The circadian clock plays a critical role in regulating plant physiology and metabolism. However, the way in which the clock impacts the regulation of lipid biosynthesis in seeds is partially understood. In the present study, we characterized the seed fatty acid (FA) and glycerolipid (GL) compositions of pseudo-response regulator mutants. Among these mutants, toc1 (timing of cab expression 1) exhibited the most significant differences compared to control plants. These included an increase in total FA content, characterized by elevated levels of linolenic acid (18:3) along with a reduction in linoleic acid (18:2). Furthermore, our findings revealed that toc1 developing seeds showed increased expression of genes related to FA metabolism. Our results show a connection between TOC1 and lipid metabolism in Arabidopsis seeds.

The objective of the present study was to analyse the fruit yield and the lipid and phenolic composition of the seeds from the edible halophyte Crithmum maritimum growing in different types of soil with variable physicochemical properties. Seed oil content ranged between 8.39-11.66 % and showed high nutritional value, and the phenolic composition (423-603 mg TPC 100 g(-1)) presented high levels of quercetin-type compounds with high pharmaceutical interest. The strong conservation of these traits, together with a high and stable fruit production, suggests that C. maritimum could be cultivated in soils with no agronomical relevance to produce seeds as an accessible and environmentally friendly source of good quality oil and phenolics for industrial purposes.

Camelina sativa is one of the preferred oil crops in plant biotechnology due to its agronomic performance, the quality of its oil and the ease with which it can be transformed. Oils with high levels of saturated fatty acids are in demand for structured lipid elaboration, whereas omega-7 fatty acids like palmitoleic or asclepic acids are of interest for other applications, such as in oleochemistry and biolubricant production. Several strategies have been followed to increase the levels of saturated and omega-7 fatty acids in the camelina plant in this work, including silencing the beta-ketoacyl-ACP synthase II (CsKASII) condensing enzyme responsible for the elongation of palmitate to stearate, the expression of exogenous thioesterases, and the overexpression of the endogenous stearoyl-ACP desaturase. The silencing of CsKASII produces an important increase in palmitate in the oil seed, whereas the expression of different alleles of sunflower FatA thioesterases favors the accumulation of both palmitate and stearate. The increase in intraplastidial desaturase activity through CsSAD co-expression forced the desaturation of palmitate, inducing the accumulation of important amounts of omega-7 fatty acids. The phenotypes of the different transformants produced were characterized by profiling the different glycerolipid classes accumulated in their seeds. These oils displayed altered physical properties that were investigated by differential calorimetry studies.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

A novel water-soluble polysaccharide named AGP1 was successfully isolated from seeds of Anethum graveolens by hot water extraction and further purified by DEAE-Sepharose chromatography. AGP1 has a relative molecular weight of 2.1 10(4) Da determined by Ultra-high-performance liquid chromatography (UHPLC). The AGP1 characterization was investigated by chemical and instrumental analysis including gas chromatography mass spectrometry (GC-MS), Fourier transform infrared (FT-IR) spectroscopy and X-ray diffraction. Results showed that AGP1 was mainly composed of glucose, galactose, mannose and arabinose in a molar percent of 54.3, 23.8, 14.7 and 7.2, respectively. The thermogravimetry analysis (TGA) and the differential scanning calorimetry (DSC) were used and showed that AGP1 has good thermal stability until 275 degrees C. Moreover, the purified polysaccharide demonstrated an appreciable in vitro antioxidant potential. The addition of the AGP1, particularly at 0.3% (w/w), in turkey sausages instead of ascorbic acid, as preservative, reduced the lipid peroxidation, preserved the pH and color and improved the bacterial stability during cold storage at 4 degrees C for 12 days. Overall, the results showed that the AGP1 deserves to be developed as functional and bioactive components for the food and nutraceutical industries. (C) 2020 Elsevier B.V. All rights reserved.

Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.

Lipoyl synthases are key enzymes in lipoic acid biosynthesis, a co-factor of several enzyme complexes involved in central metabolism. Plant pyruvate dehydrogenase complex (PDH), located in mitochondria and plastids, catalyses the first step of fatty acid biosynthesis in these organelles. Among their different components, the E2 subunit requires the lipoic acid prosthetic group to be active. De novo lipoic acid biosynthesis is achieved by the successive action of two enzymes on octanoyl-ACP: octanoyltransferase (LIP2) and lipoyl synthase (LIP1). In this study, two plastidial lipoyl synthase genes from sunflower (Helianthus annuus L.) were identified (HaLIP1p1 and HaLIP1p2), sequenced and cloned in a heterologous production system (Escherichia coli). Gene expression studies revealed similar expression patterns for both isoforms, with a slight predominance of HaLIP1p1 in vegetative tissues and mature seeds. Tertiary structural models for these enzymes indicate they both have the same theoretical catalytic sites, using lipoyl-lys and 5-deoxyadenosine as docking substrates. The fatty acid profile of E. coli cells overexpressing HaLIP1p1 and HaLIP1p2 did not present major differences, and the in vivo activity of both proteins was confirmed by complementation of an E. coli JW0623 mutant in which lipoyl synthase is defective. Although no significant differences were detected in the total fatty acid composition of transgenic Arabidopsis thaliana seeds overexpressing any of both proteins, a lipidomic analysis revealed a redistribution of the glycerolipid species, accompanied with increased phosphatidylethanolamine (PE) content and a decrease in diacyglycerols (DAG) and phosphatidylcholine (PC). Depletion of the SAM co-factor caused by HaLIP1p1 and HaLIP1p2 overexpression in transgenic plants could explain this remodelling through its effects on PC synthesis.

