Flax (Linum usitatissimum L.) is one of the richest plant sources of omega-3 fatty acids praised for their health benefits. In this study, the extent of the genetic variability for genes encoding stearoyl-ACP desaturase (SAD), fatty acid desaturase 2 (FAD2) and 3 (FAD3) was determined by sequencing the six paralogous genes from 120 flax accessions representing a broad range of germplasm including some EMS mutant lines. A total of 6 alleles for sad1 and sad2, 21 for fad2a, 5 for fad2b, 15 for fad3a and 18 for fad3b were identified. Deduced amino acid sequences of the alleles predicted 4, 2, 3, 4, 6, and 7 isoforms, respectively. Allele frequencies varied greatly across genes. Fad3a, with 110 SNPs and 19 indels, and fad3b, with 50 SNPs and 5 indels, showed the highest levels of genetic variation. While most of the SNPs and all the indels were silent mutations, both genes carried non-sense SNP mutations resulting in premature stop codons, a feature not observed in sad and fad2 genes. Some alleles and isoforms discovered in induced mutant lines were absent in the natural germplasm. Correlation of these genotypic data with fatty acid composition data of 120 flax accessions phenotyped in six field experiments revealed statistically significant correlations of some of the SAD and FAD isoforms on fatty acid composition, oil content and iodine value. The novel allelic variants and isoforms identified for the six desaturases will be a resource for the development of oilseed flax with unique and useful fatty acid profiles. / October 2015

Polyunsaturated fatty acids are major structural components of plant chloroplast and endoplasmic reticulum membranes. Two fatty acid desaturases (designated FAD2 and FAD3) desaturate 75% of the fatty acids in the endoplasmic reticulum. The w -6 fatty acid desaturase (FAD2) may be responsible for cold acclimation response, since polyunsaturated phospholipids are important in helping maintain plant viability at lowered temperatures. To study regulation of FAD2 gene expression in cotton, a FAD2 gene was isolated from two genomic libraries using an Arabidopsis FAD2 hybridization probe and a cotton FAD2 5¢ -flanking region gene-specific probe, respectively. A cotton FAD2 gene was found to be in two overlapping genomic clones by physical mapping and DNA sequencing. The cloned DNA fragments are identical in size to cotton FAD2 genomic DNA fragments shown by genomic blot hybridization. The cotton FAD2 coding region has 1,155 bp with no introns and would encode a putative polypeptide of 384 amino acids. The cotton FAD2 enzyme has a high identity of 75% with other plant FAD2 enzymes. The enzyme has three histidine-rich motifs that are conserved in all plant membrane desaturases. These histidine boxes may be the iron-binding domains for reduction of oxygen during desaturation. To confirm that this FAD2 enzyme is functional, a plasmid construct containing the cotton FAD2 coding region was transformed into Saccharomyces cerevisiae. The transformed yeast cells were able to catalyze the conversion of oleic acid (C18:1) into linoleic acid (C18:2). The FAD2 gene contains an intron of 2,967 bp in its 5¢ -flanking region, 11 bp upstream from the initiation codon. The intron could be essential for transcriptional regulation of FAD2 gene expression. Several putative promoter elements occur in the 5¢ -flanking region of this gene. A potential TATA basal promoter element occurs at 41 bp upstream from the cap site. Two presumptive helix-loop-helix (bHLH) motifs that may be seed-specific promoter elements are located at 109 bp and 135 bp upstream from the potential cap site.

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant lines harboring the cotton FAD2 genes were generated. The cotton FAD2 genes were amplified by the polymerase chain reaction (PCR) from the genomic DNAs isolated from the transgenic Arabidopsis T1 plant lines. Complementation of the putative transgenic Arabidopsis plants with the two cotton FAD2 genes was demonstrated by gas chromatography analyses of the fatty acid profiles of leaf tissues. The cellular localization of cotton FAD2-4 polypeptides with N-terminal green fluorescence protein (GFP) was visualized by confocal fluorescence microscopy. The phenotype of transgenic Arabidopsis plants transformed with the cotton FAD2-4 gene was compared to Arabidopsis knockout fad2 mutant plants and wild type Arabidopsis plants regarding their sensitivity to low temperature, and the size and height of the plants.

Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.

No description available.

