and desaturation steps performed by elongases and desaturases. The production of OLE, LIO, and LIN is successively catalyzed by the soluble, plastidial, stearoyl-ACP desaturase (SAD EC#1.14.19.1) followed by the membrane-associated fatty acid desaturases 2 and 3 (FAD2 EC#1.14.19.6 and FAD3 EC#1.14.19.-) [19]. In planta, the TAG-incorporated PUFAs serve as plant storage lipids, play key roles in the proper functioning and the structure of biological membranes [20], and have regulatory roles as precursors of signaling molecules involved in plant stress response [21]. Structurally, all reported membrane-bound desaturases have three histidine boxes located at conserved positions in the protein and are essential for creating a catalytic pocket with the ability to bind Fe ions [22]. Desaturase genes have been cloned and characterized in Arabidopsis [23, 24], soybean [25, 26], and Brassica [27]. Some genes have also been functionally characterized in yeast, such as the two microsomal oleate desaturases from olive [28]. In soybean, FAD2-1a and FAD2-1b were shown to play an important role in controlling OLE acid level in seeds and were used to produce high OLE acid soybean [26]. In flax, FAD2 and FAD3 genes are present in duplicated paralogous copies named FAD2A,

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FAD2B, FAD3A, and FAD3B. Fofana et al. [29] cloned two partial copies of FAD2 genes from a cDNA library of flax developing bolls. Khadake et al. [30] reported LUFAD2-2 gene from flax variety NL 97 and showed that it shared 81 % sequence similarity with the previously reported FAD2 gene from cultivar Nike [31]. Sequence analysis confirmed the presence of the three histidine boxes and membrane-spanning regions characteristic of plant desaturases and the conserved C-terminus amino acid sequence involved in the sub-cellular microsomal location of the encoded enzymes. Two FAD3 genes cloned from the EMS mutant line 593–708 were shown to be nonfunctional and responsible for its low LIN content [32]. Banik et al. [33] cloned FAD3A, FAD3B, and a novel FAD3C gene from cultivar AC McDuff and breeding lines UGG5-5, M5791, and SP2047 and showed some allelic diversity as well as different expression profiles of these genes. Various mutations of fatty acid desaturase genes, altering the encoded enzymes, often affecting their activity or even causing complete loss of function have been described in oilseed crops. Site-directed mutagenesis experiments demonstrated that histidine residues are essential for the enzymatic activity of D12 desaturases [34]. A similar observation was reported for FAD3B of SP2047 which had a His-box mutation causing complete loss of desaturase activity in a yeast heterologous system [33]. Point mutations leading to premature stop codons were reported for FAD3A and FAD3B of flax line 593–708 [32] and FAD3A of SP2047 [33], all resulting in loss of function. Knowledge of alleles and their predicted isoforms can be capitalized upon for altering lipid biosynthesis and producing designer oils. The genetic diversity for fatty acid desaturase genes in flax was recently uncovered [35], but the functional characterization of their encoded isoforms remains limited. Allele-specific markers could be highly useful for introgression of desirable alleles. However, the implementation of such schemes requires indepth knowledge of the genetic (allelic) diversity and of the functional characterization of the encoded isoforms. Recently, the allelic variation for SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes in flax was revealed by sequencing the six genes from 120 accessions [35]. Each gene was predicted to encode between two and seven isoforms. Here, we cloned alleles of FAD2 and FAD3 for all previously identified isoforms and expressed them in yeast (Saccharomyces cerevisiae). Enzymatic activity of each isoform was tested in the presence of exogenous substrate, and conversion was measured by gas chromatography to determine the relative activity of each isoform.

