Genomic and transcriptome analyses reveal potential contributors to erucic acid biosynthesis in seeds of rapeseed (Brassica napus).

KEY MESSAGE: Through comprehensive genomic and transcriptomic analyses, we identified a set of 23 genes that act up- or downstream of erucic acid content (EAC) production in rapeseed seeds. We selected example genes to showcase the distribution of single nucleotide polymorphisms, haplotypes associated with EAC phenotypes, and the creation of molecular markers differentiating low EAC and high EAC genotypes. Erucic acid content (EAC) is a crucial trait in rapeseed, with low LEAC oil recognized for its health benefits and high EA oil holding industrial value. Despite its significance, the genomic consequences of intensive LEAC-cultivar selection and the genetic basis underlying EA regulation remain largely unexplored. To address this knowledge gap, we conducted selective signal analyses, genome-wide association studies (GWAS), and transcriptome analyses. Our investigation unveiled the genetic footprints resulting from LEAC selection in germplasm populations, drawing attention to specific loci that contribute to enriching diversity. By integrating GWAS and transcriptome analyses, we identified a set of 23 genes that play a significant role in determining EAC in seeds or are downstream consequences of EA-level alterations. These genes have emerged as promising candidates for elucidating the potential mechanisms governing EAC in rapeseed. To exemplify the findings, we selected specific genes to demonstrate the distribution of single nucleotide polymorphisms and haplotypes associated with different EAC phenotypes. Additionally, we showcased to develop molecular markers distinguishing between LEAC and high EAC genotypes.

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement.

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co-expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

Intestinal metabolomics of juvenile lenok (Brachymystax lenok) in response to heat stress.

Changes in the metabolic profile within the intestine of lenok (Brachymystax lenok) when challenged to acute and lethal heat stress (HS) are studied using no-target HPLC-MS/MS metabonomic analysis. A total of 51 differentially expressed metabolites (VIP > 1, P < 0.05) were identified in response to HS, and 34 occurred in the positive ion mode and 17 in negative ion mode, respectively. After heat stress, changes in metabolites related to glycolysis (i.e., alpha-D-glucose, stachyose, and L-lactate) were identified. The metabolites (acetyl carnitine, palmitoylcarnitine, carnitine, and erucic acid) related to fatty acid ß-oxidation accumulated significantly, and many amino acids (L-tryptophan, D-proline, L-leucine, L-phenylalanine, L-aspartate, L-tyrosine, L-methionine, L-histidine, and L-glutamine) were significantly decreased in HS-treated lenok. The mitochondrial ß-oxidation pathway might be inhibited, while severe heat stress might activate the anaerobic glycolysis and catabolism of amino acid for energy expenditure. Oxidative damage in HS-treated lenok was indicated by the decreased glycerophospholipid metabolites (i.e., glycerophosphocholine, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dioleoyl-sn-glycero-3-phosphatidylcholine) and the increased oxylipin production (12-HETE and 9R, 10S-EpOME). The minor oxidative pathways (omega-oxidation and peroxisomal beta-oxidation) were likely to be induced in HS-treated lenok.

Diets with Higher ω-6/ω-3 Ratios Show Differences in Ceramides and Fatty Acid Levels Accompanied by Increased Amyloid-Beta in the Brains of Male APP/PS1 Transgenic Mice.

Senile plaque formation as a consequence of amyloid-ß peptide (Aß) aggregation constitutes one of the main hallmarks of Alzheimer's disease (AD). This pathology is characterized by synaptic alterations and cognitive impairment. In order to either prevent or revert it, different therapeutic approaches have been proposed, and some of them are focused on diet modification. Modification of the ω-6/ω-3 fatty acids (FA) ratio in diets has been proven to affect Aß production and senile plaque formation in the hippocampus and cortex of female transgenic (TG) mice. In these diets, linoleic acid is the main contribution of ω-6 FA, whereas alpha-linoleic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) are the contributors of ω-3 FA. In the present work, we have explored the effect of ω-6/ω-3 ratio modifications in the diets of male double-transgenic APPswe/PS1ΔE9 (AD model) and wild-type mice (WT). Amyloid burden in the hippocampus increased in parallel with the increase in dietary ω-6/ω-3 ratio in TG male mice. In addition, there was a modification in the brain lipid profile proportional to the ω-6/ω-3 ratio of the diet. In particular, the higher the ω-6/ω-3 ratio, the lower the ceramides and higher the FAs, particularly docosatetraenoic acid. Modifications to the cortex lipid profile was mostly similar between TG and WT mice, except for gangliosides (higher levels in TG mice) and some ceramide species (lower levels in TG mice).

