Omega-3 Fatty Acid Desaturase Research Articles

Enzymatic modification by point mutation and functional analysis of an omega-6 fatty acid desaturase from Arctic Chlamydomonas sp.

ABSTRACTArctic Chlamydomonas sp. is a dominant microalgal strain in cold or frozen freshwater in the Arctic region. The full-length open reading frame of the omega-6 fatty acid desaturase gene (AChFAD6) was obtained from the transcriptomic database of Arctic Chlamydomonas sp. from the KOPRI culture collection of polar micro-organisms. Amino acid sequence analysis indicated the presence of three conserved histidine-rich segments as unique characteristics of omega-6 fatty acid desaturases, and three transmembrane regions transported to plastidic membranes by chloroplast transit peptides in the N-terminal region. The AChFAD6 desaturase activity was examined by expressing wild-type and V254A mutant (Mut-AChFAD6) heterologous recombinant proteins. Quantitative gas chromatography indicated that the concentration of linoleic acids in AChFAD6-transformed cells increased more than 3-fold [6.73 ± 0.13 mg g−1 dry cell weight (DCW)] compared with cells transformed with vector alone. In contrast, transformation with Mut-AChFAD6 increased the concentration of oleic acid to 9.23 ± 0.18 mg g−1 DCW, indicating a change in enzymatic activity to mimic that of stearoyl-CoA desaturase. These results demonstrate that AChFAD6 of Arctic Chlamydomonas sp. increases membrane fluidity by enhancing denaturation of C18 fatty acids and facilitates production of large quantities of linoleic fatty acids in prokaryotic expression systems.

The Salinity Responsive Mechanism of a Hydroxyproline-Tolerant Mutant of Peanut Based on Digital Gene Expression Profiling Analysis.

Soil salinity seriously limits plant growth and yield. Strategies have been developed for plants to cope with various environmental stresses during evolution. To screen for the broad-spectrum genes and the molecular mechanism about a hydroxyproline-tolerant mutant of peanut with enhanced salinity resistance under salinity stress, digital gene expression (DGE) sequencing was performed in the leaves of salinity-resistant mutant (S2) and Huayu20 as control (S4) under salt stress. The results indicate that major transcription factor families linked to salinity stress responses (NAC, bHLH, WRKY, AP2/ERF) are differentially expressed in the leaves of peanut under salinity stress. In addition, genes related to cell wall loosening and stiffening (xyloglucan endotransglucosylase/hydrolases, peroxidases, lipid transfer protein, expansin, extension), late embryogenesis abundant protein family, fatty acid biosynthesis and metabolism (13-lipoxygenase omega-6 fatty acid desaturase, omega-3 fatty acid desaturase) and some previously reported stress-related genes encoding proteins such as defensin, universal stress protein, metallothionein, peroxidase etc, and some other known or unknown function stress related genes, have been identified. The information from this study will be useful for further research on the mechanism of salinity resistance and will provide a useful genomic resource for the breeding of salinity resistance variety in peanut.

Microsomal Omega-3 Fatty Acid Desaturase Genes in Low Linolenic Acid Soybean Line RG10 and Validation of Major Linolenic Acid QTL.

High levels of linolenic acid (80 g kg−1) are associated with the development of off-flavors and poor stability in soybean oil. The development of low linolenic acid lines such as RG10 (20 g kg−1 linolenic acid) can reduce these problems. The level of linolenic acid in seed oil is determined by the activities of microsomal omega-3 fatty acid desaturases (FAD3). A major linolenic acid QTL (>70% of variation) on linkage group B2 (chromosome Gm14) was previously detected in a recombinant inbred line population from the RG10 × OX948 cross. The objectives of this study were to validate the major linolenic acid QTL in an independent population and characterize all the soybean FAD3 genes. Four FAD3 genes were sequenced and localized in RG10 and OX948 and compared to the genes in the reference Williams 82 genome. The FAD3A gene sequences mapped to the locus Glyma.14g194300 [on the chromosome Gm14 (B2)], which is syntenic to the FAD3B gene (locus Glyma.02g227200) on the chromosome Gm02 (D1b). The location of the FAD3A gene is the same as was previously determined for the fan allele, that conditions low linolenic acid content and several linolenic acid QTL, including Linolen 3-3, mapped previously with the RG10 × OX948 population and confirmed in the PI 361088B × OX948 population as Linolen-PO (FAD3A). The FAD3B gene-based marker, developed previously, was mapped to the chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL [Linolen-RO(FAD3B)] in the RG10 × OX948 genetic map and corresponds well with the in silico position of the FAD3B gene sequences. FAD3C and FAD3D gene sequences, mapped to syntenic regions on chromosomes Gm18 (locus Glyma.18g062000) and Gm11 (locus Glyma.11g227200), respectively. Association of linolenic acid QTL with the desaturase genes FAD3A and FAD3B, their validation in an independent population, and development of FAD3 gene-specific markers should simplify and accelerate breeding for low linolenic acid soybean cultivars.