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Materials and Methods Cloning of FAD2A and FAD2B Because FAD2A and FAD2B have no introns, genomic DNA (gDNA) could be used for cloning the open reading frames. Accessions UGG5-5, SP2047, CN96845, CN97341, CN96991, and CN19158 (Table S1) were grown in a growth cabinet, and gDNA was extracted from the leaf tissue of seedlings using the DNeasy Plant kit as per manufacturer’s instructions (Qiagen, Missisauga, ON, Canada). For all genes, PCR primers were designed in-frame and spanning the initiation ATG (Fwd) and stop (Rev) codon (Table S2). A total of five independent PCR reactions were performed for each target gene. Each reaction contained 30 ng of gDNA, 0.4 lM of each primer, 1.5 mM MgCl2, 0.8 mM of total dNTPs, 0.1 ll of bovine serum albumin (1 mg/ml), and 1 unit Taq DNA polymerase in a final volume of 10 ll. The PCR reactions included an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 2 min, followed by a final extension at 72 °C for 20 min to ensure the presence of 30 A-overhangs. The five independent reactions for each target were visualized on 1 % agarose gels prior to pooling. The pooled PCR products were gel purified using the QIAEX II gel extraction kit (QIAGEN Sciences, Maryland, USA). Purified PCR products were ligated into pYES2.1/ V5-His-TOPO vector and transformed into OneShot TOP10 electrocompetent cells following manufacturer’s instructions (Invitrogen Ltd, Carlsbad, USA). Individual recombinant colonies were analyzed for the presence of the insert using a PCR fragment-specific forward primer and V5 C-terminal reverse primer from the vector that provided information about the presence/absence of the insertion as well as its orientation (Table S2). Plasmid DNA of the positive transformants was isolated using the Perfectprep Plasmid 96 VAC Direct Bind kit (Eppendorf, Hamburg, Germany) and subsequently restricted to PvuI and PstI for the FAD2A and FAD2B constructs, respectively. DNA sequencing of multiple clones was carried out [36] using Big Dye Terminator chemistry (v3.1) and sequenced on an ABI 3130xl Genetic Analyser (Applied Biosystems, Foster City, California) using the GAL1 forward primer, the V5 C-terminal reverse primer, fragment-specific forward and reverse primers, and two internal primers to ensure full coverage of the fragment (Table S2). Cloning of FAD3A and FAD3B For the cloning of FAD3A and FAD3B alleles, RNA was extracted from developing flax bolls of the accessions listed in Table S1. Flax plants were grown in a growth

chamber at temperatures of 20/18 °C (day/night) under a 16-h photoperiod. Total RNA was extracted from 200 mg tissue using an RNA extraction procedure that provided efficient removal of carbohydrates and fatty acids (Banik et al. [33]). Total RNA was treated with TurboDnase (Ambion, Austin, TX) and transcribed into first-strand cDNAs using the Oligo (dT) primer and Superscript II reverse transcriptase (Invitrogen Ltd). First-strand cDNA was used as template in quintuple-independent PCR reactions using gene-specific primers (Table S2) and subsequently cloned in pYES2.1/V5-His-TOPO reactions as described above for FAD2 genes. Transformation in Yeast Transformation of yeast strain InvSc1 with recombinant plasmids was performed using the S.c.EasyComp transformation kit (Invitrogen Ltd). Transformants, no longer uracil auxotrophic, were selected on SC minimal media lacking uracil (SC-u) and were confirmed by yeast colony PCR using the fragment-specific primers used for cloning (Table S2). Briefly, yeast colonies were stabbed with a pipette tip that was then immersed in 10 ll water, placed at -80 °C for 10 min, heated in the microwave for 90 s, and placed at -80 °C for an additional 10 min to ensure plasmid release. An aliquot of 5 ll was used as template in PCR reactions. The PCR products were visualized on agarose gels, and positive yeast colonies were streaked on selective SC-u media supplemented with 2 % glucose. Expression of Fatty Acid Desaturases in Yeast For each fatty acid desaturase, a single yeast colony containing a positive pYES2.1/V5-His-TOPO construct was inoculated in 50 ml SC-u minimal media supplemented with 2 % glucose. Pre-cultures were grown for 40 h at 30 °C with shaking at 250 rpm. The OD600 of these cultures was determined, and samples were diluted with induction media to create 30 ml aliquots having an OD600 of 0.4. These evenly diluted aliquots were centrifuged for 5 min at 1,500g at 4 °C and washed twice with 2 ml of SCu containing 1 % raffinose, and the final pellet was resuspended in 30 ml of induction media supplemented with 2 % galactose and 1 % raffinose. Each 30 ml culture was divided into three flasks to constitute the three biological replicates, and another 10 ml of induction media was added to each flask resulting in 20 ml induction culture aliquots. The cultures were induced for 6 or more hours at 20 °C with shaking at 250 rpm. Once the OD600 reached 0.7, they were supplemented with 500 lM substrate: OLE (Sigma-Aldrich, Saint-Louis, MO, USA) for FAD2A and FAD2B and LIO (Sigma-Aldrich) for FAD3A and FAD3B. The cultures were incubated in the presence of the substrate