Development of B. carinata with super-high erucic acid content through interspecific hybridization.

KEY MESSAGE: Disomic alien chromosome addition Brassica carinata lines with super-high erucic acid content were developed through interspecific hybridization with B. juncea and characterized using molecular, cytological and biochemical techniques. Brassica carinata [A.] Braun (BBCC, 2n = 34) is a climate-resilient oilseed. Its seed oil is high in erucic acid (> 40%), rendering it well suited for the production of biofuel and other bio-based applications. To enhance the competitiveness of B. carinata with high erucic B. napus (HEAR), lines with super-high erucic acid content were developed through interspecific hybridization. To this end, a fad2B null allele from Brassica juncea (AABB, 2n = 36) was introgressed into B. carinata, resulting in a B. carinata fad2B mutant with erucic acid levels of over 50%. Subsequently, the FAE allele from B. rapa spp. yellow sarson (AA, 2n = 20) was transferred to the fad2B B. carinata line, yielding lines with erucic acid contents of up to 57.9%. Molecular analysis using the Brassica 90 K Illumina Infinium™ SNP genotyping array identified these lines as disomic alien chromosome addition lines, with two extra A08 chromosomes containing the BrFAE gene. The alien chromosomes from B. rapa were clearly distinguished by molecular cytogenetics in one of the addition lines. Analysis of microspore-derived offspring and hybrids from crosses with a CMS B. carinata line showed that the transfer rate of the A08 chromosome into male gametes was over 98%, resulting in almost completely stable transmission of an A08 chromosome copy into the progeny. The increase in erucic acid levels was accompanied by changes in the proportions of other fatty acids depending on the genetic changes that were introduced in the interspecific hybrids, providing valuable insights into erucic acid metabolism in Brassica.

Functional analysis of ß-ketoacyl-CoA synthase from biofuel feedstock Thlaspi arvense reveals differences in the triacylglycerol biosynthetic pathway among Brassicaceae.

KEY MESSAGE: Differences in FAE1 enzyme affinity for the acyl-CoA substrates, as well as the balance between the different pathways involved in their incorporation to triacylglycerol might be determinant of the different composition of the seed oil in Brassicaceae. Brassicaceae present a great heterogeneity of seed oil and fatty acid composition, accumulating Very Long Chain Fatty Acids with industrial applications. However, the molecular determinants of these differences remain elusive. We have studied the ß-ketoacyl-CoA synthase from the high erucic feedstock Thlaspi arvense (Pennycress). Functional characterization of the Pennycress FAE1 enzyme was performed in two Arabidopsis backgrounds; Col-0, with less than 2.5% of erucic acid in its seed oil and the fae1-1 mutant, deficient in FAE1 activity, that did not accumulate erucic acid. Seed-specific expression of the Pennycress FAE1 gene in Col-0 resulted in a 3 to fourfold increase of erucic acid content in the seed oil. This increase was concomitant with a decrease of eicosenoic acid levels without changes in oleic ones. Interestingly, only small changes in eicosenoic and erucic acid levels occurred when the Pennycress FAE1 gene was expressed in the fae1-1 mutant, with high levels of oleic acid available for elongation, suggesting that the Pennycress FAE1 enzyme showed higher affinity for eicosenoic acid substrates, than for oleic ones in Arabidopsis. Erucic acid was incorporated to triacylglycerol in the transgenic lines without significant changes in their levels in the diacylglycerol fraction, suggesting that erucic acid was preferentially incorporated to triacylglycerol via DGAT1. Expression analysis of FAE1, AtDGAT1, AtLPCAT1 and AtPDAT1 genes in the transgenic lines further supported this conclusion. Differences in FAE1 affinity for the oleic and eicosenoic substrates among Brassicaceae, as well as their incorporation to triacylglycerol might explain the differences in composition of their seed oil.

Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver.

Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague-Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal ß-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal ß-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD+ ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal ß-oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid-induced hepatic steatosis and related metabolic disorders.

