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Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6[Q409R] and MpFADS6[M242P] abolished desaturation, while MpFADS6[F419V] and MpFADS6[A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases.

In human milk fat, palmitic acid (16:0) is esterified to the middle (sn-2 or β) position on the glycerol backbone and oleic acid (18:1) predominantly to the outer positions, giving the triacylglycerol (TG) a distinctive stereoisomeric structure that is believed to assist nutrient absorption in the infant gut. However, the fat used in most infant formulas is derived from plants, which preferentially esterify 16:0 to the outer positions. We have previously showed that the metabolism of the model oilseed Arabidopsis thaliana can be engineered to incorporate 16:0 into the middle position of TG. However, the fatty acyl composition of Arabidopsis seed TG does not mimic human milk, which is rich in both 16:0 and 18:1 and is defined by the high abundance of the TG molecular species 1,3-olein-2-palmitin (OPO). Here we have constructed an Arabidopsis fatty acid biosynthesis 1-1 fatty acid desaturase 2 fatty acid elongase 1 mutant with around 20% 16:0 and 70% 18:1 in its seeds and we have engineered it to esterify more than 80% of the 16:0 to the middle position of TG, using heterologous expression of the human lysophosphatidic acid acyltransferase isoform AGPAT1, combined with suppression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE. Our data show that oilseeds can be engineered to produce TG that is rich in OPO, which is a structured fat ingredient used in infant formulas.

Lipids are major and essential constituents of plant cells and provide energy for various metabolic processes. However, the function of the lipid signal in defence against Verticillium dahliae, a hemibiotrophic pathogen, remains unknown. Here, we characterized 19 conserved stearoyl‐ACP desaturase family proteins from upland cotton (Gossypium hirsutum). We further confirmed that GhSSI2 isoforms, including GhSSI2‐A, GhSSI2‐B, and GhSSI2‐C located on chromosomes A10, D10, and A12, respectively, played a dominant role to the cotton 18:1 (oleic acid) pool. Suppressing the expression of GhSSI2s reduced the 18:1 level, which autoactivated the hypersensitive response (HR) and enhanced cotton Verticillium wilt and Fusarium wilt resistance. We found that low 18:1 levels induced phenylalanine ammonia‐lyase‐mediated salicylic acid (SA) accumulation and activated a SA‐independent defence response in GhSSI2s‐silenced cotton, whereas suppressing expression of GhSSI2s affected PDF1.2‐dependent jasmonic acid (JA) perception but not the biosynthesis and signalling cascade of JA. Further investigation showed that structurally divergent resistance‐related genes and nitric oxide (NO) signal were activated in GhSSI2s‐silenced cotton. Taken together, these results indicate that SA‐independent defence response, multiple resistance‐related proteins, and elevated NO level play an important role in GhSSI2s‐regulated Verticillium wilt resistance. These findings broaden our knowledge regarding the lipid signal in disease resistance and provide novel insights into the molecular mechanism of cotton fungal disease resistance., GhSACPD gene family isoforms GhSSI2‐A, GhSSI2‐B, and GhSSI2‐C displayed an important role in regulating 18:1 fatty acid level and negatively regulated cotton disease resistance to Verticillium wilt.

Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography–mass spectrometry (GC–MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.

Summary Safflower (Carthamus tinctorius L.), a member of the Asteraceae, is a popular crop due to its high linoleic acid (LA) and flavonoid (such as hydroxysafflor yellow A) contents. Here, we report the first high‐quality genome assembly (contig N50 of 21.23 Mb) for the 12 pseudochromosomes of safflower using single‐molecule real‐time sequencing, Hi‐C mapping technologies and a genetic linkage map. Phyloge nomic analysis showed that safflower diverged from artichoke (Cynara cardunculus) and sunflower (Helianthus annuus) approximately 30.7 and 60.5 million years ago, respectively. Comparative genomic analyses revealed that uniquely expanded gene families in safflower were enriched for those predicted to be involved in lipid metabolism and transport and abscisic acid signalling. Notably, the fatty acid desaturase 2 (FAD2) and chalcone synthase (CHS) families, which function in the LA and flavonoid biosynthesis pathways, respectively, were expanded via tandem duplications in safflower. CarFAD2‐12 was specifically expressed in seeds and was vital for high‐LA content in seeds, while tandemly duplicated CarFAD2 genes were up‐regulated in ovaries compared to CarFAD2‐12, which indicates regulatory divergence of FAD2 in seeds and ovaries. CarCHS1, CarCHS4 and tandem‐duplicated CarCHS5˜CarCHS6, which were up‐regulated compared to other CarCHS members at early stages, contribute to the accumulation of major flavonoids in flowers. In addition, our data reveal multiple alternative splicing events in gene families related to fatty acid and flavonoid biosynthesis. Together, these results provide a high‐quality reference genome and evolutionary insights into the molecular basis of fatty acid and flavonoid biosynthesis in safflower.

Membrane-bound fatty acid desaturase (FAD) gene family plays crucial roles in regulation of fatty acid (FA) compositions in plants. Sunflower (Helianthus annuus L.) is an important oilseed crop in the world; however, no comprehensive study on exploring the role of FAD family in relation to stress tolerance in sunflower has been performed yet. In this study, we identified 40 putative FAD genes in H. annuus (HaFAD), which were unevenly distributed across 13 of the total 17 chromosomes. Phylogenetic analysis indicated that HaFAD genes were divided into four subfamilies, as supported by highly conserved gene structures and motifs. Collinearity analysis showed that tandem duplication events played a crucial role in the expansion of HaFAD gene family. In addition, tissue-specific expression showed that 32 HaFAD genes were widely expressed in various tissues or organs of sunflower. Furthermore, qRT-PCR results revealed significant expression changes of HaFAD genes in response to abiotic (cadmium, drought) and biotic (Orobanche cumana) stresses, suggesting their important functions in response to different stresses. Therefore, our results provide insights into HaFAD gene family in response to different stresses, and some specific up-regulated genes such as HaFAD3.2, HaADS8, HaFAD2.1, and HaADS9 would be the potential candidate genes for the sunflower tolerance breeding.

BACKGROUND PfFAD3 transgenic soybean expressing omega‐3 fatty acid desaturase 3 of Physaria produces increased level of α‐linolenic acid in seed. Composition data of non‐transgenic conventional varieties is important in the safety assessment of the genetically‐modified (GM) crops in the context of the natural variation. RESULTS The natural variation was characterized in seed composition of 13 Korean soybean varieties grown in three locations in South Korea for 2 years. Univariate analysis of combined data showed significant differences by variety and cultivation environment for proximates, minerals, anti‐nutrients, and fatty acids. Percent variability analysis demonstrated that genotype, environment and the interaction of environment with genotype contributed to soybean seed compositions. Principal component analysis and orthogonal projections to latent structure discriminant analysis indicated that significant variance in compositions was attributable to location and cultivation year. The composition of three PfFAD3 soybean lines for proximates, minerals, anti‐nutrients, and fatty acids was compared to a non‐transgenic commercial comparator (Kwangankong, KA), and three non‐transgenic commercial varieties grown at two sites in South Korea. Only linoleic and linolenic acids significantly differed in PfFAD3‐1 lines compared to KA, which were expected changes by the introduction of the PfFAD3‐1 trait in KA. CONCLUSION Genotype, environment, and the interaction of environment with genotype contributed to compositional variability in soybean. PfFAD3‐1 soybean is equivalent to the conventional varieties with respect to these components. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pendimethalin is a member of the dinitroaniline class herbicide. It used to control most annual grasses and many annual broad-leaved weeds. Salicylic acid acts as an endogenous signal molecule in charge of inducing environmental stress tolerance in plants. Omega-3 fatty acid desaturase is a key enzyme for α-linolenic acid biosynthesis. Here, we searched to understand the beneficial impacts of salicylic acid on fatty acid desaturase gene (FAD3 and FAD7) expression during pendimethalin stress in safflower (Carthamus tinctorious cv. “Dinçer). In this study, 0.004 and 0.01 M pendimethalin was applied to safflower plants grown under controlled climatic conditions in 36 pots. 0.05 mM salicylic acid was applied to the samples where pendimethalin was applied. After application, gene expression analyze were performed using quantitative polymerase chain reaction. The floor change between the test and control groups was calculated with the formula 2 ^ (delta delta CT). The change between gene expression levels was evaluated by t test (p, Pendimethalin dinitroanilin sınıfı bir herbisittir. Pendimethalin genellikle tek yıllık otsu bitkiler ve birçok geniş yapraklı ot ile mücadelede kullanılır. Salisilik asit bitkilerde çevresel stres toleransını indüklemekten sorumlu endojen bir sinyal molekülü olarak rol oynamaktadır. Omega-3 yağ asidi desatüraz, α-linolenik asit biyosentezi için anahtar bir enzimdir. Bu çalışmada salisilik asidin aspirde (Carthamus tinctorius L. cv. Dinçer) pendimethalin stresi altında yağ asidi desatüraz genlerinin ifade düzeyi üzerindeki etkileri araştırıldı. Çalışmada 36 saksıda kontrollü iklim koşullarında yetiştirilen aspir bitkilerine 0.004 ve 0.01 M pendimethalin uygulanmıştır. Pendimethalin uygulanan bitkilere sonrasında 0.05 mM salisilik asit uygulanmıştır. Uygulama sonrasında RNA izolasyonu, cDNA sentezi ve kantitatif polimeraz zincir reaksiyonu yapılmıştır. Test ve kontrol grupları arasındaki kat değişimi 2 ^ (delta delta CT) formülü ile hesaplanmıştır. Gen ifade düzeyleri arasındaki değişim t testi ile değerlendirildi (p