Using Caenorhabditis elegans to Uncover Conserved Functions of Omega-3 and Omega-6 Fatty Acids

The nematode Caenorhabditis elegans is a powerful model organism to study functions of polyunsaturated fatty acids. The ability to alter fatty acid composition with genetic manipulation and dietary supplementation permits the dissection of the roles of omega-3 and omega-6 fatty acids in many biological process including reproduction, aging and neurobiology. Studies in C. elegans to date have mostly identified overlapping functions of 20-carbon omega-6 and omega-3 fatty acids in reproduction and in neurons, however, specific roles for either omega-3 or omega-6 fatty acids are beginning to emerge. Recent findings with importance to human health include the identification of a conserved Cox-independent prostaglandin synthesis pathway, critical functions for cytochrome P450 derivatives of polyunsaturated fatty acids, the requirements for omega-6 and omega-3 fatty acids in sensory neurons, and the importance of fatty acid desaturation for long lifespan. Furthermore, the ability of C. elegans to interconvert omega-6 to omega-3 fatty acids using the FAT-1 omega-3 desaturase has been exploited in mammalian studies and biotechnology approaches to generate mammals capable of exogenous generation of omega-3 fatty acids.

Production of transgenic beef cattle rich in n-3 PUFAs by somatic cell nuclear transfer

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are beneficial to human health. However, the n-3 PUFAs contents of the livestock meat that we consume daily are relatively low. Utilization of transgenic technology to increase n-3 PUFAs contents in livestock may solve this problem. The omega-3 fatty acid desaturase (FAD3), encoded by fat1 gene derived from Caenorhabditis elegans (C. elegans), converts omega-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. In the study, a plasmid containing the codon-optimized C. elegans fat1 gene (mfat1) was constructed and used to produce transgenic beef cattle by somatic cell nuclear transfer. Fourteen transgenic calves were obtained, and the ratio of n-6 to n-3 PUFAs in the transgenic calves decreased from 5.33: 1 to 0.95: 1 compared with negative controls. Our results demonstrated that the codon-optimized C. elegans mfat1 gene can be functionally expressed in the beef cattle and converts n-6 PUFAs to n-3 PUFAs.

Threonine 286 of fatty acid desaturase 7 is essential for ω-3 fatty acid desaturation in the green microalga Chlamydomonas reinhardtii.

Omega-3 fatty acid desaturases catalyze the conversion of dienoic fatty acids (C18:2 and C16:2) into trienoic fatty acids (C18:3 and C16:3), accounting for more than 50% of the total fatty acids in higher plants and the green microalga Chlamydomonas reinhardtii. Here, we describe a Thr residue located in the fourth transmembrane domain of fatty acid desaturase 7 (FAD7) that is essential for the biosynthesis of ω-3 fatty acids in C. reinhardtii. The ω-3 fatty acid deficiency in strain CC-620, which contains a putative missense mutation at Thr286 of CrFAD7, was recovered by the overexpression of CC-125 CrFAD7. A Ser substitution in position 286 was able to partially complement the phenotype of the ω-3 fatty acid deficiency, but other substitution variants, such as Tyr, His, Cys, and Gly, failed to do so. Prediction of the phosphorylation target site revealed that Thr286 may be phosphorylated. Analysis of the structural conformation of CC-620 CrFAD7 via topology prediction (and bends in the helix) shows that this missense mutation may collapse the catalytic structure of CrFAD7. Taken together, this study suggests that Thr286 is essential for the maintaining the catalytic structure of CrFAD7.