Conversion of waste materials into very long chain fatty acids by the recombinant yeast Yarrowia lipolytica.

Erucic acid (C22:1Δ13) has several industrial applications including its use as a lubricant, surfactant and biodiesel and composite material constituent. It is produced by plants belonging to the Brassicaceae family, especially by the high erucic acid rapeseed. The ability to convert oleic acid into erucic acid is facilitated by FAE1. In this study, FAD2 (encoding Δ12-desaturase) was deleted in the strain Po1d to increase oleic acid content. Subsequently, FAE1 from Thlaspi arvense was overexpressed in Yarrowia lipolytica with the Δfad2 genotype. This resulted in the YL10 strain producing very long chain fatty acids, especially erucic acid. The YL10 strain was cultivated in media containing crude glycerol and waste cooking oil as carbon substrates. The cells grown using glycerol produced microbial oil devoid of linoleic acid, which was enriched with very long chain fatty acids, mainly erucic acid (9% of the total fatty acids). When cells were grown using waste cooking oil, the highest yield of erucic acid was obtained (887 mg L-1). However, external linoleic and α-linolenic were accumulated in cellular lipids when yeasts were grown in an oil medium. This study describes the possibility of conversion of waste material into erucic acid by a recombinant yeast strain.

High-throughput method for Antibiotic Susceptibility Testing based on Fluorescein Quenching by Bacteria: Application to Urinary Tract Infection.

We recently reported a sugar-induced bacterial release of 13-Docosenamide and its ability to quench fluorescein. This simple handle to monitor bacterial growth is readily applicable to develop a quicker antibiotic sensitivity testing method along with a low-cost field-use optical instrumentation. Conditions were standardized to perform this new procedure in the most preferred and CLSI-recommended microdilution format in 12-well strips. A simple and portable optoelectronic prototype was used to capture the image and read the fluorescence signal of the culture medium of the 12-well strips. This new Fluorescence Quenching Method along with the device enabled the choice of the right antibiotic within 8 h of sample collection from the patient. It was compliant to the Clinical Laboratory Standard Institute's quality control guidelines. Clinical assessment of the method using 440 urine samples from Urinary Tract Infection patients against 21 routinely used antibiotics showed a 94.3% match with the results of the Standard Disk Diffusion method. This new method saves the precious time taken for and the cost of antibiotic susceptibility testing for quicker and effective treatment with better compliance.

Erucic acid, a nutritional PPARδ-ligand may influence Huntington's disease pathogenesis.

Increasing recent evidence suggests a key role of oligodendroglial injury and demyelination in the pathophysiology of Huntington's Disease (HD) and the transcription factor PPARδ is critical for oligodendroglial regeneration and myelination. PPARδ directly involves in the pathogenesis of HD and treatment with a brain-permeable PPARδ-agonist (KD3010) alleviates its severity in mice. Erucic acid (EA) is also a PPARδ-ligand ω9 fatty acid which is highly consumed in Asian countries through ingesting cruciferous vegetables such as rapeseed (Brassica napus) and indian mustard (Brassica juncea). EA is also an ingredient of Lorenzo's oil employed in the medical treatment of adrenoleukodystrophy and can be converted to nervonic acid, a component of myelin. HD pathogenesis also involves oxidative and inflammatory injury and EA exerts antioxidative and antiinflammatory efficacies including inhibition of thrombin and elastase. Consumption of rapeseed, indian mustard, and Canola oils (containing EA) improves cognitive parameters in animal models, as well as treatment with pure EA. Moreover, erucamide, an endogenous EA-amide derivative regulating angiogenesis and water balance, exerts antidepressive and anxiolytic effects in mice. Hitherto, no study has investigated the therapeutic potential of EA in HD and we believe that it strongly merits to be studied in animal models of HD as a potential therapeutic.

Isoforms of Acyl-CoA:Diacylglycerol Acyltransferase2 Differ Substantially in Their Specificities toward Erucic Acid.

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.

The long-chain monounsaturated cetoleic acid improves the efficiency of the n-3 fatty acid metabolic pathway in Atlantic salmon and human HepG2 cells.