Psychrophilic bacteria, living at low and mild temperatures, can contribute significantly to our understanding of microbial responses to temperature, markedly occurring in the bacterial membrane. Here, a newly isolated strain, Pseudomonas sp. B14-6, was found to dynamically change its unsaturated fatty acid and cyclic fatty acid content depending on temperature which was revealed by phospholipid fatty acid (PLFA) analysis. Genome sequencing yielded the sequences of the genes Δ-9-fatty acid desaturase (desA) and cyclopropane-fatty acid-acyl-phospholipid synthase (cfa). Overexpression of desA in Escherichia coli led to an increase in the levels of unsaturated fatty acids, resulting in decreased membrane hydrophobicity and increased fluidity. Cfa proteins from different species were all found to promote bacterial growth, despite their sequence diversity. In conclusion, PLFA analysis and genome sequencing unraveled the temperature-related behavior of Pseudomonas sp. B14-6 and the functions of two membrane-related enzymes. Our results shed new light on temperature-dependent microbial behaviors and might allow to predict the consequences of global warming on microbial communities.

Fatty acid desaturases, especially Δ12 fatty acid desaturases, are key enzymes for the production of unsaturated fatty acids in oleaginous yeasts. In this study, we identified and characterized a gene encoding Δ12 fatty acid desaturase of Pseudozyma antarctica named PaFAD2. Almost all oleic acid (C18:1) was converted to linoleic acid by the heterologous expression of the PaFAD2 gene in Saccharomyces cerevisiae and Lipomyces starkeyi oleaginous yeast. Notably, PaFad2 converted not only oleic acid to linoleic acid, but also palmitoleic acid (C16:1) to 9,12-hexadecadienoic acid (C16:2). These results indicated that the PaFAD2 gene was very useful for the production of polyunsaturated fatty acids in yeast, including oleaginous yeast.

RNA interference is a powerful tool for dissecting gene function. In Caenorhabditis elegans, ingestion of double stranded RNA causes strong, systemic knockdown of target genes. Further insight into gene function can be revealed by tissue-specific RNAi techniques. Currently available tissue-specific C. elegans strains rely on rescue of RNAi function in a desired tissue or cell in an otherwise RNAi deficient genetic background. We attempted to assess the contribution of specific tissues to polyunsaturated fatty acid (PUFA) synthesis using currently available tissue-specific RNAi strains. We discovered that rde-1 (ne219), a commonly used RNAi-resistant mutant strain, retains considerable RNAi capacity against RNAi directed at PUFA synthesis genes. By measuring changes in the fatty acid products of the desaturase enzymes that synthesize PUFAs, we found that the before mentioned strain, rde-1 (ne219) and the reported germline only RNAi strain, rrf-1 (pk1417) are not appropriate genetic backgrounds for tissue-specific RNAi experiments. However, the knockout mutant rde-1 (ne300) was strongly resistant to dsRNA induced RNAi, and thus is more appropriate for construction of a robust tissue-specific RNAi strains. Using newly constructed strains in the rde-1(null) background, we found considerable desaturase activity in intestinal, epidermal, and germline tissues, but not in muscle. The RNAi-specific strains reported in this study will be useful tools for C. elegans researchers studying a variety of biological processes.

Teleost fish can synthesize one of the major omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), docosahexaenoic acid (DHA, 22:6n-3), from dietary α-linolenic acid (ALA; 18:3n-3), via elongase of very long chain fatty acid (Elovl) and fatty acid desaturase (Fads). However, it remains unclear which elongase is responsible for the endogenous synthesis of DHA. Here in this study, the knockout models of the two major elongases, Elovl2 and Elovl5, were generated by CRISPR/Cas9 approach in zebrafish and comparatively analyzed. The homozygous mutants were validated by Sanger sequencing, mutation-mediated PCR and whole-mount in situ hybridization analysis of the endogenous target genes. Compared with wildtype (WT) counterparts, the content of DHA was significantly reduced by 67.1% (pelovl2 mutant, but not of the elovl5 mutant. Further study revealed that elovl2 and fads2 was upregulated by 9.9-fold (pelovl5 mutant, and elovl5 and fads2 was upregulated by 15.1-fold (pelovl2 mutant. Our study indicates that although both Elovl2 and Elovl5 have the elongase activity toward C20, the upregulation of elovl2 could completely replace the genetic depletion of elovl5, but upregulation of elovl5 could not compensate the endogenous deficiency of elovl2 in mediating DHA synthesis. In conclusion, the endogenous synthesis of DHA in is mediated by Elovl2 but not Elovl5 in teleost, and a DHA-deficient genetic model of zebrafish has been generated.

Alpha-linolenic acid (ALA, 18:3Δ9,12,15) and γ-linolenic acid \ (GLA, 18:3Δ6,9,12) are important trienoic fatty acids, which are beneficial for human health in their own right, or as precursors for the biosynthesis of long-chain polyunsaturated fatty acids. ALA and GLA in seed oil are synthesized from linoleic acid (LA, 18:2Δ9,12) by the microsomal ω-3 fatty acid desaturase (FAD3) and Δ6 desaturase (D6D), respectively. Cotton (Gossypium hirsutum L.) seed oil composition was modified by transforming with an FAD3 gene from Brassica napus and a D6D gene from Echium plantagineum, resulting in approximately 30% ALA and 20% GLA, respectively. The total oil content in transgenic seeds remained unaltered relative to parental seeds. Despite the use of a seed-specific promoter for transgene expression, low levels of GLA and increased levels of ALA were found in non-seed cotton tissues. At low temperature, the germinating cottonseeds containing the linolenic acid isomers elongated faster than the untransformed controls. ALA-producing lines also showed higher photosynthetic rates at cooler temperature and better fiber quality compared to both untransformed controls and GLA-producing lines. The oxidative stability of the novel cottonseed oils was assessed, providing guidance for potential food, pharmaceutical and industrial applications of these oils.