Indole-3-acetic acid (IAA) induced changes in oil content, fatty acid profiles and expression of four fatty acid biosynthetic genes in Chlorella vulgaris at early stationary growth phase

Microalgae lipids and oils are potential candidates for renewable biodiesel. Many microalgae species accumulate a substantial amount of lipids and oils under environmental stresses. However, low growth rate under these adverse conditions account for the decrease in overall biomass productivity which directly influence the oil yield. This study was undertaken to investigate the effect of exogenously added auxin (indole-3-acetic acid; IAA) on the oil content, fatty acid compositions, and the expression of fatty acid biosynthetic genes in Chlorella vulgaris (UMT-M1). Auxin has been shown to regulate growth and metabolite production of several microalgae. Results showed that oil accumulation was highest on days after treatment (DAT)-2 with enriched levels of palmitic (C16:0) and stearic (C18:0) acids, while the linoleic (C18:2) and α-linolenic (C18:3n3) acids levels were markedly reduced by IAA. The elevated levels of saturated fatty acids (C16:0 and C18:0) were consistent with high expression of the β-ketoacyl ACP synthase I (KAS I) gene, while low expression of omega-6 fatty acid desaturase (ω-6 FAD) gene was consistent with low production of C18:2. However, the increment of stearoyl-ACP desaturase (SAD) gene expression upon IAA induction did not coincide with oleic acid (C18:1) production. The expression of omega-3 fatty acid desaturase (ω-3 FAD) gene showed a positive correlation with the synthesis of PUFA and C18:3n3.

Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: Role for omega-3 epoxides

Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3-derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3-dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic diseases.

Genome-wide analysis of the omega-3 fatty acid desaturase gene family in Gossypium.

BackgroundThe majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation.ResultsEleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings.ConclusionsThe omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. Collectively, these data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0312-5) contains supplementary material, which is available to authorized users.

Analysis of expressed sequence tags from a normalized cDNA library of perilla (Perilla frutescens)

Perilla (Perilla frutescens), an herbal plant belonging to the Lamiaceae family, has long been cultivated in Asia. Perilla is notable as an aroma-rich leaf vegetable and as the oilseed crop richest in omega-3 fatty acids. However, molecular analysis of this herbal plant is lacking due to insufficient genetic resources. Here, we constructed a normalized cDNA library from whole young perilla plants and analyzed expressed sequence tags (ESTs). A total of 4,582 uniESTs were generated from analysis of 5,435 ESTs. Among these, 307 uniESTs (6.7%) were identified as unique in perilla and 3,625 uniESTs were assigned at least one GO term through similarity searches in public databases. The most frequent GO terms were related to abiotic and biotic stress responses. In addition, we identified 141 uniESTs involved in lipid metabolism, of which four genes encoded fatty acid desaturases. We found one new candidate omega-3 fatty acid desaturase gene, in addition to the four that were previously reported. This analysis of uniESTs from perilla provides a valuable genetic resource to elucidate the molecular underpinnings of lipid metabolism and for molecular breeding of perilla species.

New layers in understanding and predicting α-linolenic acid content in plants using amino acid characteristics of omega-3 fatty acid desaturase

