Oleic Acid Biosynthesis Research Articles

Comparative analysis of lipid and flavonoid biosynthesis between Pongamia and soybean seeds: genomic, transcriptional, and metabolic perspectives

BackgroundSoybean (Glycine max) is a vital oil-producing crop. Augmenting oleic acid (OA) levels in soybean oil enhances its oxidative stability and health benefits, representing a key objective in soybean breeding. Pongamia (Pongamia pinnata), known for its abundant oil, OA, and flavonoid in the seeds, holds promise as a biofuel and medicinal plant. A comparative analysis of the lipid and flavonoid biosynthesis pathways in Pongamia and soybean seeds would facilitate the assessment of the potential value of Pongamia seeds and advance the genetic improvements of seed traits in both species.ResultsThe study employed multi-omics analysis to systematically compare differences in metabolite accumulation and associated biosynthetic genes between Pongamia seeds and soybean seeds at the transcriptional, metabolic, and genomic levels. The results revealed that OA is the predominant free fatty acid in Pongamia seeds, being 8.3 times more abundant than in soybean seeds. Lipidomics unveiled a notably higher accumulation of triacylglycerols (TAGs) in Pongamia seeds compared to soybean seeds, with 23 TAG species containing OA. Subsequently, we identified orthologous groups (OGs) involved in lipid biosynthesis across 25 gene families in the genomes of Pongamia and soybean, and compared the expression levels of these OGs in the seeds of the two species. Among the OGs with expression levels in Pongamia seeds more than twice as high as in soybean seeds, we identified one fatty acyl-ACP thioesterase A (FATA) and two stearoyl-ACP desaturases (SADs), responsible for OA biosynthesis, along with two phospholipid:diacylglycerol acyltransferases (PDATs) and three acyl-CoA:diacylglycerol acyltransferases (DGATs), responsible for TAG biosynthesis. Furthermore, we observed a significantly higher content of the flavonoid formononetin in Pongamia seeds compared to soybean seeds, by over 2000-fold. This difference may be attributed to the tandem duplication expansions of 2,7,4ʹ-trihydroxyisoflavanone 4ʹ-O-methyltransferases (HI4ʹOMTs) in the Pongamia genome, which are responsible for the final step of formononetin biosynthesis, combined with their high expression levels in Pongamia seeds.ConclusionsThis study extends beyond observations made in single-species research by offering novel insights into the molecular basis of differences in lipid and flavonoid biosynthetic pathways between Pongamia and soybean, from a cross-species comparative perspective.

Effects of gut bacterial community on differential growth of sea cucumber (Apostichopus japonicus): Molecular mechanisms based on host-gut microbiome co-metabolism

Gut microbiome and their metabolites are increasingly recognized for their pivotal role in regulating the health and growth of the host. The mechanism by which the host-gut microbiome co-metabolism affects the growth rate of sea cucumber (Apostichopus japonicus) remains unclear. In this study, the intestinal wall tissues of fast-growing (FG) and slow-growing (SG) A. japonicus were collected from the full-sib family for 16S amplicon sequencing, metagenomics, metabolomics, and transcriptomics analyses. The results showed a marked variation in the gut bacterial community of A. japonicus with different growth rates. Compared with SG group, FG A. japonicus had a higher abundance of Bacteroidetes. Many of these bacteria were associated with the degradation of polysaccharides, which could potentially affect growth performance. The abundance of Proteobacteria, however, was found to be higher in SG A. japonicus, thereby increasing the potential risk of disease. The metabolome results were further supported by the predicated KEGG functions of the gut microbiomes, lots of metabolites related to the metabolism and biosynthesis of fructose 6-phosphate, arachidonic acid, oleic acid, and dihydroxyacetone phosphate were upregulated in the FG A. japonicus. Furthermore, the genes (IGF1, HK, PK, PCK1) were significantly upregulated in the FG A. japonicus, which were mainly enriched in the metabolic pathways of lipids, amino acids, and carbohydrates. Gut bacterial community via their unique metabolic pathways, which in turn affected the metabolic phenotypes of the organism and eventually led to differences in growth performance of A. japonicus. These findings might help understand the molecular mechanisms of growth gap in sea cucumber, thereby increasing the mariculture production.