Background Unexplained heterogeneity in clinical trials has resulted in questions regarding the effectiveness of ɣ-linolenic acid (GLA)-containing botanical oil supplements. This heterogeneity may be explained by genetic variation within the fatty acid desaturase (FADS) gene cluster that is associated with circulating and tissue concentrations of arachidonic acid (ARA) and dihomo-ɣ-linolenic acid (DGLA), both of which may be synthesized from GLA and result in proinflammatory and anti-inflammatory metabolites, respectively. Objectives The objective of this study was to prospectively compare the capacity of a non-Hispanic white cohort, stratified by FADS genotype at the key single-nucleotide polymorphism (SNP) rs174537, to metabolize 18-carbon omega-6 (n-6) PUFAs in borage oil (BO) and soybean oil (SO) to GLA, DGLA, and ARA. Methods Healthy adults (n = 64) participated in a randomized, double-blind, crossover intervention. Individuals received encapsulated BO (Borago officinalis L.; 37% LA and 23% GLA) or SO [Glycine max (L.) Merr.; 50% LA and 0% GLA] for 4 wk, followed by an 8-wk washout period, before consuming the opposite oil for 4 wk. Serum lipids and markers of inflammation (C-reactive protein) were assessed for both oil types at baseline and during weeks 2 and 4 of the intervention. Results SO supplementation failed to alter circulating concentrations of any n-6 long-chain PUFAs. In contrast, a modest daily dose of BO elevated serum concentrations of GLA and DGLA in an rs174537 genotype-dependent manner. In particular, DGLA increased by 57% (95% CI: 0.38, 0.79) in GG genotype individuals, but by 141% (95% CI: 1.03, 2.85) in TT individuals. For ARA, baseline concentrations varied substantially by genotype and increased modestly with BO supplementation, suggesting a key role for FADS variation in the balance of DGLA and ARA. Conclusions The results of this study clearly suggest that personalized and population-based approaches considering FADS genetic variation may be necessary to optimize the design of future clinical studies with GLA-containing oils. This trial was registered at clinicaltrials.gov as NCT02337231.

Background Walnut kernels contain a large amount of unsaturated fatty acids, such as linoleic acid and linolenic acid, which are essential fatty acids for humans and have important effects on growth and health. The main function of fatty acid desaturase (FAD), which is widely distributed in organisms, is to remove hydrogen from carbon chains in the biosynthesis of unsaturated fatty acids to generate C=C bonds. Results By performing a series of bioinformatics analysis, 24 members of the JrFAD gene family were identified from the genome database of walnut, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that JrFADs were classified into four subfamilies: the SAD desaturase subfamily, Δ7/Δ9 desaturase subfamily, Δ12/ω-3 desaturase subfamily and “front-end” desaturase subfamily. Meanwhile, the expression of fatty acid synthesis genes in walnut kernels at different developmental stages was analysed by transcriptome sequencing, with expression of JrFAD3-1, which encodes an enzyme involved in linolenic acid synthesis, being particularly prominent. The relative expression level of JrFAD3-1 changed dramatically with the kernel development stages and exhibited a Bell-Shaped Curve. A significant positive correlation was observed between the expression of JrFAD3-1 during 70–100 DAF (Days after flowering) and the content of alpha-linolenic acid during 100–130 DAF, with a correlation coefficient of 0.991. Additionally, JrFAD3-1 was proved closely related to homologous genes in Betula pendula and Corylus heterophylla, indicating that the conserved structure of FADs is consistent with classical plant taxonomy. Conclusion Twenty-four members JrFADs in walnut were identified and classified into four subfamilies. JrFAD3-1 may play significant roles in the biosynthesis of polyunsaturated fatty acids in walnut.

Botryococcus braunii, a green alga that can produce a variety of secondary metabolites such as lipids, protein, and carotenoid, is considered a potential biodiesel feedstock for industrial commercialization. This study investigated the effects of exogenous plant growth regulators (PGRs) (6-benzylaminopurine (6-BA), abscisic acid (ABA), 2,4-epibrassinolide (EBR), ethephon (ETH), gibberellic acid (GA3), 1-naphthaleneacetic acid (NAA), salicylic acid (SA), and spermidine (SPD)) on the growth, protein, carotenoid and lipid biosynthesis, fatty acid composition, and expression of fatty acid biosynthetic genes in Botryococcus braunii B12 (B. braunii B12). NAA was the most effective inducer of microalgae biomass, which increased approximately 2-fold compared with that of the control. The maximum lipid accumulation and content were found under 15 mg/L GA3 treatment; the lipid accumulation reached 0.27 g/L, and the lipid content reached 60.25%. Moreover, the soluble protein content reached 28.49 mg/g DCW under 0.05 mg/L 6-BA treatment. Similarly, the highest carotenoid content (108.70 mg/g DCW) was observed under 50 mg/L NAA treatment. Furthermore, all PGRs altered the fatty acid composition of B. braunii B12, particularly the C16-C18 content (%). Finally, the effects of eight PGRs on seven lipid-related genes (acyl-acyl carrier protein (ACP), biotin carboxylase (BC), ω-3 fatty acid desaturase (FAD), acyl carrier protein thioesterase (FATA), 3-ketoacyl carrier protein synthase gene (KAS), malonyl-CoA:ACP transacylase (MCTK), and stearoyl-ACP-desaturase (SAD)) were studied by qRT-PCR, and an apparent correlation was found between gene expression and lipid synthesis in the microalgae.

The replication of positive strand RNA viruses in plant cells is markedly influenced by the desaturation status of fatty acid chains in lipids of intracellular plant membranes. At present, little is known about the role of lipid desaturation in the replication of tobamoviruses. Therefore, we investigated the expression of fatty acid desaturase (FAD) genes and the fatty acid composition of pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation induced a hypersensitive reaction (incompatible interaction) while Pepper mild mottle virus (PMMoV) inoculation caused a systemic infection (compatible interaction). Changes in the expression of 16 FADs were monitored in pepper leaves following ObPV and PMMoV inoculations. ObPV inoculation rapidly and markedly upregulated seven Δ12-FADs that encode enzymes putatively located in the endoplasmic reticulum membrane. In contrast, PMMoV inoculation resulted in a weaker but rapid upregulation of two Δ12-FADs and a Δ15-FAD. The expression of genes encoding plastidial FADs was not influenced neither by ObPV nor by PMMoV. In accordance with gene expression results, a significant accumulation of linoleic acid was observed by gas chromatography-mass spectrometry in ObPV-, but not in PMMoV-inoculated leaves. ObPV inoculation led to a marked accumulation of H2O2 in the inoculated leaves. Therefore, the effect of H2O2 treatments on the expression of six tobamovirus-inducible FADs was also studied. The expression of these FADs was upregulated to different degrees by H2O2 that correlated with ObPV-inducibility of these FADs. These results underline the importance of further studies on the role of pepper FADs in pepper-tobamovirus interactions.

In plants, submergence from flooding causes hypoxia, which impairs energy production and affects plant growth, productivity, and survival. In Arabidopsis, hypoxia induces nuclear localization of the group VII ethylene-responsive transcription factor RELATED TO AP2.12 (RAP2.12), following its dissociation from the plasma membrane-anchored ACYL-COA BINDING PROTEIN1 (ACBP1) and ACBP2. Here, we show that polyunsaturated linolenoyl-CoA (18:3-CoA) regulates RAP2.12 release from the plasma membrane. Submergence caused a significant increase in 18:3-CoA, but a significant decrease in 18:0-, 18:1-, and 18:2-CoA. Application of 18:3-CoA promoted nuclear accumulation of the green fluorescent protein (GFP) fusions RAP2.12-GFP, HYPOXIA-RESPONSIVE ERF1-GFP, and RAP2.3-GFP, and enhanced transcript levels of hypoxia-responsive genes. Plants with decreased ACBP1 and ACBP2 (acbp1 ACBP2-RNAi, produced by ACBP2 RNA interference in the acbp1 mutant) had reduced tolerance to hypoxia and impaired 18:3-CoA-induced expression of hypoxia-related genes. In knockout mutants and overexpression lines of LONG-CHAIN ACYL-COA SYNTHASE2 (LACS2) and FATTY ACID DESATURASE 3 (FAD3), the acyl-CoA pool size and 18:3-CoA levels were closely related to ERF-VII-mediated signaling and hypoxia tolerance. These findings demonstrate that polyunsaturation of long-chain acyl-CoAs functions as important mechanism in the regulation of plant hypoxia signaling, by modulating ACBP-ERF-VII dynamics.

Summary In cold marine environments, the obligate hydrocarbon‐degrading psychrophile Oleispira antarctica RB‐8, which utilizes aliphatic alkanes almost exclusively as substrates, dominates microbial communities following oil spills. In this study, LC–MS/MS shotgun proteomics was used to identify changes in the proteome induced during growth on n‐alkanes and in cold temperatures. Specifically, proteins with significantly higher relative abundance during growth on tetradecane (n‐C14) at 16°C and 4°C have been quantified. During growth on n‐C14, O. antarctica expressed a complete pathway for the terminal oxidation of n‐alkanes including two alkane monooxygenases, two alcohol dehydrogenases, two aldehyde dehydrogenases, a fatty‐acid‐CoA ligase, a fatty acid desaturase and associated oxidoreductases. Increased biosynthesis of these proteins ranged from 3‐ to 21‐fold compared with growth on a non‐hydrocarbon control. This study also highlights mechanisms O. antarctica may utilize to provide it with ecological competitiveness at low temperatures. This was evidenced by an increase in spectral counts for proteins involved in flagella structure/output to overcome higher viscosity, flagella rotation to accumulate cells and proline metabolism to counteract oxidative stress, during growth at 4°C compared with 16°C. Such species‐specific understanding of the physiology during hydrocarbon degradation can be important for parameterizing models that predict the fate of marine oil spills.