The present study aimed to determine if the long-chain MUFA cetoleic acid (22 : 1n-11) can improve the capacity to synthesise the health-promoting n-3 fatty acids EPA and DHA in human and fish models. Human hepatocytes (HepG2) and salmon primary hepatocytes were first enriched with cetoleic acid, and thereafter their capacities to convert radio-labelled 18 : 3n-3 (α-linolenic acid, ALA) to EPA and DHA were measured. Increased endogenous levels of cetoleic acid led to increased production of radio-labelled EPA + DHA in HepG2 by 40 % and EPA in salmon hepatocytes by 12 %. In order to verify if dietary intake of a fish oil rich in cetoleic acid would have the same beneficial effects on the n-3 fatty acid metabolic pathway in vivo as found in vitro, Atlantic salmon were fed four diets supplemented with either sardine oil low in cetoleic acid or herring oil high in cetoleic acid at two inclusion levels (Low or High). The diets were balanced for EPA + DHA content within the Low and within the High groups. The salmon were fed these diets from 110 to 242 g. The level of EPA + DHA in liver and whole-body retention of docosapentaenoic acid and EPA + DHA relative to what was eaten, increased with increased dietary cetoleic acid levels. Thus, it is concluded that cetoleic acid stimulated the synthesis of EPA and DHA from ALA in human HepG2 and of EPA in salmon hepatocytes in vitro and increased whole-body retention of EPA + DHA in salmon by 15 % points after dietary intake of cetoleic acid.

PPAR-δ and erucic acid in multiple sclerosis and Alzheimer's Disease. Likely benefits in terms of immunity and metabolism.

The transcription factor, PPARδ is involved in suppressing inflammation, stimulating oligodendroglial biogenesis and myelination. Furthermore, activation of PPARδ directly protects mitochondria against noxious stimuli and stimulates biogenesis of new mitochondria. PPARδ activation directly inhibits neuronal cell death and reduces both the level and neurotoxicity of Amyloid-ß fibers in Alzheimer's Disease (AD) models. Among the important ligands of PPARδ is erucic acid (EA, 22:1 n9), an edible omega-9 fatty acid and a component of Lorenzo's oil, which is used in the treatment of adrenoleukodystrophy (ALD). Nonetheless, the feature of PPARδ-erucic acid interaction has not been extensively studied. EA can also be converted to nervonic acid, an important component of myelin. Hence, EA may act as an anti-inflammatory and remyelinating agent, which might be important in the management of another demyelinating disease, multiple sclerosis (MS). Oxidative injury and mitochondrial damage are among the features of ALD. Direct inhibitory effects of EA was observed on lipid peroxidation and inflammatory enzymes, neutrophil elastase and thrombin. EA also induces catalase, a potent antioxidant peroxisomal enzyme. However, EA is claimed to be a cardiotoxic molecule, yet these studies were mostly performed on rats, which do not efficiently metabolize EA. Further, EA is largely consumed by Asian population and Greenland Eskimos with no signs of cardiac damage. In this review, we shed light on the potential theraputic role of EA in MS and AD by blocking neural cell death, mitigating neuroinflammation and/or inducing myelination.

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop.

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyl-triacylglycerol-containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress' rapid domestication to meet the growing sustainable food and fuel demands.

13-Docosenamide release by bacteria in response to glucose during growth-fluorescein quenching and clinical application.

Our investigations on extracellular biochemical events to find readily and sensitively detectable/measurable molecular targets for developing easier, simpler, and quicker diagnostic methods and tools for bacterial pathogens led to the observation that bacteria grown in the presence of glucose produced a compound capable of quenching fluorescein. Under the experimental conditions, among various sugars, glucose was found to induce maximum amount of the quencher when Escherichia coli was grown in presence of 50 mM glucose in rarified LB. The release of quencher closely following bacterial growth significantly from fourth hour after moderate inoculation. This fluorescein-quencher was purified using TLC and HPLC and identified using GC-MS as 13-docosenamide or erucamide, originally known as plant lipid, is a neuroactive compound in human and animals. Fluorescence and UV-absorption spectral analysis showed that the compound formed stable adduct with fluorescein in the ground state. Commercial 13-docosonamide enabled quantitation of the compound produced in micromolar quantities during glucose utilization from the medium. Twenty-seven different commonly encountered bacteria, pathogens or otherwise, could produce the quencher. A simple microplate-based growth monitoring method was developed exploiting quenching as an easily and readily measurable signal, either using a reader or an imager. While 13-docosenamide release by bacteria may be relevant in host-bacteria interactions, especially when growing under conditions that provide glucose, the new approach with inexpensive reagents can provide a new antibiogram technique.