α-linolenic acid (ALA) is the most frequent omega-3 in plants. The content of ALA is highly variable, ranging from 0 to 1% in rice and corn to >50% in perilla and flax. ALA production is strongly correlated with the enzymatic activity of omega-3 fatty acid desaturase. To unravel the underlying mechanisms of omega-3 diversity, 895 protein features of omega-3 fatty acid desaturase were compared between plants with high and low omega-3. Attribute weighting showed that this enzyme in plants with high omega-3 content has higher amounts of Lys, Lys-Phe, and Pro-Asn but lower Aliphatic index, Gly-His, and Pro-Leu. The Random Forest model with Accuracy criterion when run on the dataset pre-filtered with Info Gain algorithm was the best model in distinguishing high omega-3 content based on the frequency of Lys–Lys in the structure of fatty acid desaturase. Interestingly, the discriminant function algorithm could predict the level of omega-3 only based on the six important selected attributes (out of 895 protein attributes) of fatty acid desaturase with 75% accuracy. We developed “Plant omega3 predictor” to predict the content of α-linolenic acid based on structural features of omega-3 fatty acid desaturase. The software calculates the 6 key structural protein features from imported Fasta sequence of omega-3 fatty acid desaturase or utilizes the imported features and predicts the ALA content using discriminant function formula. This work unravels an underpinning mechanism of omega-3 diversity via discovery of the key protein attributes in the structure of omega-3 desaturase offering a new approach to obtain higher omega-3 content.

Transcriptome analysis of the oil-rich tea plant, Camellia oleifera, reveals candidate genes related to lipid metabolism.

BackgroundRapidly driven by the need for developing sustainable sources of nutritionally important fatty acids and the rising concerns about environmental impacts after using fossil oil, oil-plants have received increasing awareness nowadays. As an important oil-rich plant in China, Camellia oleifera has played a vital role in providing nutritional applications, biofuel productions and chemical feedstocks. However, the lack of C. oleifera genome sequences and little genetic information have largely hampered the urgent needs for efficient utilization of the abundant germplasms towards modern breeding efforts of this woody oil-plant.ResultsHere, using the 454 GS-FLX sequencing platform, we generated approximately 600,000 RNA-Seq reads from four tissues of C. oleifera. These reads were trimmed and assembled into 104,842 non-redundant putative transcripts with a total length of ∼38.9 Mb, representing more than 218-fold of all the C. oleifera sequences currently deposited in the GenBank (as of March 2014). Based on the BLAST similarity searches, nearly 42.6% transcripts could be annotated with known genes, conserved domains, or Gene Ontology (GO) terms. Comparisons with the cultivated tea tree, C. sinensis, identified 3,022 pairs of orthologs, of which 211 exhibited the evidence under positive selection. Pathway analysis detected the majority of genes potentially related to lipid metabolism. Evolutionary analysis of omega-6 fatty acid desaturase (FAD2) genes among 20 oil-plants unexpectedly suggests that a parallel evolution may occur between C. oleifera and Olea oleifera. Additionally, more than 2,300 simple sequence repeats (SSRs) and 20,200 single-nucleotide polymorphisms (SNPs) were detected in the C. oleifera transcriptome.ConclusionsThe generated transcriptome represents a considerable increase in the number of sequences deposited in the public databases, providing an unprecedented opportunity to discover all related-genes associated with lipid metabolic pathway in C. oleifera. It will greatly enhance the generation of new varieties of C. oleifera with increased yields and high quality.

Double transgenesis of humanized fat1 and fat2 genes promotes omega-3 polyunsaturated fatty acids synthesis in a zebrafish model.

Omega-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential nutrients for human health. However, vertebrates, including humans, have lost the abilities to synthesize EPA and DHA de novo, majorly due to the genetic absence of delta-12 desaturase and omega-3 desaturase genes. Fishes, especially those naturally growing marine fish, are major dietary source of EPA and DHA. Because of the severe decline of marine fishery and the decrease in n-3 LC-PUFA content of farmed fishes, it is highly necessary to develop alternative sources of n-3 LC-PUFA. In the present study, we utilized transgenic technology to generate n-3 LC-PUFA-rich fish by using zebrafish as an animal model. Firstly, fat1 was proved to function efficiently in fish culture cells, which showed an effective conversion of n-6 PUFA to n-3 PUFA with the n-6/n-3 ratio that decreased from 7.7 to 1.1. Secondly, expression of fat1 in transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.8- and 2.4-fold, respectively. Third, co-expression of fat2, a fish codon-optimized delta-12 desaturase gene, and fat1 in fish culture cell significantly promoted n-3 PUFA synthesis with the decreased n-6/n-3 ratio from 7.7 to 0.7. Finally, co-expression of fat1 and fat2 in double transgenic zebrafish increased the 20:5n-3 and 22:6n-3 contents to 1.7- and 2.8-fold, respectively. Overall, we generated two types of transgenic zebrafish rich in endogenous n-3 LC-PUFA, fat1 transgenic zebrafish and fat1/fat2 double transgenic zebrafish. Our results demonstrate that application of transgenic technology of humanized fat1 and fat2 in farmed fishes can largely improve the n-3 LC-PUFA production.