Analysis of Delta(9) fatty acid desaturase gene family and their role in oleic acid accumulation in Carya cathayensis kernel.

Carya cathayensis, commonly referred to as Chinese hickory, produces nuts that contain high-quality edible oils, particularly oleic acid (18:1). It is known that stearoyl-ACP desaturase (SAD) is the first key step converting stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the aminolevulinic acid (ALA) biosynthetic pathway and play an important role in OA accumulation. Thus far, there is little information about SAD gene family in C. cathayensis and the role of individual members in OA accumulation. This study searched the Chinese Hickory Genome Database and identified five members of SAD genes, designated as CcSADs, at the whole genome level through the comparison with the homologous genes from Arabidopsis. RNA-Seq analysis showed that CcSSI2-1, CcSSI2-2, and CcSAD6 were highly expressed in kernels. The expression pattern of CcSADs was significantly correlated with fatty acid accumulation during the kernel development. In addition, five full-length cDNAs encoding SADs were isolated from the developing kernel of C. cathayensis. CcSADs-green fluorescent protein (GFP) fusion construct was infiltrated into tobacco epidermal cells, and results indicated their chloroplast localization. The catalytic function of these CcSADs was further analyzed by heterologous expression in Saccharomyces cerevisiae, Nicotiana benthamiana, and walnut. Functional analysis demonstrated that all CcSADs had fatty acid desaturase activity to catalyze oleic acid biosynthesis. Some members of CcSADs also have strong substrate specificity for 16:0-ACP to synthesize palmitoleic acid (C16:1, PA). Our study documented SAD gene family in C. cathayensis and the role of CcSSI2-1, CcSSI2-2, and CcSAD6 in OA accumulation, which could be important for future improvement of OA content in this species via genetic manipulation.

Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax (Linum usitatissimum L.) is a versatile crop and its seeds are a major source of unsaturated fatty acids. Stearoyl-acyl carrier protein desaturase (SAD) is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses. However, the function of SAD orthologs from L. usitatissimum has not been assessed. Here, we found that two LuSAD genes, LuSAD1 and LuSAD2, are present in the genome of L. usitatissimum cultivar ‘Longya 10’. Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds. Interestingly, ectopic expression of LuSAD2 in A. thaliana caused altered plant architecture. Similarly, the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds. Furthermore, we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels, as well as reducing membrane damage. These findings not only broaden our knowledge of the LuSAD functions in plants, but also offer promising targets for improving the quantity and quality of oil, and the abiotic stress tolerance of oil-producing crops, through molecular manipulation.

Dietary oleic acid contributes to the regulation of food intake through the synthesis of intestinal oleoylethanolamide.

Among the fatty acid ethanolamides (FAEs), oleoylethanolamide (OEA), linoleoylethanolamide (LEA), and palmitoylethanolamide (PEA) are reported to be involved in feeding regulation. In particular, OEA is well characterized as a satiety signal. Following food consumption, OEA is synthesized from oleic acid (OA) via an N-acyl phosphatidylethanolamine-specific phospholipase D-dependent pathway in the gastroenterocytes, and OEA induces satiety by recruiting sensory fibers. Thus, we hypothesized that dietary OA is an important satiety-inducing molecule. However, there has been no direct demonstration of the effect of dietary OA on satiety induction without the influence of the endogenous biosynthesis of OA from stearic acid (SA) or other FAEs. In this study, we used two experimental diets to test our hypothesis: (i) an OA diet (OAD; 38.4 mg of OA/g and 7.2 mg of SA/g) and (ii) a low OA diet (LOAD; 3.1 mg of OA/g and 42.4 mg of SA/g). Relative to mice fed the OAD, mice fed the LOAD for two weeks exhibited reduced levels of jejunal OEA but not jejunal LEA and PEA. The LOAD-fed mice showed an increase in food intake and body weight gain. Moreover, LOAD-induced increase in food intake was immediately observed after the switch from the OAD, whereas these effects were diminished by the switch back to the OAD. Furthermore, treatment with OA and OEA diminished the effects of LOAD on food intake. Collectively, these results show that dietary OA is a key factor in the reduction of food intake and increase in satiety mediated by OEA signaling.