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid-associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild-type levels of 16:1t. The FAD4-PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co-production of PRXQ with FAD4 was required to produce Δ3-desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site-directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.

The synthesis of α-linolenic acid (ALA) requires the activity of ω-3 fatty acid desaturases (ω-3 FADs). The quality of peanut oil would be much improved if the content of ALA could be increased. A scan of the peanut genome revealed that it harbored 36 FAD genes, mapping to 16 of the species’ 20 chromosomes. A phylogenetic analysis concluded that these genes belonged to six sub-families, namely stearoyl-acyl-acyl carrier protein desaturases (SAD), FAD2, FAD3, FAD4/5, FAD6 and FAD7/8. Of these, FAD3 and FAD7/8 encoded ω-3 FADs, while genes belonging to the other four sub-families encoded ω-6 FADs. Based on RNA-Seq data, each of the 36 FAD genes was shown to be transcribed in non-stressed plants, but there was variation between them with respect to which organs they were transcribed in. Four ω-3 AhFAD3 genes were functionally characterized; when expressed in Arabidopsis thaliana protoplasts, each was localized mainly in the endoplasmic reticulum, while within peanut, the genes were more strongly transcribed in the developing seed than in either the root or the leaf. When constitutively expressed in Arabidopsis thaliana, both the total fatty acid content of the seed and the relative contribution of ALA were increased. The transgenic seedlings also exhibited an improved level of survival when challenged by salinity stress.

The diversity of 11 fatty acid desaturase (fad2) genes has not been investigated between cultivated and wild species in the Carthamus genus. In this study, 17 C. tinctorius accessions and 28 accessions from other Carthamus species were subjected to sequence analyses of this fad2 gene family. Results showed that among these genes, fad2-1 had a major role in the conversion of oleic acid to linoleic acid. Grouping of all studied wild polyploid species and the wild diploid C. leucocaulos suggested that C. lanatus transferred its fad2-1 gene to C. turkestanicus and C. lanatus. A phylogenetic tree based on fad2-1 gene sequences also showed that C. palaestinus and C. oxyacanthus grouped with C. tinctorius individuals, suggesting that C. tinctorius is closely related to both wild species. A one base pair deletion at position 604 in the fad2-1 gene coding region correlated with high levels of oleic acid content in five mutant phenotypes of the evaluated C. tinctorius accessions. Grouping of fad2-1 and fad2-8 (Ctfad2-10) indicated that both of these genes are involved in oleate desaturases activity. The fad2-3 (Ctfad2-3) and Ctfad2-4 had the highest sequence similarity among the other fad2 genes, indicating the conservative nature of these two genes among all the studied species. Our results suggest that C. lanatus is the likely progenitor of C. turkestanicus and C. creticus (Synonym C. baeticus). Also, C. palaestinus is genetically closer to C. tinctorius but the involvement of C. oxyacanthus cannot be excluded and, this requires further investigation.

In this study, the impacts of exogenous melatonin on freezing stress mitigation in pistachio (Pistacia vera L.) seedlings (30-days old) were investigated. Our results showed that in pistachio seedlings under freezing stress antioxidant enzyme activity, malondialdehyde (MDA) hydrogen peroxide (H2O2), electrolyte leakage, chlorophyll degradation, proline and soluble sugar and γ-aminobutyric acid (GABA) content increased. Phenolic component, phenylalanine ammonia lyase (PAL) activity and the expression of fatty acid desaturase (FAD2) gene increased whilst ascorbate and glutathione concentration significantly decreased in comparison with control. In this research, application of melatonin significantly reduced the antioxidant enzyme activity, MDA, H2O2, chlorophyll degradation, GABA concentration, soluble sugar, and proline contents under stress condition. In addition electrolyte leakage, PAL activity and transcripts of FAD2 gene markedly decreased when the leaves of pistachio seedlings sprayed with melatonin. The results of this study showed that MEL could alleviate the oxidative damages induced by freezing stress and to maintain the structure and mobility of cell membrane in pistachio seedlings under stress conditions. Considering that MEL is an inexpensive and safe bio-stimulator, it seems that MEL is a potential candidate that could be applied used to increase the resistant pistachio trees against variety of biotic and abiotic stresses in the field conditions.

Aim This study investigated the effect of several metabolic enhancers on the expression of fatty acid biosynthetic genes and their influence on the production of high-value PUFA in the marine microalgae Isochrysis sp., CASA CC 101. Methods and Results The effect of the presence of iron (Fe), nicotinic acid (NIC), methyl jasmonate (MJ) and thidiazuron (TDZ) on the expression of the fatty acid desaturase genes Δ6Des, Δ5Des and Δ4Des was studied in cultures of the marine microalga Isochrysis sp., CASA CC 101. The production of high-value PUFA like γ-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was correlated with these gene expressions. The results showed that MJ, Fe and TDZ significantly increased the lipid content than the control. MJ specifically up-regulated ∆6Des gene expression and thereby increased GLA production. Whereas Fe specifically increased ∆5Des gene expression and thereby increased EPA production. However, Fe and TDZ-treated cells effectively upregulated the expression of ∆4Des and increased the production of DHA when compared with control cells. Conclusions Our findings suggest that addition of Fe and MJ in the culture medium triggers the expression of PUFA biosynthetic genes, especially ∆6Des and ∆4Des, in marine microalga Isochrysis sp., CASA CC 101 their presence resulted in increased production of the PUFAs GLA, EPA and DHA. Significance and Impact of the Study This study shows that the addition of Fe and MJ to the culture media of Isochrysis sp., CASA CC 101 results in up-regulation of its genes Δ4Des, Δ6Des and Δ5Des, and improves the production of PUFA. Therefore, the addition of Fe and MJ to the culture medium is useful to increase the production of high-value PUFA in Isochrysis sp., CASA CC 101 and also to the other micro algal species.

Perilla (Perilla frutescens), a traditional medicinal and oilseed crop in Asia, contains extremely high levels of polyunsaturated α-linolenic acid (ALA) (up to 60.9%) in its seeds. ALA biosynthesis is a multistep process catalyzed by fatty acid desaturases (FADs), but the FAD gene family in perilla has not been systematically characterized. Here, we identified 42 PfFADs in the perilla genome and classified them into five subfamilies. Subfamily members of PfFADs had similar exon/intron structures, conserved domain sequences, subcellular localizations, and cis-regulatory elements in their promoter regions. PfFADs also possessed various expression patterns. PfFAD3.1 was highly expressed in the middle stage of seed development, whereas PfFAD7/8.3 and PfFAD7/8.5 were highly expressed in leaf and later stages of seed development, respectively. Phylogenetic analysis revealed that the evolutionary features coincided with the functionalization of different subfamilies of PUFA desaturase. Heterologous overexpression of PfFAD3.1 in Arabidopsis thaliana seeds increased ALA content by 17.68%–37.03%. These findings provided insights into the characteristics and functions of PfFAD genes in perilla.

Fatty acid desaturases add a second bond into a single bond of carbon atoms in fatty acid chains, resulting in an unsaturated bond between the two carbons. They are classified into soluble and membrane-bound desaturases, according to their structure, subcellular location, and function. The orthologous genes in Camelina sativa were identified and analyzed, and a total of 62 desaturase genes were identified. It was revealed that they had the common fatty acid desaturase domain, which has evolved separately, and the proteins of the same family also originated from the same ancestry. A mix of conserved, gained, or lost intron structure was obvious. Besides, conserved histidine motifs were found in each family, and transmembrane domains were exclusively revealed in the membrane-bound desaturases. The expression profile analysis of C. sativa desaturases revealed an increase in young leaves, seeds, and flowers. C. sativa ω3-fatty acid desaturases CsaFAD7 and CsaDAF8 were cloned and the subcellular localization analysis showed their location in the chloroplast. They were transferred into Arabidopsis thaliana to obtain transgenic lines. It was revealed that the ω3-fatty acid desaturase could increase the C18:3 level at the expense of C18:2, but decreases in oil content and seed weight, and wrinkled phenotypes were observed in transgenic CsaFAD7 lines, while no significant change was observed in transgenic CsaFAD8 lines in comparison to the wild-type. These findings gave insights into the characteristics of desaturase genes, which could provide an excellent basis for further investigation for C. sativa improvement, and overexpression of ω3-fatty acid desaturases in seeds could be useful in genetic engineering strategies, which are aimed at modifying the fatty acid composition of seed oil.