Identification of a strawberry flavor gene candidate using an integrated genetic-genomic-analytical chemistry approach.

BackgroundThere is interest in improving the flavor of commercial strawberry (Fragaria × ananassa) varieties. Fruit flavor is shaped by combinations of sugars, acids and volatile compounds. Many efforts seek to use genomics-based strategies to identify genes controlling flavor, and then designing durable molecular markers to follow these genes in breeding populations. In this report, fruit from two cultivars, varying for presence-absence of volatile compounds, along with segregating progeny, were analyzed using GC/MS and RNAseq. Expression data were bulked in silico according to presence/absence of a given volatile compound, in this case γ-decalactone, a compound conferring a peach flavor note to fruits.ResultsComputationally sorting reads in segregating progeny based on γ-decalactone presence eliminated transcripts not directly relevant to the volatile, revealing transcripts possibly imparting quantitative contributions. One candidate encodes an omega-6 fatty acid desaturase, an enzyme known to participate in lactone production in fungi, noted here as FaFAD1. This candidate was induced by ripening, was detected in certain harvests, and correlated with γ-decalactone presence. The FaFAD1 gene is present in every genotype where γ-decalactone has been detected, and it was invariably missing in non-producers. A functional, PCR-based molecular marker was developed that cosegregates with the phenotype in F1 and BC1 populations, as well as in many other cultivars and wild Fragaria accessions.ConclusionsGenetic, genomic and analytical chemistry techniques were combined to identify FaFAD1, a gene likely controlling a key flavor volatile in strawberry. The same data may now be re-sorted based on presence/absence of any other volatile to identify other flavor-affecting candidates, leading to rapid generation of gene-specific markers.

A tomato endoplasmic reticulum (ER)-type omega-3 fatty acid desaturase (LeFAD3) functions in early seedling tolerance to salinity stress

Transgenic tomato plants overexpressing LeFAD3 sense and antisense sequences were generated. Salt stress suppressed the growth of WT and antisense plants to a higher extent than that in sense plants. In this study, we investigated the role of the LeFAD3-encoding ER-type omega-3 fatty acid desaturase in salt tolerance in tomato plants. We created transgenic tomato plants by overexpressing its sense and antisense sequences under the control of the cauliflower mosaic virus 35S promoter. Based on the results of northern and western blotting as well as quantitative reverse transcription-polymerase chain reaction, sense plants expressed more desaturase than wild-type (WT) plants, whereas antisense plants expressed less desaturase than WT. Salt stress suppressed the growth of both WT and antisense plants to a higher extent than that in sense plants, which can be attributed to the fact that sense plants performed better in maintaining the integrity of the membrane system, as revealed by electron microscopy. The concomitant increase in superoxide dismutase (EC 1.15.1.1) and ascorbate peroxidase (EC 1.11.1.7) may have alleviated the photoinhibition caused by the increased level of ROS in sense plants. Our results suggest that LeFAD3 overexpression can enhance the tolerance of early seedlings to salinity stress.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica

BackgroundCrambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed.ResultsThe individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil.ConclusionsCaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.

Isolation and characterization of the omega-6 fatty acid desaturase (FAD2) gene family in the allohexaploid oil seed crop Crambe abyssinica Hochst