Salt stress affects the fruit quality of Lycium ruthenicum Murr.

Halophytic plant Lycium ruthenicum is rich with anthocyanin in fruit, and has thus attracted considerable attention due to its health-promoting effects. However, the effect of salt stress on fruit quality remains unknown. In this study, RNA-seq, LC-MS/MS coupling with TMT labeling, and GC-MS approaches were respectively used to examine the transcriptomic, proteomic, and metabolomic profiles in ripening fruit of 3-year-old trees treated with 0 or 300 mM NaCl, which resulting in 5608 differentially expressed genes (DEGs), 201 differentially expressed proteins (DEPs), and 23 differentially accumulated metabolites (DAMs). KEGG enrichment analysis showed that DEGs related to amino acid metabolism, the TCA cycle, flavonoid biosynthesis, inositol phosphate metabolism, and alpha-linolenic acid metabolism were significantly enriched. DEPs related to protein homeostasis and redox homeostasis were enriched. The contents of anthocyanins, proline, glucose, and glutamine are up-regulated in the salt-treated fruit. Our results indicate that the increased fruit anthocyanin might attribute to the up-regulated MYB component of the MYB-bHLH-WD40 complex and the post-transcriptionally up-regulated proteins encoded by anthocyanin structural genes under salt stress condition. Additionally, weighted gene correlation network analysis (WGCNA) results show that anthocyanin content is highly associated with three modules including multiple transcription factors, genes related to reactive oxygen species homeostasis and phytohormones (abscisic acid and ethylene), and that three FADs genes are involved in the biosynthesis of oleic acid and linoleic acid. Taken together, these findings provide a comprehensive understanding of salt stress-mediated regulation of fruit quality, thereby facilitating the breeding of high anthocyanin cultivars of L. ruthenicum in future.

Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica.

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Sequence characterization and temporal expression analysis of different SADs and FAD2-2 genes in two Iranian olive cultivars

Stearoyl-acyl carrier protein (ACP) desaturases (SADs) and fatty acid desaturases (FADs) are the main genes catalyzing the first and the second desaturation steps throughout the biosynthesis of oleic and linoleic acids, respectively, hence, the major determinants of oil quality and composition. To uncover the molecular mechanisms behind differential oil content and composition in olive fruit, the nucleotide and amino acid sequences of FAD2-2 and three SAD genes were characterized in 'Mari' and 'Shengeh' as two Iranian olive cultivars displaying high contrasting oil composition. In addition, the expression levels of these genes were screened at different time points during fruit development and ripening. Despite detection of a number of nucleotide sequence variations in four characterized genes, the results of amino acid analyses have shown most of them were synonymous and caused no differences at protein level. However, some of the single nucleotide variations caused non cognate amino acid substitution indicating possible conformational changes in the resultant peptide. In particular, regarding OeFAD2-2, a nonsynonymous SNP was detected in 'Shengeh' compared with other olive's FAD2-2 causing an amino acid lysine residue substitution for a glutamic acid, as well as a nonsynonymous substitution in 'Mari' (threonine to serine). The in silico prediction of three-dimensional structure of FAD2-2 revealed these substitutions may lead to structural changes in the final protein structure and function, hence, may contribute to the higher activity of FAD2-2 protein in mesocarp of 'Shengeh' than 'Mari' cultivars. According to results obtained from sequence analysis of oil biosynthesis genes, two Iranian olive cultivars were the most similar and had high sequence differences with international varieties. Results of temporal transcript abundance revealed that OeSAD2 had higher expression in 'Mari' (the high quality oil cultivar) than 'Shengeh' during the critical period of oil biosynthesis. Results of our data corroborates the previous records suggesting that OeSAD2 as the most pronounced isoform of stearoyl-acyl carrier protein desaturases responsible for oleic acid biosynthesis in olive mesocarp. In addition our results also suggest that OeSAD3 may have contributed to the higher oil quality of 'Mari' than 'Shengeh'. Based on our results, OeSAD2 and OeFAD2-2 are suggested as the possible targets for engineering fatty acid composition to help pyramid genes to improve quality and quantity of olive oil and creating high quality olive cultivars.