Jasmonates are fatty acid-derived hormones that regulate multiple aspects of plant development, growth and stress responses. Bioactive jasmonates, defined as the ligands of the conserved COI1 receptor, differ between vascular plants and bryophytes (jasmonoyl-l-isoleucine (JA-Ile) and dinor-12-oxo-10,15(Z)-phytodienoic acid (dn-OPDA), respectively). The biosynthetic pathways of JA-Ile in the model vascular plant Arabidopsis thaliana have been elucidated. However, the details of dn-OPDA biosynthesis in bryophytes are still unclear. Here, we identify an orthologue of Arabidopsis fatty-acid-desaturase 5 (AtFAD5) in the model liverwort Marchantia polymorpha and show that FAD5 function is ancient and conserved between species separated by more than 450 million years (Myr) of independent evolution. Similar to AtFAD5, MpFAD5 is required for the synthesis of 7Z-hexadecenoic acid. Consequently, in Mpfad5 mutants, the hexadecanoid pathway is blocked, dn-OPDA concentrations are almost completely depleted and normal chloroplast development is impaired. Our results demonstrate that the main source of wounding-induced dn-OPDA in Marchantia is the hexadecanoid pathway and the contribution of the octadecanoid pathway (i.e. from OPDA) is minimal. Remarkably, despite extremely low concentrations of dn-OPDA, MpCOI1-mediated responses to wounding and insect feeding can still be activated in Mpfad5, suggesting that dn-OPDA may not be the only bioactive jasmonate and COI1 ligand in Marchantia.

SUMMARYJasmonates are fatty acid derived hormones that regulate multiple aspects of plant development, growth and stress responses. Bioactive jasmonates, defined as the ligands of the conserved COI1 receptor, differ between vascular plants and bryophytes (using jasmonoyl-L-isoleucine; JA-Ile and dinor-12-oxo-10,15(Z)-phytodienoic acid; dn-OPDA, respectively). Whilst the biosynthetic pathways of JA-Ile in the model vascular plantArabidopsis thalianahave been elucidated, the details of dn-OPDA biosynthesis in bryophytes are still unclear. Here, we identify an ortholog ofArabidopsisFatty Acid Desaturase 5 (AtFAD5) in the model liverwortMarchantia polymorphaand show that FAD5 function is ancient and conserved between species separated by more than 450 million years of independent evolution. Similar to AtFAD5, MpFAD5 is required for the synthesis of 7Z-hexadecenoic acid. Consequently, in Mpfad5mutants the hexadecanoid pathway is blocked, dn-OPDA levels almost completely depleted and normal chloroplast development is impaired. Our results demonstrate that the main source of dn-OPDA inMarchantiais the hexadecanoid pathway and the contribution of the octadecanoid pathway, i.e. from OPDA, is minimal. Remarkably, despite extremely low levels of dn-OPDA, MpCOI1-mediated responses to wounding and insect feeding can still be activated in Mpfad5, suggesting that dn-OPDA is not the only bioactive jasmonate and COI1 ligand inMarchantia.

Fatty acid desaturation is a highly complex and regulated process involving different molecular and genetic actors. Ultimally, the fatty acid desaturase enzymes are responsible for the introduction of double bonds at different positions of specific substrates, resulting in a wide variety of mono- and poly-unsaturated fatty acids. This substrate-specificity makes it possible to meet all the functional needs of the different tissues against a wide variety of internal and external conditions, giving rise to a varied profile of expression and functionality of the different desaturases in the body. Being our main interest to study and characterize at the molecular level the fatty acid desaturation process in fishes, we have focused our effort on characterizing SCD 1b from European sea bass (Dicentrarchus labrax, L.). In this work, we have characterized a tearoyl-CoA Desaturase cDNA that codes a protein of 334 amino acids, which shares the greatest homology to marine fish SCD 1b. Northern blot analysis showed two transcripts of 3.5kb and 1.4kb. Two putative cis-acting conserved motifs are localized in the cDNA 5'-end: a polypyrimidine CT dinucleotide repeat tract and two non-palindromic putative NRL-response elements (NREs). The deduced protein presents two Delta9 FADs like domain, three His-rich motifs, a total of nine His residues acting as di‑iron coordination ligands. The SCD 1b 3D protein modelling shows a structure made up primarily of alpha-helices, four of which could be transmembrane helices. The catalytic region is oriented to the cytosolic side of the Endoplasmic Reticulum membrane, where the 9-histidine residues are arranged coordinated to two non-heme Fe2+ ions. A new His-containing motif NX3H-like includes an Asn residue that participates in the coordination of Fe2+1 through a water molecule. The protein has a large pocket with a large opening to the outside. It includes a tunnel in which the substrate-binding site is located. The external shape is reminiscent of a boathook. It shows group specificity, although a greater preference for 18C substrates. The length of the tunnel, delimited by seven amino acids that forms a pocket at the end of the tunnel, the possibility that the substrates adopt different conformations inside the tunnel as well as and the movement of acyl chain inside the tunnel, could explain the high preference for 18C fatty acids and the group specificity of the enzyme. The cDNA encodes a functional SCD enzyme, whose subcellular localization is the Endoplasmic Reticulum, which complements the ole1Delta gene-disrupted gene in DTY-11A Saccharomyces cerevisiae strain and produces an increment of palmitoleic and oleic acids. The scd 1b gene is expressed in all tested tissues, showing the liver and adipose tissue a higher level of expression against the brain, heart, gonad and intestine. Scd 1b expression was always bigger than those of the Delta6 fad gene, being especially significant in adipose tissue and liver. From our data, we conclude that, in contrast to the functional significance of SCD 1b in adipose tissue, liver and heart, Delta6 FAD seems to play a more determining role in the biosynthesis of unsaturated fatty acids in the intestine, brain and gonad in fish.

Oilseed crop oils contain a variety of unsaturated fatty acids that are synthesized and regulated by fatty acid desaturases (FADs). In this study, 14 FAD3 (ω3 desaturase) protein sequences from oilseeds are analyzed and presented through the application of several computational tools. The results indicated a close relationship between Brassica napus and Camelina sativa, as well as between Salvia hispanica and Perilla frutescens FAD3s, due to a high similarity in codon preferences in codon usage clusters and the phylogenetic tree. The cis-acting element results reveal that the seed-specific promoter region of BnFAD3 contains the critical conserved boxes such as HSE and ABRE, which are involved in responsiveness to heat stress and abscisic acid. The presence of the aforementioned conserved boxes may increase cold acclimation as well as tolerance to drought and high salinity. Omega(ω)3 desaturases contain a Skn-1 motif which is a cis-acting regulatory element required involved in endosperm development. In oilseed FAD3s, leucine is the most repeated amino acid in FAD3 proteins. The study conveyed that B. napus, Camelina sativa, Linum usitatissimum, Vernicia fordii, Gossypium hirsutum, S. hispanica, Cannabis sativa, and P. frutescens have retention signal KXKXX/XKXX at their c-terminus sites, which is one of the most important characteristics of FADs. Additionally, it was found that BnFAD3 is a transmembrane protein that can convert ω6 to ω3 fatty acids and may simultaneously act as a potassium ion channel in the ER.