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Fig. 1 Multiple alignment of deduced amino acid sequences of isoforms. a FAD2A and FAD2B, b FAD3A and FAD3B. Large boxes identify the three His-boxes. Small boxes frame the amino acid substitutions characterizing the isoforms

for 3 days and then pelleted for 5 min at 1,500 g. The pellets were washed once with 2 ml SC-u containing 1 % raffinose and once with 2 ml cold water prior to storage at -80 °C until extraction for GC analysis. Fatty acid methyl esters were extracted and analyzed by GC as previously described [29]. FAD2 conversion rates were calculated as the ratio of C18:2/(C18:1 ? C18:2) averaged over the three biological replicates and expressed

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as percentage. FAD3 conversion rates were similarly calculated (C18:3/(C18:2 ? C18:3) 9 100). One-way analyses of variance were performed independently for each desaturase using the SAS program (SAS Institute Inc, NC) where isoforms were fixed effect. The mean squares were calculated using PROC ANOVA. The mean percentages of fatty acids were obtained from three measurements for each isoform and the empty vector

Mol Biotechnol Fig. 1 continued

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Mol Biotechnol Table 1 Description of the fatty acid desaturase isoforms including their amino acid substitutions and respective positions Isoform Position Residue

34 A

318 T

FAD2A-A FAD2A-B

V

FAD2A-C

M Position Residue

49 P

281 Y

337 P

FAD2B-A FAD2B-B

H

FAD2B-C

F

FAD2B-D

L Position Residue

10 A

77 T

85 W

291 R

330 H

369 V

P

I

P

I

283 G

380 R

FAD3A-A FAD3A-B

T

FAD3A-C

T

FAD3A-D

T

A STOP

FAD3A-E FAD3A-F

STOP

Position Residue FAD3B-A FAD3B-B FAD3B-C

53 W

106 H

112 I

STOP Y

FAD3B-D

S

FAD3B-E

S

FAD3B-F

S

FAD3B-G

S

controls. The mean conversion rates of three replicates were calculated and are presented with their standard errors.

Results FAD2A and FAD2B Sequence analysis was performed on multiple cDNA clones, and only clones that were a complete and perfect match to the expected predicted isoforms were transformed in yeast. Full-length FAD2A and FAD2B genes generated fragments of 1,137 and 1,149 bp, respectively. The FAD2A and FAD2B genes have no introns and encode 378 and 382 amino acid membrane-bound D12 desaturases with three

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223 A

V E P

and four isoforms, respectively. Compared to FAD2A-A, FAD2A-B had an alanine-to-valine substitution at position 34, while FAD2A-C displayed a threonine-to-methionine substitution soon after the third histidine box (Fig. 1a; Table 1). All three FAD2A isoforms displayed D12 desaturase activity, while yeast cells transformed with the empty pYES2.1 construct did not show any C18:2 peak (Fig. 2a). All three FAD2A isoforms had significantly different conversion rates (P \ 0.0001). The highest conversion rate of 66.27 % was observed for the FAD2A-B isoform (Table 2, Fig. 3a). Sequence variations among the four FAD2B isoforms were also all amino acid substitutions (Fig. 1a; Table 1). Conversion rates for FAD2B-A, FAD2B-B, and FAD2B-D were not significantly different from one another and