Mycobacterium tuberculosis transcriptional regulator Rv1019 is upregulated in hypoxia, and negatively regulates Rv3230c-Rv3229c operon encoding enzymes in the oleic acid biosynthetic pathway.

The main obstacle in eradicating tuberculosis is the ability of Mycobacterium tuberculosis to remain dormant in the host, and then to get reactivated even years later under immunocompromised conditions. Transcriptional regulation in intracellular pathogens plays an important role in their adapting to the challenging environment inside the host cells. Previously, we demonstrated that Rv1019, a putative transcriptional regulator of M.tuberculosis H37Rv, is an autorepressor. We showed that Rv1019 is cotranscribed with Rv1020 (mfd) and Rv1021 (mazG) which encode DNA repair proteins and negatively regulates the expression of these genes. In the present study, we show that Rv1019 regulates the expression of the genes Rv3230c and Rv3229c (desA3) alsowhich form a two-gene operon in M.tuberculosis. Overexpression of Rv1019 in M.tuberculosis significantly downregulated the expression of these genes. Employing Wayne's hypoxia-induced dormancy model of M.tuberculosis, we show that Rv1019 is upregulated three-fold under hypoxia. Finally, by reporter assay, using Mycobacterium smegmatis as a model, we validate that Rv1019 is recruited to the promoter of Rv3230c-Rv3229c during hypoxia, and negatively regulates this operon which is involved in the biosynthesis of oleic acid.

High oleic acid biosynthesis and its absolute quantification by GC/MS in oleaginous Scenedesmus sp. SVMIICT1 cultivated in dual stress phase

High photosynthetic rates and high lipid content make microalgae a promising source for the industrial production of edible oils. Enhancement of oleic acid production in microalgae is an important area in the algae-based edible oils research. This study employed the mixotrophic mode of nutrition with a nutrient-rich growth phase (GP) followed by two different stress phases (SPs), including nitrate depletion (single stress phase; SSP) and nitrate depletion with salinity (dual stress phase; DSP). In DSP, higher oleic acid (C18:1) content was observed and considered the same for autotrophic mode. In addition to DSP, it evaluated the role of nitrate to phosphate ratio in GP towards fatty acid biosynthesis. The relative and absolute quantities of fatty acids were measured in mixotrophic and autotrophic cultivations. Higher oleic acid was obtained at 250 mM NaCl DSP condition in mixotrophic cultivation compared to other conditions. Whereas autotrophic mode microalgae cultivated in dual stress phase (DSP for A-GP-1N1P, DSP for A-GP-1N2P, DSP for A-GP-1N4P, and DSP for A-GP-1N6P) resulted in ~30 % (w/w) of total fatty acids with 55 % of Oleic acid in absolute quantity. Overall, the study depicted a distinct influence of nitrate, nitrate to phosphate ratio, nitrate depletion (single stress), and dual stress on the oleic acid yield. The application of dual stress to microalgae cultivation is a new approach to oleic acid enhancement. • Nitrate depletion and salinity in stress phase influenced oleic acid biosynthesis. • Study achieved 30 % of total fatty acids with 55 % of Oleic acid in absolute quantity. • Nitrates and phosphates in growth phase also influenced fatty acids production. • Nitrates increment in mixotrophic growth phase negligibly affected oleogenesis. • Biosynthesis of fatty acids is interdependent with growth phase and stress phase.

Age Evolution of Lipid Accretion Rate in Boars Selected for Lean Meat and Duroc Barrows.