This study investigated the effects of coconut oil as a dietary supplement on the growth, lipid metabolism and related gene expressions of juvenile orange‐spotted grouper Epinephelus coioides. Coconut oil at concentrations of 0, 10, 30 and 50 g/kg was used to replace dietary lipids in a basal diet containing 150 g/kg lipids. The four experimental diets were, respectively, fed to triplicate groups of juvenile groupers (initial weight: 8.53 ± 0.13 g) in a recirculating system for 8 weeks. Fish fed the diet containing 50 g/kg coconut oil exhibited lower (p

Hot air (HA) treatment and ultraviolet C (UV-C) irradiation have been demonstrated to control chilling injury in peaches. However, little is known about the effects of HA and UV-C treatments on volatiles changes in peach fruit during cold storage. In this study, peaches were treated with HA at 40 °C for 4 h or irradiated with UV-C lamp (1.5 kJ/m2), and then stored at 1 °C for 35 days plus 3 days of shelf life at 20 °C. Results showed that HA and UV-C suppressed chilling injury significantly, exhibiting lower internal browning index. Also, the two treatments enhanced emissions of esters and lactones in peaches by increasing enzymes of alcohol acyltransferase (AAT), fatty acid desaturase (FAD) acetyl coenzyme A transferase (ACX). These suggested that the increase of fruity note aromas in peaches by the two treatments is closely associated with aroma-related metabolism (LOX pathway and lactone pathway).

Polyacetylenes (PAs) are bioactive, specialized plant defense compounds produced by some species in the eudicot clade campanulids. Early steps of PA biosynthesis are catalyzed by Fatty Acid Desaturase 2 (FAD2). Canonical FAD2s catalyze desaturation, but divergent forms can catalyze hydroxylation, conjugation, acetylenation, and epoxygenation. These alternate reactions give rise to valuable unusual fatty acids, including the precursors to PAs. The extreme functional diversity of FAD2 enzymes and the origin of PA biosynthesis are poorly understood from an evolutionary perspective. We focus here on the evolution of the FAD2 gene family. We uncovered a core eudicot-wide gene duplication event giving rise to two lineages: FAD2-α and FAD2-β. Independent neofunctionalizations in both lineages have resulted in functionally diverse FAD2-LIKEs involved in unusual fatty acid biosynthesis. We found significantly accelerated rates of molecular evolution in FAD2-LIKEs and use this metric to provide a list of uncharacterized candidates for further exploration of FAD2 functional diversity. FAD2-α has expanded extensively in Asterales and Apiales, two main clades of campanulids, by ancient gene duplications. Here, we detected positive selection in both Asterales and Apiales lineages, which may have enabled the evolution of PA metabolism in campanulids. Together, these findings also imply that yet uncharacterized FAD2-α copies are involved in later steps of PA biosynthesis. This work establishes a robust phylogenetic framework in which to interpret functional data and to direct future research into the origin and evolution of PA metabolism.

Lesquerella (Physaria fendleri) contains a major unusual hydroxy fatty acid, lesquerolic acid (14-hydroxy-eicos-cis-11-enoic acid, C20:1-OH), at 55–60% of seed oil which has industrial value. The remaining seed oil comprises mainly common fatty acids including α-linolenic acid (octadec-cis-9,12,15-enoic acid, C18:3) at 10.7–15.8%. C18:3 is produced from linoleic acid (octadec-cis-9,12-enoic acid, C18:2) by FATTY ACID DESATURASE3. Previous seed transcriptome analysis uncovers two fatty acid desaturase 3 (FAD3) transcripts, PfFAD3-1 and PfFAD3-2. To determine the activity of PfFAD3-1 and PfFAD3-2, PfFAD3-1 and PfFAD3-2 were introduced into an Arabidopsis FAD3-deficient mutant (fad3-2) which has reduced C18:3 from 20.0% in wild-type to 1.6% in fad3-2. Among 20 T2 transgenic lines expression PfFAD3-1, C18:3 increased variably from 2.5 to 29.9% demonstrating that PfFAD3-1 acted as a functional FAD3. Among 32 T2 transgenic lines expressing PfFAD3-2, C18:3 content ranged from 1.0 to 3.6%, showing that PfFAD3-2 failed to recover the loss of C18:3 in fad3-2. Sequence comparison among known FAD3s revealed putative variation in PfFAD3-2 which might cause the absence of PfFAD3-2. In addition, lesquerella accumulates a minor hydroxy fatty acid, densipolic acid (12-hydroxy-octadec-cis-9,15-enoic acid, C18:2 OH) at about 1%. C18:2 OH has been shown to be produced by a FAD3 in Arabidopsis (AtFAD3) using ricinoleic acid (12-hydroxy-9-cis-octadecenoic acid, 18:1-OH) as substrate. To test if either of PfFAD3s is able to convert C18:1-OH to C18:2-OH, PfFAD3-1 or PfFAD3-2 was transferred into a CL37 Arabidopsis which already expresses a castor (Ricinus communis) fatty acid hrdroxylase FAH12 gene (RcFAH12) and consequently accumulates C18:1-OH and C18:2-OH at 13.7% and 3.4%, respectively. Among 43 transgenic CL37 lines expressing PfFAD3-1, C18:2-OH level varied from 0.2 to 7.2%, and four of these lines exceeded to the background level of 3.4% in CL37. Whereas among 23 transgenic CL37 lines expressing PfFAD3-2, C18:2-OH level ranged from 0.4 to 3.4%, none exceeding 3.4%. The results consist with our notion that PfFAD3-1, not PfFAD3-2, exerts FAD3 function which includes converting C18:1-OH to C18:2-OH. Factors limiting PfFAD3s function in CL37 are discussed.

Perilla (Perilla frutescens (L.) var. frutescens) is an oilseed crop that produces a large amount of α-linolenic acid (ALA, 18:3), an ω-3 fatty acid in seeds. Perilla is also a fragrant leafy herb with great health benefits. To develop value-added perilla crop, we surveyed the fatty acid composition and oil content of 87 germplasms accessions maintained at Republic of Korea. Our results show that ALA is the major fatty acid, ranging from 47 to 64%, followed by linoleic acid (LA, 18:2, an ω-6 fatty acid) from 10 to 24% and the oleic acid (OA, 18:1, an ω-9 fatty acid) from 9 to 20%. The seed oil content among accessions ranges from 17 to 42.7%. To access if the different fatty acid profile and oil content among germplasms are associated with changes in key genes, we compare the sequences encoding fatty acid desaturase 2 (FAD2) and fatty acid desaturase 3 (FAD3) responsible for LA and ALA synthesis, respectively. Besides, we examine the sequence of diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in determining oil content. We do not find major changes in FAD2 or FAD3 associated with different fatty acid profiles. We discover a change in DGAT1 that might associate a low oil content in seeds.

The membrane-bound fatty acid desaturases (FADs) play vital roles in regulating plant fatty acid (FA) compositions which directly affect oil quality in rapeseed (Brassica napus L.). In the present study, a total of 68 putative FAD genes were identified in the crop and were phylogenetically clustered into four subfamilies. Genomic localization revealed that the genes were distributed across 21 chromosomes at various densities. The structures of paralogous genes were considerably conserved in the number of exons and in the length of introns. It was found that the expansion of the B. napus FAD family was mainly attributable to whole genome duplication and segmental duplication. Rapeseed seedlings were grown hydroponically under three levels (0, 250, and 500 μM) of cadmium (Cd) and three levels (0, 50, and 100 mM) of salinity. After one week of treatment, the roots and leaves of seedlings were harvested to study the expression patterns of 68 selected BnFADs in response to the different stresses. The results from qRT-PCR analyses suggested that the FAD genes in rapeseed were differentially expressed under various abiotic stresses. The transcript levels of the FAD genes ADS4.8, ADS4.9, ADS4.10, ADS9.1 and SLD8 in the leaves of Cd-tolerant cultivar ZD619 were significantly higher than those in the leaves of sensitive cultivar ZD622 under the 250 μM Cd stress condition. Moreover, the expression levels of 8FAD genes (ADS4.1, ADS4.4, ADS4.9, ADS9.1, ADS9.2, SLD8, FAD7.4 and FAD7.5) in ZD619 roots were obviously enhanced with the increase of salinity concentrations. These results indicate the likelihood of FAD genes involved in Cd and salinity tolerance. This study provides insight on membrane-bound FAD genes involvement in tolerance to abiotic stresses and would provide some more candidate genes for the crop improvement.