One-Step Bioconversion of Fatty Acids into C8-C9 Volatile Aroma Compounds by a Multifunctional Lipoxygenase Cloned from Pyropia haitanensis.

The multifunctional lipoxygenase PhLOX cloned from Pyropia haitanensis was expressed in Escherichia coli with 24.4 mg·L-1 yield. PhLOX could catalyze the one-step bioconversion of C18-C22 fatty acids into C8-C9 volatile organic compounds (VOCs), displaying higher catalytic efficiency for eicosenoic and docosenoic acids than for octadecenoic acids. C20:5 was the most suitable substrate among the tested fatty acids. The C8-C9 VOCs were generated in good yields from fatty acids, e.g., 2E-nonenal from C20:4, and 2E,6Z-nonadienal from C20:5. Hydrolyzed oils were also tested as substrates. The reactions mainly generated 2E,4E-pentadienal, 2E-octenal, and 2E,4E-octadienal from hydrolyzed sunflower seed oil, corn oil, and fish oil, respectively. PhLOX showed good stability after storage at 4 °C for 2 weeks and broad tolerance to pH and temperature. These desirable properties of PhLOX make it a promising novel biocatalyst for the industrial production of volatile aroma compounds.

Dissection of the genetic architecture of three seed-quality traits and consequences for breeding in Brassica napus.

Genome-wide association studies (GWASs) combining high-throughput genome resequencing and phenotyping can accelerate the dissection of genetic architecture and identification of genes for plant complex traits. In this study, we developed a rapeseed genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs. GWAS was performed for three seed-quality traits, including erucic acid content (EAC), glucosinolate content (GSC) and seed oil content (SOC) using 3.82 million polymorphisms in an association panel. Six, 49 and 17 loci were detected to be associated with EAC, GSC and SOC in multiple environments, respectively. The mean total contribution of these loci in each environment was 94.1% for EAC and 87.9% for GSC, notably higher than that for SOC (40.1%). A high correlation was observed between phenotypic variance and number of favourable alleles for associated loci, which will contribute to breeding improvement by pyramiding these loci. Furthermore, candidate genes were detected underlying associated loci, based on functional polymorphisms in gene regions where sequence variation was found to correlate with phenotypic variation. Our approach was validated by detection of well-characterized FAE1 genes at each of two major loci for EAC on chromosomes A8 and C3, along with MYB28 genes at each of three major loci for GSC on chromosomes A9, C2 and C9. Four novel candidate genes were detected by correlation between GSC and SOC and observed sequence variation, respectively. This study provides insights into the genetic architecture of three seed-quality traits, which would be useful for genetic improvement of B. napus.

Fish oil feeding attenuates neuroinflammatory gene expression without concomitant changes in brain eicosanoids and docosanoids in a mouse model of Alzheimer's disease.

BACKGROUND: Neuroinflammation is a recognized hallmark of Alzheimer's disease, along with accumulation of amyloid-ß plaques, neurofibrillary tangles and synaptic loss. n-3 polyunsaturated fatty acids (PUFA) and molecules derived from them, including eicosapentaenoic acid-derived eicosanoids and docosahexaenoic acid-derived docosanoids, are known to have both anti-inflammatory and pro-resolving properties, while human observational data links consumption of these fatty acids to a decreased risk of Alzheimer's disease. Few studies have examined the neuroinflammation-modulating effects of n-3 PUFA feeding in an Alzheimer's disease-related model, and none have investigated whether these effects are mediated by changes in brain eicosanoids and docosanoids. Here, we use both a fat-1 transgenic mouse and a fish oil feeding model to study the impact of increasing tissue n-3 PUFA on neuroinflammation and the production of pro-inflammatory and pro-resolving lipid mediators. METHODS: Fat-1 mice, transgenic animals that can convert n-6 to n-3 PUFA, and their wildtype littermates were fed diets containing either fish oil (high n-3 PUFA) or safflower oil (negligible n-3 PUFA) from weaning to 12 weeks. Animals then underwent intracerebroventricular infusion of either amyloid-ß 1-40 or a control peptide. Hippocampi were collected from non-surgery and surgery animals 10 days after infusion. Microarray was used to measure enrichment of inflammation-associated gene categories and expression of genes involved in the synthesis of lipid mediators. Results were validated by real-time PCR in a separate cohort of animals. Lipid mediators were measured via liquid chromatography tandem mass spectrometry. RESULTS: Fat-1 and wildtype mice fed fish oil had higher total hippocampal DHA than wildtype mice fed the safflower oil diet. The safflower-fed mice, but not the fat-1 or fish oil-fed mice, had significantly increased expression in gene ontology categories associated with inflammation in response to amyloid-ß infusion. These effects were independent of changes in the expression of genes involved in the synthesis of eicosanoids or docosanoids in any group. Gene expression was replicated upon validation in the wildtype safflower and fish oil-fed, but not the fat-1 mice. Protectin, maresin and D and E series resolvins were not detected in any sample. There were no major differences in levels of other eicosanoids or docosanoids between any of the groups in response to amyloid-ß infusion. CONCLUSIONS: Fish oil feeding decreases neuroinflammatory gene expression in response to amyloid-ß. Neither amyloid-ß infusion or increasing brain DHA affects the brain concentrations of specialized pro-resolving mediators in this model, or the concentrations of most other eicosanoids and docosanoids.