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Fig. 2 Gas chromatograms of fatty acid profiles of transformed yeast where substrate is OLE acid (a-d) or LIO acid (e-j) and construct is pYES 2.1 empty vector (a, c, e, h), FAD2A-B (b), FAD2B-D (d),

FAD3A-C (f), FAD3A-E (g), FAD3B-D (i), or FAD3B-B (j). X-axes represent retention time in min, and Y-axes represent detector response in lV

varied from 74.33 to 76.19 % (Table 2; Fig. 3b). With a conversion rate of only 71.16 %, FAD2B-C was significantly lower than the other three isoforms. Overall, FAD2B activity (*71–76 %) was approximately 10 % higher than FAD2A (*61–66 %) (Table 2; Fig. 3a, b).

exception of isoforms D and E, which were predicted to be truncated at amino acid 85 and 291, respectively, due to the presence of premature stop codons (Fig. 1b; Table 1). Yeast transformed with empty pYES2.1 vector did not convert the LIO substrate into LIN as evidenced by the absence of a peak at 2.99 min (Fig. 2c). D15 desaturase activity was also not detected for the truncated D and E isoforms (Fig. 3c; Table 2). The other four isoforms were functional D15 desaturases with significantly different (P \ 0.0001) mean conversion rates (Table 2). Isoform FAD3A-C had the highest conversion rate of 15.48 %,

FAD3A and FAD3B A total of six isoforms encoded by FAD3A genes were identified. The deduced amino acid sequences of the FAD3A genes had 392 amino acid residues with the

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Mol Biotechnol Table 2 Linoleic and linolenic acid conversion rates of OLE acid to LIO acid for FAD2A, FAD2B, and of LIO acid to LIN acid for FAD3A and FAD3B isoforms expressed in yeast LIO (%)

LIN (%)

Conversion (%)1

SE

pYES2.1 construct

Substrate

OLE (%)

Empty vector

OLE

42.437

FAD2A-A

OLE

17.773

28.604

61.68c

0.396

FAD2A-B

OLE

15.671

30.792

66.27a

0.374

FAD2A-C

OLE

16.763

30.043

64.19b

0.132

Empty vector

OLE

40.860

FAD2B-A

OLE

11.066

0d

0

0c

0 35.415

76.19a

0.453

a

FAD2B-B FAD2B-C

OLE OLE

11.918 13.356

34.506 32.954

74.33 71.16b

0.834 0.786

FAD2B-D

OLE

11.270

34.577

75.42a

0.859

Empty vector

LIO

34.632

0

FAD3A-A

LIO

29.751

0.838

2.74d

0.127

c

0.220 0.776

0e

FAD3A-B

LIO

26.489

2.188

7.63

FAD3A-C

LIO

24.992

4.577

15.48a

FAD3A-D

LIO

29.169

0

0e

FAD3A-E

LIO

28.204

0

0e

FAD3A-F

LIO

28.571

3.612

11.22b

Empty vector

LIO

33.890

0

0b

FAD3B-A

LIO

31.043

0.882

FAD3B-B

LIO

31.493

0

0b

FAD3B-C

LIO

31.965

0

0b

FAD3B-D

LIO

31.843

0.938

2.86a

0.187

FAD3B-E FAD3B-F

LIO LIO

32.644 32.381

0.894 0

2.67a 0b

0.292

FAD3B-G

LIO

31.495

0.960

2.96a

0.281

1

2.76a

0.205 0.085

superscript letters represent statistically significant conversion rate according to Duncan’s multiple range tests (P \ 0.0001)