Seed yield and nutritional quality are dependent on seed development in oil crops. In a previous study, we showed that light shading improved seed yield and quality in oilseed peony, but the underlying mechanisms driving light shading impacts on seed yield and nutritional quality as mediated through seed development remain unclear. To develop an understanding of these mechanisms, oilseed peony trees were shaded by different density polyethylene nets starting at 28 days after pollination (DAP) and continuing until harvest. The seed volume, weight, and nutritional quality parameters, as well as the expression of genes related to the synthesis of unsaturated fatty acids (UFAs), were examined at 14-day intervals. Results showed that hundred-seed volume (Vh), fresh weight (Wf), and dry weight (Wd) increased by 8.2, 15.2, and 33.0 times, respectively, from 14 to 70 DAP. During the same period, the contents of starch, soluble protein, crude oil, and UFAs increased by 2.8, 3.7, 20.4, and 21.1 times, respectively. From 71 to 98 DAP, Vh, Wf, and Wd increased by 4.1, 5.4, and 20.8%, respectively. During this period, the contents of soluble protein, crude oil, and UFAs increased by 36.8, 51.4, and 49.5%, respectively. From 99 to 112 DAP, Vh, Wf, and Wd slightly increased, and the content of soluble protein increased by 25.2%. In contrast, the contents of soluble sugar, starch, and crude oil decreased by 28.3, 15.1, and 6.3%, respectively. Thus, seed development of oilseed peony can be divided into three periods of rapid growth (before 70 DAP), slow growth (71–98 DAP), and maturation (99–112 DAP). At 98 DAP, light shading increased the content of soluble sugar by 8.3%. At 112 DAP, light shading increased Vh, Wf, Wd, the content of crude oil, and UFAs by 6.9, 8.3, 8.3, 6.6, and 9.6%, respectively. The expression level of FAD2 in peony seed was up-regulated by 87.1 and 219.3% at 98 and 112 DAP, respectively. The expression level of FAD8 was up-regulated by 10.8, 71.7, 162.1, and 371.3% at 70, 84, 98, and 112 DAP, respectively. The expression level of SAD was up-regulated by 53.5 and 150.3% at 98 and 112 DAP, respectively. It is thus concluded that light shading significantly improved seed yield and nutritional quality parameters via increased seed growth and the expression of fatty acid desaturase genes during the slow growth and the maturation periods of seed development.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential w-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization., This project was supported by JSPS KAKENHI grants 15H02418 and 23113007 (to J.K.), grant 16H06279 (to A.T.), and grants 26870824 and 16K07469 (to A.I.); the Asahi Glass Foundation (to J.K.); the Sumitomo Foundation (to A.I.); and JSPS postdoctoral fellowships 11J04816 to A.I. and 16J06812 to N.K. The work by N.L. and C.J.S. was funded by internal Eawag funds. B.M. was supported by SNF grant 31003A 175614. O.S. was supported by SNSF grant 31003A_163338. D.M. was supported by SNSF grant PDAMP3_123135 (to O.S).

Due to the preeminence of reductionist approaches, understanding of plant responses to combined stresses is limited. We speculated that light‐quality signals of neighboring vegetation might increase susceptibility to heat shocks because shade reduces tissue temperature and hence the likeness of heat shocks. In contrast, plants of Arabidopsis thaliana grown under low red/far‐red ratios typical of shade were less damaged by heat stress than plants grown under simulated sunlight. Neighbor signals reduce the activity of phytochrome B (phyB), increasing the abundance of PHYTOCHROME INTERACTING FACTORs (PIFs). The phyB mutant showed high tolerance to heat stress even under simulated sunlight and a pif multiple mutant showed low tolerance under simulated shade. phyB and red/far‐red ratio had no effects on seedlings acclimated with non‐stressful warm temperatures before the heat shock. The phyB mutant showed reduced expression of several fatty acid desaturase (FAD) genes, and less proportion of fully unsaturated fatty acids and electrolyte leakage of membranes exposed to heat shocks. Red‐light‐activated phyB also reduced thermotolerance of dark‐grown seedlings but not via changes in FADs expression and membrane stability. We propose that the reduced photosynthetic capacity linked to thermotolerant membranes would be less costly under shade, where the light input limits photosynthesis. Fil: Arico, Denise Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Legris, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Castro, Luciana Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Garcia, Carlos Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Laino, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Casal, Jorge José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina. Fundación Instituto Leloir; Argentina Fil: Mazzella, Maria Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina

Tree peony (Paeonia section Moutan DC.) has recently been recognised as an important woody oil plant of high economic value, with its seeds containing high levels of α-linolenic acids (ALA,...

Background Polyunsaturated fatty acids such as linoleic acid (LA) and α-linolenic acid (ALA) are abundant in vegetable oils and are important for human health. In the body, LA and ALA are respectively converted to the omega-6 fatty acid γ-linolenic acid (GLA) and the omega-3 fatty acid stearidonic acid (SDA) by Δ6 desaturase (D6DES). Currently, dietary GLA and SDA are mainly obtained from marine organisms, but given their benefits to human health, many studies have aimed to enhance their accumulation in transgenic crops. Perilla frutescens (perilla) accumulates more ALA in its seed oil compared to other oilseed crops, making it a good candidate for the production of fatty acids via the fatty acid desaturase D6DES. Results In this study, we cloned the D6DES gene from Phytophthora citrophthora and confirmed its function in budding yeast. We then transformed the functional D6DES gene under the control of the seed-specific vicilin promoter into the perilla cultivar Yeobsil. The resulting transgenic perilla seeds accumulated significant levels of GLA and SDA, as well as putative C18:2Δ6,9 at minor levels. Developing seeds and leaves also accumulated GLA and SDA, although PcD6DES expression and GLA and SDA levels were much lower in leaves compared to developing seeds. GLA and SDA accumulated in both polar lipids and neutral lipids in mature perilla seeds expressing PcD6DES, especially in neutral lipids. Although the seed weight in PcD6DES perilla was 87–96% that of wild type, the total oil content per seed weight was similar between lines. The PcD6DES perilla plants contained very high content (over 45%) of both GLA and SDA in seed oil. Conclusions Thus, PcD6DES perilla plants may represent a feasible alternative to traditional marine sources for the production of omega-3 oil capsules and to evening primrose seed oil for GLA as health food. In addition, these plants can be used to create other transgenic lines harboring additional genes to produce other desirable fish-oil like oils. Electronic supplementary material The online version of this article (10.1186/s12870-019-1713-2) contains supplementary material, which is available to authorized users.

Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil-triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37g/L and 73.3mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.

In this study, the effect of freezing stress on expression of fatty acid desaturases (FAD2-2, FAD6 and FAD7) and beta-glucosidase (BGLC) genes was investigated in freezing- tolerant (“Fishomi”) and sensitive (“Zard”) olive (Olea europaea L.) cultivars. The olive cultivars were exposed to freezing stress (–10°C) at three different times points (1, 4 and 8 h), followed by a recovery stage. The degree of cell membrane damage was determined by measurement of leaves ion leakage of two olive cultivars both at freezing and recovery condition. The results indicated that ion leakage of cv. “Fishomi” was significantly lower than cv. “Zard” when exposed to the freezing stress and recovery condition. Furthermore, BGLC gene expression level in cv. “Zard” significantly suppressed 8 h after freezing stress. At the recovery period, its expression increased about three-fold at cv. “Fishomi”. However, it was suppressed in cv. “Zard” at recovery period. FAD genes expression pattern in cv. “Fishomi” was different from that in cv. “Zard”. FAD2 expression level in cv. “Fishomi” initially increased at the first 1 h and 4 h of freezing stress time and thereafter, decreased to control level over 8 h. Following recovery period, FAD2 gene expression level in cv. “Fishomi” increased up 5-fold. While, no expression detected in “Zard” at any time point or recovery period. The expression of FAD6 and FAD7 genes showed similar patterns at both cultivars. FAD6 and FAD7 genes expression significantly up-regulated 5- and 4.5-fold, respectively, following recovery period in “Fishomi”. Overall, the results suggested that FADs and BGLC could be involved in the increasing of freezing tolerance in olive cultivars, particularly in plants recovery from freezing stress.