Depressed expression of FAE1 and FAD2 genes modifies fatty acid profiles and storage compounds accumulation in Brassica napus seeds.

In plants, the enzymes fatty acid dehydrogenase 2 (FAD2) and fatty acid elongase 1 (FAE1) have been shown in previous studies to play important roles in the de novo biosynthesis of fatty acids. However, the effects of depressed expression of FAD2 and FAE1 on seed storage compounds accumulation remains to be elucidated. In this study, we produced RNA interfering transgenic rapeseeds lines, BnFAD2-Ri, BnFAE1-Ri and BnFAD2/BnFAE1-Ri, which exhibited depressed expression of the BnFAD2 and BnFAE1 genes under the control of seed-specific napin A promoter. These transgenic rapeseeds showed normal growth and development as compared with the wild type (CY2). Depressed expression of BnFAD2 and BnFAE1 genes modified fatty acid profiles, leading to increased oleic acid and decreased erucic acid contents in transgenic seeds. Consistent with these results, the ratios of C18:1/C18:2 and C18:1/C18:3 in C18 unsaturated fatty acids were greatly increased due to increased oleic acid content in transgenic seeds. Moreover, depressed expression of BnFAD2 and BnFAE1 genes resulted in slightly decreased oil contents and increased protein contents in transgenic seeds. Our results demonstrated that depressed expression of BnFAD2 and BnFAE1 greatly improves seed nutritional quality by modulating the fatty acid metabolism and storage products accumulation and that BnFAD2 and BnFAE1 are reliable targets for genetic improvement of rapeseed in seed nutritional quality.

Regulation of FATTY ACID ELONGATION1 expression and production in Brassica oleracea and Capsella rubella.

MAIN CONCLUSION: The contribution of variations in coding regions or promoters to the changes in FAE1 expression levels have be quantified and compared in parallel by specifically designed swapping constructs. FATTY ACID ELONGATION1 (FAE1) is a key gene in control of erucic acid synthesis in plant seeds. The expression of FAE1 genes in Brassica oleracea and Capsella rubella, representatives of high and low erucic acid species, respectively, was characterized to provide insight into the regulation of very long-chain fatty-acid biosynthesis in seeds. Virtually, no methylation was detected either in B. oleracea or in C. rubella, suggesting that modification of promoter methylation might not be a predominant mechanism. Swapping constructs were specifically designed to quantify and compare the contribution of variations in coding regions or promoters to the changes in FAE1 expression levels in parallel. A significantly higher fold change in erucic acid content was observed when swapping coding regions rather than when swapping promoters, indicating that the coding region is a major determinant of the catalytic power of ß-ketoacyl-CoA synthase proteins. Common motifs have been proposed as essential for the preservation of basic gene expression patterns, such as seed-specific expression. However, the occurrence of variation in common cis-elements or the presence of species-specific cis-elements might be plausible mechanisms for changes in the expression levels in different organisms. In addition, conflicting observations in previous reports associated with FAE1 expression are discussed, and we suggest that caution should be taken when selecting a plant transformation vector and in interpreting the results obtained from vectors carrying the CaMV 35S promoter.