Contexte : La dysfonction du tissu adipeux blanc (TAB) favorise les facteurs de risque de diabète de type 2 (DbT2), c’est-à-dire la résistance à l’insuline (RI), l’hyper sécrétion d’insuline glucostimulée (SIGS), le délai de clairance des gras et les concentrations élevées d’apoBlipoprotéines (apoB plasmatique) incluant les lipoprotéines de faible densité (LDL). De récentes études de notre laboratoire et d’autres suggèrent que le niveau élevé d’apoB plasmatique (hyperapoB) est une cause et non seulement une conséquence de la dysfonction du TAB. De plus, une internalisation augmentée d’apoB-lipoprotéines via les récepteurs tels que le récepteur aux LDLs (LDLR) et le cluster de différenciation 36 (CD36), favorise le risque de DbT2. Cependant, les mécanismes sous-jacents de même que les interventions nutritionnelles pour les cibler demeurent incertains. L'activation de la voie de l’inflammasome NLRP3/ interleukine (IL) -1β favorise la dysfonction du TAB et les facteurs de risque de DbT2 et est activée par les LDLs oxydées dans les cellules immunitaires. L'acide eicosapentaénoïque (AEP) et l'acide docosahexaénoïque (ADH) réduisent l'hyperapoB, l'activité de l’inflammasome NLRP3 dans les cellules immunitaires et les facteurs de risque de DbT2 chez l’humain. Ils sont synthétisés de façon endogène par l’entremise des désaturases d’acides gras δ-5 (D5D) et δ-6 (D6D). Chez l’humain, de faibles niveaux d’AEP et d’ADH circulants et d’activité de la D5D et une activité élevée de la D6D prédisent l'incidence de DbT2 et la RI par des mécanismes inconnus. Objectifs : L'hypothèse de ma thèse est que l'AEP et l’ADH améliorent les facteurs de risque de DbT2, soit la dysfonction du TAB, le délai de clairance des gras, la RI et l’hyper SIGS, ceci via une baisse de l'apoB plasmatique et de l’activité de l’inflammasome NLRP3 dans le TAB. Les objectifs sont d'examiner si: 1) les associations entre les activités de la D5D et de la D6D et les facteurs de risque de DbT2 dépendent de l'apoB plasmatique; 2) la supplémentation en AEP+ADH réduit l'apoB plasmatique, l'expression du LDLR et du CD36 dans le TAB, l'activité de l’inflammasome NLRP3 dans le TAB et les facteurs de risque de DbT2; 3) l’AEP+ADH inhibe la sécrétion d'IL-1β par le TAB humain stimulée par des signaux canoniques ou les LDLs natives. Méthodes: Des hommes et des femmes postménopausées normoglycémiques ont été testés à l’état basal et après une supplémentation en AEP (1,8 g/jour) et ADH (0,9 g/jour) de 12 semaines. Les activités de la D5D et de la D6D ont été estimées à partir des acides gras produits/précurseurs dans les phospholipides plasmatiques. Nous avons mesuré la SIGS, la RI et le disposition index lors d’un clamp Botnia. Après un repas à 66% de gras, le délai de clairance des gras a été mesuré par l’aire sous la courbe (sur 6 h) des triglycérides (TG) ou de l’apoB48 (chylomicrons) plasmatiques. Ex vivo dans une biopsie de TAB, nous avons mesuré l'expression de surface du LDLR et du CD36 par immunohistochimie, l'ARNm de NLRP3 et IL1B par RT-qPCR et la sécrétion d'IL-1β par alpha-LISA en l’absence ou en présence d’une stimulation par le lipopolysaccharide (LPS), l'adénosine triphosphate (ATP) et/ou les LDLs humaines natives et lors d’une co-incubation avec l’AEP+ADH. Résultats: À l’état basal (N=98), l'activité de la D5D corrélait négativement avec l'apoB plasmatique, la 2e phase de SIGS, la RI et le délai de clairance des chylomicrons et ces associations étaient dépendantes de l'apoB plasmatique. Inversement, l'activité de la D6D corrélait positivement avec la SIGS, la RI et le délai de clairance des chylomicrons indépendamment de l'apoB plasmatique. Chez les sujets ayant complété la supplémentation en AEP+ADH (N=30), on notait une amélioration de la 1e phase de SIGS, du disposition index et de la clairance des TGs. Des niveaux initiaux plus élevés d'apoB plasmatique, de TGs postprandiaux plasmatiques et de RI, et dans le TAB d'expression du LDLR et du CD36, de sécrétion d’IL-1β et d'ARNm de NLRP3 prédisaient une plus grande réduction de ces paramètres. En comparaison à l'acide palmitique, l’AEP+ADH inhibait la sécrétion d'IL-1β par le TAB, en l’abscence ou en présence d’une stimulation par le LPS, l'ATP et/ou les LDLs natives de ces sujets. Conclusion: Les associations inverses entre l'activité de la D5D avec les facteurs de risque de DbT2 sont dépendantes de l'apoB plasmatique. Les meilleurs répondants à la supplémentation en AEP et ADH, en termes de réduction d'apoB plasmatique, d’expression du LDLR et du CD36 dans le TAB, d'activité de l’inflammasome NLRP3 dans le TAB, de TGs postprandiaux