High oleate is an important seed quality trait frequently incorporated in peanut varieties. Crop wild relatives (CWR) are potentially useful genetic resources for cultivar improvement through genetic introgression; but for wild peanut species, many chemical or nutritional traits are not well characterized. A mutant from Arachis veigae S. H. Santana & Valls (2n = 2x = 20), with increased oleic and very long chain (C ≥ 22) fatty acid content was identified from screening 209 accessions of 45 species using gas chromatography (GC). The A. veigae (formerly A. sylvestris) accession, VVeSv 8373 (PI 688970) contained 55.5% oleic acid in seeds, significantly higher than the average (18.3%) of other accessions within the same species and also significantly higher than the average (37.0%) of all wild peanut accessions evaluated. A C37T substitution was identified by sequencing the coding region of FAD2H, resulting in the nonsense mutation of Q13* (a premature stop codon). This functional mutation may significantly reduce the fatty acid desaturase (FAD) activity and result in the enhanced oleate level. Arachis veigae also contained a high percentage of very long-chain (C ≥ 22) fatty acids, and their variation identified in this study is also discussed and compared with other species. The mutant with such an altered fatty acid composition may be useful for potentially improving seed or food nutrition quality.

To investigate the effects of five different foods on fatty acid metabolism in the monogonont marine rotifer Brachionus koreanus, we measured life cycle parameters, analyzed the area of Nile red staining, fatty acid composition, and mRNA expression of fatty acid desaturase (Fads) and elongation of very long chain fatty acid (Elovl) genes. Among the five different foods (Tetraselmis suecica, Chlorella sp., Isochrysis galbana, Chaetoceros sp., and Nannochloropsis oculata), life span of I. galbana-fed group was significantly increased compared to the other groups. However, the reproduction parameters (cumulative offspring and daily reproduction) were the highest in T. suecica-fed group, and the lowest in Chlorella sp.-fed group. The area of Nile red staining analysis showed similar trends, in respect to the content of total fatty acid. The single fatty acid analysis result clearly indicated that not only the most of the content of fatty acid was highly dependent on the food types, especially in omega-6 fatty acids group, but also, B. koreanus could newly synthesize some specific fatty acids by themselves (e.g. omega-3 and -9 families). Lastly, the mRNA expression of Elovl and Fads genes were modulated by food types indicating the lipid metabolism could be controlled by food in B. koreanus and probably very small amount of 18:3n-3 fatty acids was utilized and synthesized omega-3 fatty acids through the increase in Elovl and Fads gene expression in Chaetoceros sp. and N. oculata-fed groups. In conclusion, this study revealed that although B. koreanus could modulate fatty acid metabolism by itself, some nutrient values (e.g. omega-6 fatty acids) should be considered, therefore, it is important to understand the essential nutrient through both the molecular and quantitative analysis.

Salt stress can trigger several physiological responses in microorganisms such as the increasing accumulation of unsaturated fatty acid, which was biosynthesized by delta-9 fatty acid desaturases (D9D) at the first step. In the present study, two D9D genes, designated AoD9D1 and AoD9D2, were isolated from Aspergillus oryzae. The expression analysis showed that AoD9D1 and AoD9D2 were upregulated under salt stress. To investigate the function of AoD9D, transgenic Saccharomyces cerevisiae strains that heterologously expressed AoD9D were exposed to salinity condition. These transgenic strains exhibited greater tolerance to salt stress than wild-type strains, and the heterologous expression of AoD9D increased the content in unsaturated fatty acids as compared to control cells. Moreover, AoD9D1 and AoD9D2 both contained fatty acid desaturase (FAD) and cytochrome b5-like Heme/Steroid-binding domains (Cyt-b5). S. cerevisiae separately transformed with the gene fragments coding for the FAD and Cyt-b5 domains in the AoD9D1 protein grew better and accumulated a higher concentration of unsaturated FAs than the control. Altogether, the heterologous expression of AoD9D enhanced the tolerance of transgenic S. cerevisiae to high salinity stress with increased accumulation of unsaturated fatty acid. The results provide some practical basis for the successful development of salt-tolerant fermentation microorganisms.

The cDNA of the delta-12 fatty acid desaturase gene, IgFAD2, was cloned from the marine microalgae Isochrysis galbana, a species capable of producing docosahexaenoic acid. Sequence analysis indicated that the open reading frame measured a length of 1 158 bp and encoded 386 amino acids with a predicted molecular weight of 42.8 kDa and an isoelectric point of 9.2. Computational analysis of the protein sequence of IgFAD2 showed typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that were universally present among plant desaturases. Quantitative real-time PCR results showed that the abundance of IgFAD2 transcript was significantly upregulated under different environmental stresses including low temperature (15°C), high salinity (salinity of 62 and 93), and nitrogen starvation (220 μmol/L). Heterologous expression indicated that yeast cells transformed with a plasmid construct containing IgFAD2 could convert endogenous oleic acid (18:1Δ9, OA) into linoleic acid (18:2Δ9, 12, LA). These findings confirm that I. galbana IgFAD2 plays important roles in the biosynthetic pathways of unsaturated fatty acids.

Fatty acid desaturases catalyze the formation of a C=C double bond from a C–C single bond in fatty acids, leading to diversification of the fatty acid pool. In this study, we identified the fatty acid desaturase genes in the monogonont marine rotifer Brachionus koreanus, measured the messenger RNA (mRNA) expression, and quantified fatty acid composition under feeding with the alga Tetraselmis suecica. Eight fatty acid desaturase genes were identified and classified by phylogenetic analysis. Fatty acid desaturases in the rotifer B. koreanus were categorized into two Δ4 desaturases, five Δ5/6 desaturases, and one Δ9 desaturase. mRNA expression of B. koreanus under the alga T. suecica-fed condition clearly indicated that transcriptional levels of desaturase genes were increased compared to the non-T. suecica-fed group. Also, the distribution of fatty acids of B. koreanus fed T. suecica was more balanced compared to that of T. suecica. These results provide a better understanding of the role of fatty acid desaturases in B. koreanus fed the alga T. suecica.

Fatty acid desaturases are key enzymes in the biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) via conversion of n-6 polyunsaturates to their n-3 counterparts. In this study, we reported the characterization and molecular docking of Δ17 desaturases from Rhizophagus irregularis and Octopus bimaculoides. These two new desaturase genes were screened using the known Δ17 desaturase gene (oPaFADS17) from Pythium aphanidermatum as a template. Analysis of their genes revealed that the sequences of oRiFADS17 and oObFADS17 contained the typical His-rich motifs (one HXXXH and two HXXHH). They were then expressed in Saccharomyces cerevisiae INVSc1 to examine their activities and substrate preferences. Our results show that the two candidate n-3 desaturases possess a strong Δ17 desaturase activity, exhibiting remarkable increase in desaturation activity on C20 fatty acids compared to C18 fatty acids. To the best of our knowledge, oRiFADS17 desaturase has greater (3–4 fold) catalytic activity for C18 substrates than other reported Δ17 desaturases and oObFADS17 is the first reported Δ17 desaturase in sea mollusks. Characterization of these two new desaturases will be of greater value for genetic engineering in industrial production of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). Due to lack of crystal structure information about n-3 desaturases, for the first time, the view of their predicted structures, binding pockets and substrate tunnels was clearly observed based on molecular docking. This will contribute to strengthening our understanding of the structure–function relationships of n-3 fatty acid desaturases.

The plastidial delta-12 fatty acid desaturase (FAD6) plays a key role in linoleic acid (LA) and α-linolenic acid (ALA) biosynthesis. Perilla (Perilla frutescens) is an important oilseed crop with the highest ALA level. Here, using RACE PCR, two FAD6 cDNAs were cloned from perilla: the 1726 bp PfFAD6 and the 1881 bp PfFAD6’. PfFAD6 encodes a 443 aa protein, while PfFAD6’ is a pseudogene caused by premature stop codon mutation. N-terminal of PfFAD6 has a chloroplast transit peptide. PfFAD6 harbors an omega-6 FAD conserved domain, 2 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis revealed some important evolutionary features of PfFAD6 and other plant FAD6 genes. Heterologous yeast expression showed that protein encoded by PfFAD6 can catalyze the production of LA. PfFAD6 was expressed in various organs at different levels, and responded to multiple biotic/abiotic stresses. The qRT-PCR assay and fluorescence imaging showed the correlation of PfFAD6 transcripts and photosynthetic activity under Sclerotinia infection. This study will lay a foundation for further function study of PfFAD6 in quality improvement in oilseed crops and plant-stress response.