Simple SummaryFat deposition profile and fatty acid biosynthesis in adipose tissues are two relevant metabolic processes for breeding companies as the fat content of meat relates to its technological, organoleptic, and nutritional quality. Age, breed, and sex have a great impact on fat metabolism. Therefore, in the present experiment, a labeled saturated fatty acid was added to the diet of pigs belonging to three different producing types during two growth phases, to define the rate of its incorporation into body fat. In addition, the activity of the enzyme stearoyl-CoA desaturase, which promotes the conversion of some saturated fatty acids into monounsaturated, was analyzed. In the growing phase, direct incorporation of fat from diet was higher than biosynthesis in most adipose tissues, whereas in the fattening phase it was higher only in the liver, revealing the changing roles of organs in fat metabolism with age. The leaner pigs also obtained a higher fat incorporation, denoting higher reliance on dietary fatty acids for fat deposition; overall, real stearoyl-CoA desaturase activity was higher in the fattening phase.Fatty acid (FA) deposition in growing–fattening pigs is mainly based on endogenous lipid synthesis, but also direct FA incorporation from the diet. To evaluate the direct fat incorporation rates and the endogenous desaturation action of the stearoyl-CoA desaturase (SCD) enzyme, a deuterium (D)-labeled saturated FA (d35-C18:0) was added to the diet. Sixteen three-way (3W) crossbred boars, and thirty-two purebred Duroc barrows homozygous for the SCD single nucleotide polymorphism rs80912566 (16 CC/16 TT), were used. Half of the animals of each genotype belonged to the growing and fattening phases. The fractional incorporation rate (FIR) of dietary fat in growing pigs was generally higher in adipose tissues, whereas in fattening pigs it was higher in the liver. Duroc pigs exhibited lower FIRs than 3W pigs, suggesting lower rates of endogenous synthesis by 3W pigs. Real fractional unsaturation rates (FURs) increased with age by the higher FIRs in 3W pigs and the de novo synthesis pathway in Duroc genotypes. Moreover, pigs carrying the SCD_T allele showed more enhanced oleic acid biosynthesis than Duroc CC pigs. In conclusion, suitable feeding protocols should be designed for each pig type to optimize production traits, considering that the metabolic pathway of FA for its deposition may differ.

Targeting stearoyl-coa desaturase enhances radiation induced ferroptosis and immunogenic cell death in esophageal squamous cell carcinoma

ABSTRACT Overcoming resistance to radiation is a major challenge in cancer treatment. Stearoyl-coa desaturase (SCD1) is the enzyme responsible for oleic acid (OA) and palmitoleic acid (POA) formation. Here, we provided evidence that targeting SCD1 was capable of inducing ferroptosis and immunogenic cell death (ICD), thereby improving the radiation sensitivity of esophageal squamous cell carcinoma (ESCC). ESCC cell lines with high SCD1 expression were treated with MF-438 (SCD1 inhibitor) to determine cell viability. Colony formation assay was performed to evaluate the radiation sensitization of SCD1 inhibitor. Tumor cell ferroptosis and ICD was analyzed in MF-438, radiation therapy (RT) and the combination treatment group. The potential molecular mechanisms underlying MF-438 as a novel radiation sensitizer in ESCC were explored. We concluded by assessing SCD1 as a prognostic factor in ESCC. MF-438 exhibited antitumor activity in ESCC cells. Our outcomes revealed significant improvement of radiation sensitivity by MF-438. Moreover, the combination treatment enhanced tumor cell ferroptosis and ICD. Further analyses revealed SCD1 conferred radiation resistance via alleviating ferroptosis in tumor cells; targeting SCD1 inhibited the biosynthesis of OA and POA, and improved radiation induced ferroptosis in ESCC cells. Clinical analysis indicated high expression of SCD1 was associated with unfavorable survival in patients of ESCC. In summary, our results demonstrated that MF-438 acted as a ferroptosis inducer. Targeting SCD1 conferred the immunogenicity of ferroptotic cancer cells and increased the effectiveness of RT in ESCC. SCD1 could be considered as a useful prognostic indicator of survival in ESCC.