et de RI, sont les sujets avec des niveaux initiaux élevés de ces paramètres. L’AEP et l’ADH inhibent directement la sécrétion d'IL-1β par le TAB humain induite par les LDLs natives ou d'autres signaux. Nous proposons que la supplémentation en AEP et ADH puisse cibler l'activité de l’inflammasome NLRP3 dans le TAB, induite par un niveau élevé d’apoB-lipoprotéines plasmatiques ou internalisées par les récepteurs, et ainsi aider à prévenir le DbT2. / Background: White adipose tissue (WAT) dysfunction promotes risk factors for type 2 diabetes (T2D), namely insulin resistance (IR), high glucose-stimulated insulin secretion (GIIS), delayed fat clearance and high concentrations of apoB-lipoproteins (measured as plasma apoB) including low density lipoproteins (LDL). Recent studies from our lab and others suggest that high plasma apoB (hyperapoB) is a cause and not only a consequence of WAT dysfunction. Moreover, upregulated receptor-mediated uptake of apoB-lipoproteins via LDL receptor (LDLR) and cluster of differentiation 36 (CD36), promotes the risk for T2D. However, underlying mechanisms as well as nutritional interventions to target them remain unclear. Activation of the NLRP3 inflammasome/interleukin (IL)-1β pathway promotes WAT dysfunction and risk factors for T2D and is activated by oxidized LDLs in immune cells. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) reduce hyperapoB, NLRP3 inflammasome activity in immune cells and risk factors for T2D in humans. They are synthesized endogenously through δ-5 (D5D) and δ-6 (D6D) fatty desaturases. In humans, low levels of circulating EPA and DHA and D5D activity and high D6D activity predict the incidence of T2D and IR by unknown mechanisms. Objectives: The hypothesis of my thesis is that EPA and DHA improve T2D risk factors, namely WAT dysfunction, delayed fat clearance, IR and high GIIS, this via a reduction of plasma apoB and WAT NLRP3 inflammasome activity. The objectives are to examine whether: 1) the associations between the levels of D5D and D6D activities and the risk factors for T2D are dependent on plasma apoB; 2) supplementation with EPA+DHA reduces plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity and T2D risk factors; 3) EPA+DHA directly inhibits IL-1β secretion from human WAT stimulated by canonical signals or native LDLs. Methods: Normoglycemic men and postmenopausal women were tested at baseline and after supplementation with EPA (1.8 g/day) and DHA (0.9 g/day) for 12 weeks. The activities of D5D and D6D were estimated from the product/precursor fatty acids in plasma phospholipids. We measured GIIS, IR and disposition index by a Botnia clamp. Following a 66% fat meal, delayed fat clearance was measured as the area under the curve (over 6 h) of plasma triglycerides (TG) or apoB48 (chylomicrons). Ex vivo in a WAT biopsy, we measured LDLR and CD36 surface expression by immunohistochemistry, NLRP3 and IL1B mRNA by RT-qPCR, and IL-1β secretion by alpha-LISA either unstimulated or stimulated by lipopolysaccharide (LPS), adenosine triphosphate (ATP), and/or native human LDLs, and during co-incubation with EPA+DHA. Results: At baseline (N=98), D5D activity correlated negatively with plasma apoB, 2nd phase GIIS, IR and delayed chylomicron clearance and these associations were dependent on plasma apoB. Conversely, D6D activity correlated positively with GIIS, IR, and delayed chylomicron clearance independently of plasma apoB. In subjects who completed the EPA+DHA supplementation (N=30), there was an amelioration in 1st phase GIIS, disposition index and TG clearance. Higher baseline levels of plasma apoB, plasma postprandial TGs, IR, WAT LDLR and CD36 surface expression, WAT IL-1β secretion and WAT NLRP3 mRNA predicted a greater reduction of these parameters. In comparison with palmitic acid, EPA+DHA inhibited IL-1β secretion from WAT, either unstimulated or stimulated by LPS, ATP and/or subjects’ native LDLs. Conclusion: The negative associations of D5D activity with risk factors for T2D are dependent on plasma apoB. Best responders to EPA and DHA supplementation to reduce plasma apoB, WAT LDLR and CD36 expression, WAT NLRP3 inflammasome activity, delayed TG clearance, and IR are subjects with elevated baseline levels of these parameters. EPA and DHA directly inhibit IL-1β secretion from human WAT induced by native LDLs or other signals. We propose that EPA and DHA supplementation may target upregulated WAT NLRP3 inflammasome activity induced by high plasma concentrations, or receptor-mediated uptake, of apoB-lipoproteins, and thus help prevent T2D.

No description available.