followed by FAD3A-F, FAD3A-B, and FAD3A-A with conversion rates of 11.22, 7.63, and 2.74, respectively (Fig. 3c; Table 2). The deduced amino acid sequence of FAD3B had 391 residues. The seven FAD3B isoforms had single or double amino acid substitutions with the exception of FAD3B-B predicted to be truncated at amino acid residue 60 due to a premature stop codon (Fig. 1b; Table 1). The conversion rates for the functional FAD3B desaturases were generally much lower than FAD3A ranging from 2.67 to 2.96 % for the four isoforms where D15 desaturase activity was detected (Fig. 3c, d). Aside from the predicted truncated isoform FAD3B-B, isoforms FAD3B-C and FAD3B-F also had no detectable D15 desaturase activity even though they did not have predicted premature stop codons (Fig. 3d; Table 2). FAD3B-C had a histidine-to-tyrosine substitution located in the first His-box, while FAD3B-F had two amino acid substitutions, but the only unique change resided in a glycine to glutamic acid residue at position 283, i.e., between the second and third His-box (Fig. 1b; Table 1). No significant differences in conversion rates were

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detected for the four FAD3B active isoforms, namely isoforms A, D, E, and G (Fig. 3d; Table 2).

Discussion Linseed varieties contain up to 50 % oil, and the nutritional value and end-use properties are largely dictated by the fatty acid composition of this oil [29, 37, 38]. Fatty acid biosynthesis is a complex process involving intersecting pathways that involve a series of desaturation and elongation steps [39, 40]. FADs insert double bonds into fatty acid acyl chains [41, 42]. FAD2 and FAD3 are membrane desaturases with eight conserved histidine residues comprising a tripartite motif, while SAD is a soluble desaturase with only two histidine residues that are both interacting with iron ions [41, 43–45]. Fatty acid desaturases SAD, FAD2, and FAD3 are a diverse gene family responsible in flax for the production of OLE, LIO, and LIN acids, respectively. In this study, we described cloning of different isoforms of four fatty acid desaturase genes (FAD2A,

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Fig. 3 Conversion rate of flax fatty acid desaturase isoforms transformed in yeast. a FAD2A, b FAD2B, c FAD3A, and d FAD3B. Standard errors are shown, and letters above each bar represent values that are significantly different according to the Duncan’s multiple range test

FAD2B, FAD3A, and FAD3B) and their functional analysis in yeast. In plants, the conversion of OLE acid to LIO acid is catalyzed by the fatty acid desaturase 2 (FAD2) that

introduces a double bond at the D12 position of the C18 fatty acyl chain [41, 42]. Since the cloning of the first plant FAD2 gene in Arabidopsis thaliana [46], its ortholog has been cloned from various crop plants such as soybean [26], rapeseed [47], cotton [48], flax [29], and olive [28]. Here, we cloned, sequenced, and functionally characterized three isoforms of FAD2A and four isoforms of FAD2B. Flax FAD2 enzymes have conserved desaturase domains, such as three His-rich motifs, four membranespanning domains, and the sequence ‘‘YNNKL’’ at the C terminus of the protein needed for ER localization of the enzyme [30]. The deduced amino acid sequence of the flax FAD2A and FAD2B genes predicted the three histidine boxes HECGHH, HRRHH, and HVAHH [30, 44]. These membrane-bound desaturase histidine boxes are essential for coordinating the diiron cofactor required for catalysis [49]. FAD3 truncated for the latter motif had reduced activity caused by dislocation of the protein [49]. Functional expression of FAD2 from A. thaliana in S. cerevisiae resulted in accumulation of LIO acid [50]. Two closely related FAD2 genes (FAD2A and FAD2B) encoding FAD2 desaturase have been previously cloned and characterized in flax [30, 31]. In flax, FAD2 genes were found to be the rate-limiting genes in the fatty acid biosynthesis pathway, and hence, genetic variation in FAD2 might have significant impact on fatty acid composition [51]. Similarly, the functional expression of FAD2A- and FAD2B-predicted isoforms revealed significantly different enzyme activities with FAD2B being the most efficient. Genetic variation in FAD2 was also shown to be associated with consequent changes in fatty acid profiles in other crops such as soybean and peanuts [52, 53]. Thambugala et al. [35] showed that among flax desaturases, FAD2B was highly conserved, in line with its physiological importance. The positive selection of FAD2B in flax over the process of domestication also corroborates this assumption [54]. However, Tang et al. [55] reported that expression of soybean FAD2-1A isoform (FAD2A ortholog) in yeast was more unstable than FAD2-1B (FAD2B ortholog) particularly when cultures were maintained at elevated growth temperatures. In flax, FAD3A and FAD3B are the major genes responsible for the desaturation of LIO into LIN. In the present study, we cloned and characterized six isoforms of FAD3A and seven isoforms of FAD3B in flax. The nucleotide sequence analysis of FAD3A-D from SP2047 and FAD3A-E from E1747 confirmed the presence of single point mutations that created premature stop codons, a feature only observed in FAD3 desaturases of EMS mutant flax accessions [35]. These proteins were truncated at amino acids 85 and 291, respectively. Truncated proteins were nonfunctional, i.e., incapable of any conversion of LIO to LIN. Mutant solin line 593–708 [32] with premature