Effect of dietary fats on endogenous oleic acid biosynthesis in rat liver

Aim: Determine the effect of dietary fats with different fatty acid composition on the biosynthesis of oleic acid and its metabolic precursors in the liver .
 Methods: High linoleic sunflower oil (HLSO), high oleic sunflower oil (HOSO) and palm oil (PO) were used. Rats were fed a semi-synthetic fat-free diet (FFD) and fat diets containing 5 % of the above oils (instead of starch) for 30 days. Liver lipids were divided into 3 fractions: neutral lipids (NL), phospholipids (PL) and free fatty acids (FFA). The fatty acid composition of the fractions was determined by gas chromatography. The “activity” of fatty acid synthase was determined from the total content of the products of this reaction (C16:0 and C16:1). The “activity” of palmitic acid elongase was determined by the ratio С18:0/С16:0, as well as by the formula (С18:0+С18:1)/(С16:0–С16:1). The “activity” of stearic acid desaturase (SCD1) was determined by the ratio C16:1/C16:0 (SCD16) and by the ratio C18:1/C18:0 (SCD18).
 Results: In rats treated with fat diets, the content of palmitic and oleic acids is reduced only in the NL fraction, and to the greatest extent when consuming the diet with HLSO. The “activity” of palmitic acid elongase increases significantly with the consumption of a diet with HLSO. SCD16 desaturase “activity” decreases with fat diet, while SCD18 desaturase “activity” increases. The level of SCD18 is significantly higher than the level of SCD16. Consumption of HLSO reduces the content of ω-3 PUFA in rat liver lipids, while the intake of HOSO increases it.
 Conclusions: HLSO diet reduces the endogenous biosynthesis of oleic and palmitic acids, as determined by the analysis of the rat liver NL fraction. A fat diet reduces SCD16 “activity” but increases SCD18 “activity”, especially when fed a diet with HOSO. The diet with HLSO reduces the content of ω-3 PUFA in liver lipids.

Understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides

BackgroundRhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive.ResultsWe present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA).ConclusionOur work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.

The Corylus mandshurica genome provides insights into the evolution of Betulaceae genomes and hazelnut breeding

Hazelnut is popular for its flavor, and it has also been suggested that hazelnut is beneficial to cardiovascular health because it is rich in oleic acid. Here, we report the first high-quality chromosome-scale genome for the hazelnut species Corylus mandshurica (2n = 22), which has a high concentration of oleic acid in its nuts. The assembled genome is 367.67 Mb in length, and the contig N50 is 14.85 Mb. All contigs were assembled into 11 chromosomes, and 28,409 protein-coding genes were annotated. We reconstructed the evolutionary trajectories of the genomes of Betulaceae species and revealed that the 11 chromosomes of the hazelnut genus were derived from the most ancestral karyotype in Betula pendula, which has 14 protochromosomes, by inferring homology among five Betulaceae genomes. We identified 96 candidate genes involved in oleic acid biosynthesis, and 10 showed rapid evolution or positive selection. These findings will help us to understand the mechanisms of lipid synthesis and storage in hazelnuts. Several gene families related to salicylic acid metabolism and stress responses experienced rapid expansion in this hazelnut species, which may have increased its stress tolerance. The reference genome presented here constitutes a valuable resource for molecular breeding and genetic improvement of the important agronomic properties of hazelnut.