Crambe (Crambe abyssinica Hochst ex. R. E. Fries) is an ideal crop for industrial oil production because of its high erucic acid content (C22:1, approx. 60 %) in its seed oil. The value of crambe oil can be improved by increasing C22:1 content or reducing polyunsaturated fatty acids (PUFA). The FAD2 gene plays a critical role in PUFA biosynthesis. To identify targets for breeding, we characterized FAD2 in crambe for copy number and expression profile. Seven copies of FAD2 were detected in allohexaploid crambe. Three genes (CaFAD2-C1, -C2 and -C3) were expressed in multiple tissues, including root, seedling, leaf, flower, bud and developing seeds. In developing seeds, the expression of these genes was upregulated with CaFAD2-C3, being expressed predominantly with a peak at 20 days after pollination. This gene is therefore a promising candidate gene for determining PUFA levels in seed oil. Four other FAD2 genes were considered to be “pseudo-genes” as they harbour internal stop codons and were not expressed. Among the six crambe varieties with consistent variation in oil composition, no nucleotide polymorphisms were found in the CaFAD2-C1, -C2 and -C3 genes. In seeds at 30 days after pollination, statistically significant expression level polymorphisms for only one gene, CaFAD2-C2, was found among the varieties. However, although significantly different, the difference in expression was small and did not explain the variation in oil composition. Given the absence of genetic variation in CaFAD2 genes in crambe breeding lines, breeding for high erucic acid content calls for a molecular breeding approach whereby mutations are chemically induced to increase the genetic variation.

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (PN) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.

Activity of Brassica napus and Perilla frutescens microsomal ω-3 desaturases expressed in yeast (Saccharomyces cerevisiae)

Yeast transformations with Brassica napus and Perilla frutescens \omega-3 desaturases were carried out. Feeding experiments on exogenous 18:2 at 10 mg/L and 100 mg/L for both genes under this investigation showed the same activity levels over the 3 days of the incubation, producing almost the same amount of 18:3. To further investigate the characteristics of B. napus and P. frutescens \omega -3 desaturases, a time-course in vivo activity study was conducted. Both enzymes showed the same behavior over the time course, desaturating 18:2 into 18:3. Moreover, the use of 2 different temperatures for incubation (30 °C and 20 °C) did not affect levels of 18:3 in either \omega -3 desaturase-expressing yeast lines. The 18:2 caused a change in yeast fatty acid profiles (compared to the wild yeast type), and 16:1 was highly reduced in 18:2-fed yeast lines, especially in the yeast lines with the higher 18:2 level. Amino acid sequence comparisons for some higher plant \omega -3 desaturases show that \omega -3 desaturases, including B. napus and P. frutescens \omega -3 desaturases, are highly conserved amino acid sequences. This is the first report of P. frutescens \omega -3 desaturase expression in yeast. All \omega -3 desaturases may have the same behaviors if they are expressed under the control of the same promoter and the same conditions.

Contribution of the different omega-3 fatty acid desaturase genes to the cold response in soybean

This study analysed the contribution of each omega-3 desaturase to the cold response in soybean. Exposure to cold temperatures (5 °C) did not result in great modifications of the linolenic acid content in leaf membrane lipids. However, an increase in the GmFAD3A transcripts was observed both in plant leaves and soybean cells whereas no changes in GmFAD3B or GmFAD3C expression levels were detected. This increase was reversible and accompanied by the accumulation of an mRNA encoding a truncated form of GmFAD3A (GmFAD3A-T), which originated from alternative splicing of GmFAD3A in response to cold. When the expression of plastidial omega-3 desaturases was analysed, a transient accumulation of GmFAD7-2 mRNA was detected upon cold exposure in mature soybean trifoliate leaves while GmFAD7-1 transcripts remained unchanged. No modification of the GmFAD8-1 and GmFAD8-2 transcripts was observed. The functionality of GmFAD3A, GmFAD3B, GmFAD3C and GmFAD3A-T was examined by heterologous expression in yeast. No activity was detected with GmFAD3A-T, consistent with the absence of one of the His boxes necessary for desaturase activity. The linolenic acid content of Sacharomyces cerevisiae cells overexpressing GmFAD3A or GmFAD3B was higher when the cultures were incubated at cooler temperatures, suggesting that reticular desaturases of the GmFAD3 family, and more specifically GmFAD3A, may play a role in the cold response, even in leaves. The data point to a regulatory mechanism of omega-3 fatty acid desaturases in soybean affecting specific isoforms in both the plastid and the endoplasmic reticulum to maintain appropriate levels of linolenic acid under low temperature conditions.