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stop codons in FAD3A and FAD3B genes also encoded nonfunctional enzymes, but line 593–708 still produced a small amount of LIN (\2 %). We hypothesized that the truncated FAD3A and FAD3B may still retain low levels of desaturase activities. Banik et al. [33] later showed that the SP2047 FAD3A-D, which also had a premature stop codon, was expressed at very low level throughout seed development, but enzymatic activity was not shown. Based on the results presented herein, we hypothesize that the small fraction of LIN in lines with defective FAD3A and FAD3B could also be the results of other minor FAD3 enzymes such as FAD3C [33]. FAD3A-C enzyme activity was more than fivefold higher than isoform A and twice that of isoform B, indicative of the selection potential for the development of flax varieties with altered fatty acid composition. Functional FAD3B isoforms were not significantly different in their activity, with an average conversion rate of 2.81 %, and considerably lower activity than three of the four FAD3A isoforms. Banik et al. [33] studied the expression of FAD3A and FAD3B genes during seed development and reported that FAD3B expression was approximately threefold higher than FAD3A. Hence, FAD3A and FAD3B contribute differently to the production of LIN: FAD3A through a higher enzymatic activity potential and FAD3B through a higher level of expression. As such, altering fatty acid composition through FAD3 may be addressed through various strategies including the selection of higher activity isoforms and by increasing gene expression.

Conclusions Genetic diversity in flax for fatty acid desaturase genes generated many isoforms with significant functional differences. D12 desaturation activity by FAD2 enzymes exceeded D15 desaturation activity by FAD3 enzymes as measured by heterologous expression in yeast. Paralogous FAD2 and FAD3 differed significantly in activity, with FAD2B having greater activity than FAD2A, and FAD3A being generally more active than FAD3B. Differences in activity were observed among isoforms of each desaturase. Genes with premature stop codons predicted to produce truncated proteins were only found in FAD3, and none showed D15 desaturase activity. Two other FAD3B isoforms also displayed no enzymatic activity as a result of single amino acid substitutions. Functional characterization of desaturase isoforms combined with knowledge of genetic diversity and expression analysis provide breeders with the knowledge necessary to make educated decisions in designing crosses for the purposes of developing flax germplasm with tailored desirable fatty acid composition.

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Studies have showed that manipulation of single genes in the FA biosynthetic pathways can create significant differences in FA profiles and alter oil content, the most important quality traits of oilseed flax. Acknowledgments This work was conducted as part of the Total Utilization Flax Genomics (TUFGEN) project funded by Genome Canada and co-funded by the Government of Manitoba, the Flax Council of Canada, the Saskatchewan Flax Development Commission, Agricultural Development Fund and the Manitoba Flax Growers Association. Project management and support by Genome Prairie are also gratefully acknowledged.

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