Characterization of the stearoyl-ACP desaturase gene (PoSAD) from woody oil crop Paeonia ostii var. lishizhenii in oleic acid biosynthesis

Paeonia ostii var. lishizhenii has been approved as a woody oil crop with the outstanding characteristic of abundant α-linolenic acid (C18:3, ALA) in its seed oil. The stearoyl-ACP desaturase gene (SAD) regulates the first key step from stearic acid (C18:0, SA) to oleic acid (C18:1, OA) in the ALA biosynthetic pathway, but its functional characterization in P. ostii var. lishizhenii is absent to date. In this study, a key PoSAD gene (1719 bp in length) was acquired from endosperm of P. ostii var. lishizhenii by transcriptome sequencing analysis and the RACE (rapid-amplification of cDNA ends) method. Bioinformatic analysis of the PoSAD protein showed high homology (ranging from 90.4% to 94.4%) and similar physical and chemical properties to SAD from other higher plants, indicating that it encodes a putative stearoyl-ACP desaturase. Analysis of cis-acting elements found several endosperm tissue-specific motifs; i.e., one Prolamin box, thirteen DOFCOREs and one RY repeat in its promoter. The results of the qRT-PCR experiments verified that PoSAD was most highly expressed in developing endosperm at 59 days after pollination (53.7 times that in shoots) compared with that in roots (1.4 times), stems (2.5 times), leaves (3.1 times), petals (13.1 times) and stamens (46.0 times). Meanwhile, the fatty acid contents in P. ostii var. lishizhenii endosperm at seven growth stages were compared with variation in PoSAD expression. Heterologous expression of PoSAD significantly decreased SA and increased OA content, which effectively reduced the ratios of SA to OA in Saccharomyces cerevisiae and Arabidopsis thaliana. However, contents and ratios of palmitic acid (C16:0) and palmitoleic acid (C16:1) were stable in transgenic yeast, and palmitoleic acid remained absent in transgenic A. thaliana seeds. These results illustrate that PoSAD plays an essential role in endosperm development of P. ostii var. lishizhenii, strictly in catalysis of SA desaturation and OA biosynthesis but without functioning in PA desaturation. The results contribute to our understanding of the characterization of PoSAD in OA biosynthesis in P. ostii var. lishizhenii.

SncRNA-1 Is a Small Noncoding RNA Produced by Mycobacterium tuberculosis in Infected Cells That Positively Regulates Genes Coupled to Oleic Acid Biosynthesis.

Nearly one third of the world’s population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different Mtb encoded proteins in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. We performed RNA sequencing and identified a subset of Mtb H37Rv-encoded small RNAs (<30 nts in length) that were produced in infected macrophages. Designated as smaller noncoding RNAs (sncRNAs), three of these predominated the read counts. Each of the three, sncRNA-1, sncRNA-6, and sncRNA-8 had surrounding sequences with predicted stable secondary RNA stem loops. Site-directed mutagenesis of the precursor sequences suggest the existence of a hairpin loop dependent RNA processing mechanism. A functional assessment of sncRNA-1 suggested that it positively regulated two mycobacterial transcripts involved in oleic acid biosynthesis. Complementary loss- and gain- of-function approaches revealed that sncRNA-1 positively supports Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Overall, the findings reveal that Mtb produces sncRNAs in infected cells, with sncRNA-1 modulating mycobacterial gene expression including genes coupled to oleic acid biogenesis.

Comparative transcriptomic analysis of high- and low-oil Camellia oleifera reveals a coordinated mechanism for the regulation of upstream and downstream multigenes for high oleic acid accumulation.

Tea oil camellia (Camellia oleifera) is an important woody oil tree in southern China. However, little is known regarding the molecular mechanisms that contribute to high oleic acid accumulation in tea oil camellia. Herein, we measured the oil content and fatty acid compositions of high- and low-oil tea oil camellia seeds and investigated the global gene expression profiles by RNA-seq. The results showed that at the early, second and third seed developmental stages, a total of 64, 253, and 124 genes, respectively, were significantly differentially expressed between the high- and low-oil cultivars. Gene ontology (GO) enrichment analysis of the identified differentially expressed transcription factors (TFs; ABI3, FUS3, LEC1, WRI1, TTG2 and DOF4.6) revealed some critical GO terms associated with oil biosynthesis and fatty acid accumulation, including glycolysis, zinc ion binding, positive regulation of fatty acid biosynthetic process, triglyceride biosynthetic process, seed coat development, abscisic acid-mediated signaling pathway and embryo development. Comprehensive comparisons of transcriptomic profiles and expression analysis of multigenes based on qRT-PCR showed that coordinated high expression of the upstream genes HAD, EAR and KASI directly increased the relative levels of C16:0-ACP, which provided enough precursor resources for oleic acid biosynthesis. Continuous high expression of the SAD gene accelerated oleic acid synthesis and accumulation, and coordinated low expression of the downstream genes FAD2, FAD3, FAD7, FAD8 and FAE1 decreased the consumption of oleic acid for conversion. The coordinated regulation of these multigenes ensures the high accumulation of oleic acid in the seeds of tea oil camellia. Our data represent a comprehensive transcriptomic study of high- and low-oil tea oil camellia, not only increasing the number of sequences associated with lipid biosynthesis and fatty acid accumulation in public resource databases but also providing a scientific basis for genetic improvement of the oleic acid content in woody oil trees.

ITRAQ-based quantitative proteomics analysis of an immature high-oleic acid near-isogenic line of rapeseed

High-oleic acid rapeseed is a new and promising rapeseed variety because it produces oil with high-quality health and nutrient properties. To identify differentially expressed proteins in a high-oleic acid rapeseed line, self-bred seeds (20 to 35 days after pollination) of a high- and a low-oleic acid rapeseed near-isogenic line (oleic acid contents of 81.4 and 56.2%, respectively) were used as raw materials for iTRAQ (isobaric tags for relative and absolute quantitation) analysis. In total, 24,755 peptides, 14,455 unique peptides, and 4726 proteins were identified. Among them, 137 differentially expressed proteins were assigned pathway annotations, accounting for 3.84% of the total proteins (3568). Three differentially expressed proteins, PSaB (gi|383930478), Lhcb6 (gi|50313237), and ALD (gi|15227981) were subjected to western blotting analyses. The results were consistent with the iTRAQ data, demonstrating the satisfactory quality of the iTRAQ experimental procedures and data. The transcript levels of 19 genes involved in fatty acid metabolism and photosynthesis were analyzed by qPCR. The transcript levels were consistent with the protein levels for 10 of these genes, but inconsistent for the other nine genes. Among the differentially expressed proteins were key enzymes in fatty acid biosynthesis and polyunsaturated fatty acid biosynthesis (acyl-CoA thioesterase, 3-ketoacyl-CoA reductase, omega 6 reductase, and acyl-CoA dehydrogenase), oleic acid biosynthesis (acyl-CoA thioesterase and omega 6 reductase), and seed lipid content (long-chain acyl-CoA synthetase). These results provide fundamental functional proteomics information for further breeding work on high-oleic acid rapeseed. This information will also be useful for research on the molecular mechanism of oleic acid synthesis.

Conserved Function of ACYL-ACYL CARRIER PROTEIN DESATURASE 5 on Seed Oil and Oleic Acid Biosynthesis between Arabidopsis thaliana and Brassica napus.

Previous studies have shown that several ACYL–ACYL CARRIER PROTEIN DESATURASE (AtAAD) members in Arabidopsis thaliana are responsible for oleic acid (C18:1) biosynthesis. Limited research has been conducted on another member, AtAAD5, and its paralog BnAAD5 in the closely related and commercially important plant, Brassica napus. Here, we found that AtAAD5 was predominantly and exclusively expressed in developing embryos at the whole seed developmental stages. The aad5 mutation caused a significant decrease in the amounts of oil and C18:1, and a considerable increase in the content of stearic acid (C18:0) in mature seeds, suggesting that AtAAD5 functioned as an important facilitator of seed oil biosynthesis. We also cloned the full-length coding sequence of BnAAD5-1 from the A3 subgenome of the B. napus inbred line L111. We showed that ectopic expression of BnAAD5-1 in the A. thaliana aad5-2 mutant fully complemented the phenotypes of the mutant, such as lower oil content and altered contents of C18:0 and C18:1. These results help us to better understand the functions of AAD members in A. thaliana and B. napus and provide a promising target for genetic manipulation of B. napus.