The distribution of Ruppia cirrhosa meadow density and its epiphytic organisms in relation with environmental factors were studied in summer 2011 at five stations in the Ghar El Melh lagoon (GML; southern Mediterranean Sea). Eleven epiphytic groups were recognised among which diatoms and dinoflagellates were the dominant groups and greatest contributors to temporal dissimilarity. An overwhelming concentrations of harmful microalgae was recorded, mainly represented by the toxic dinoflagellate Prorocentrum lima with maximal concentrations attaining 6 × 105 cells 100 g−1 of Ruppia fresh weight. The epifauna community accounted for only 1.4% of total epiphyte abundance and was comprised predominantly of nematodes (47.51%), ciliates (32.59%), fish eggs (7.2%) and larvae (4.95%). PERMANOVA analyses revealed a significant spatio-temporal variation of all epiphytic groups (p < 0.01). In this study, R. cirrhosa and its epiphytes were studied as potential early warning indicators of the health status of GML waters.

Palm oil (PO), although subject of controversies, is the most consumed oil and the first source of oil widely produced. In this review, we discussed its biochemical composition in fatty acids, carotenoids, vitamin E, its phenolic compounds, and its nutritional benefits. We addressed its biochemical properties in relation with the stereospecific distribution of its unsaturated fatty acids at the sn-2 position in triacylglycerols. PO is one of the most stable oils, which help it prolong food storability mostly due not only to its content of saturated fatty acids, but also to its antioxidant compounds. PO plays an important role in the prevention of many pathologies (diabetes, cardiovascular diseases, obesity and cancers). It is widely use in nutrition especially in the food industry and in biodiesel industry. Faced with attacks from environmentalists who blame PO for destorying biodiversity, there is an urgent need to develop a sustainable PO production plan. Compliance with sustainable PO goals would help ease those controversies. The use and consumption of PO in normal or moderate amounts in a varied, balanced and adequate diet does not present any known health risk. Education campaigns on the nutritional benefits of PO should be promoted.

Past research has sought to improve the production of cyclopropane fatty acids by the oleaginous yeast Yarrowia lipolytica by heterologously expressing the E. coli fatty acid synthase gene and improving cultivation processes. Cyclopropane fatty acids display properties that hold promise for biofuel applications. The E. coli fatty acid synthase gene was introduced into several genetic backgrounds of the yeast Y. lipolytica to optimize lipid synthesis; the mean cyclopropane fatty acid productivity was 43 mg L−1 h−1 on glucose, and the production rate reached its maximum (3.06 g L−1) after 72 h of cultivation in a bioreactor. The best strain (JMY6851) overexpressed simultaneously the E. coli cyclopropane fatty acid synthase gene under a hybrid promoter (hp8d) and Y. lipolytica LRO1 gene. In fed-batch process using crude glycerol as carbon source, JMY6851 strain displayed high lipid accumulation (78% of dry cell weight) and high biomass production (56 g L−1). After 165 h of cultivation, cyclopropane fatty acids represented 22% of the lipids produced; cyclopropane fatty acid productivity (103.3 mg L−1 h−1) was maximal at 72.5 h of cultivation.

Flax (Linum usitatissinum L.) oil is an important source of α-linolenic (C18:3 ω-3). This polyunsaturated fatty acid is well known for its nutritional role in human and animal diets. Understanding storage lipid biosynthesis in developing flax embryos can lead to an increase in seed yield via marker-assisted selection. While a tremendous amount of work has been done on different plant species to highlight their metabolism during embryo development, a comprehensive analysis of metabolic flux in flax is still lacking. In this context, we have utilized in vitro cultured developing embryos of flax and determined net fluxes by performing three complementary parallel labeling experiments with 13C-labeled glucose and glutamine. Metabolic fluxes were estimated by computer-aided modeling of the central metabolic network including 11 cofactors of 118 reactions of the central metabolism and 12 pseudo-fluxes. A focus on lipid storage biosynthesis and the associated pathways was done in comparison with rapeseed, arabidopsis, maize and sunflower embryos. In our hands, glucose was determined to be the main source of carbon in flax embryos, leading to the conversion of phosphoenolpyruvate to pyruvate. The oxidative pentose phosphate pathway (OPPP) was identified as the producer of NADPH for fatty acid biosynthesis. Overall, the use of 13C-metabolic flux analysis provided new insights into the flax embryo metabolic processes involved in storage lipid biosynthesis. The elucidation of the metabolic network of this important crop plant reinforces the relevance of the application of this technique to the analysis of complex plant metabolic systems.

The effect of Pimpinella saxifraga essential oil (PSEO) addition (1-3%) in sodium alginate coating on the bacterial and oxidative stability of cheese was studied during refrigerated storage. The GC-HRMS analysis of PSEO showed that anethole, pseudoisoeugenol and p-anisaldehyde were the main components. The PSEO exhibited strong in vitro DPPH radical scavenging activity (IC50 = 6.81 mu g/mL), beta-carotene bleaching inhibition (IC50 = 206 mu g/mL), ferric reducing power (EC50 = 35.20 mu g/mL), total antioxidant activity (213.96 +/- 11.12 mu mol/mL alpha-tocopherol equivalent) and notable DNA protection potential. Additionally, PSEO displayed potent antibacterial activity against 3 Gram-positive and 3 Gram-negative bacteria (MICs = 0.78-3.12 mg/mL). The acute toxicity of PSEO was determined using mice model (LD50 = 976.2 mg/kg). The enrichment of sodium alginate coating with PSEO, particularly at 3%, improved cheese preservation by reducing the weight loss, preserving the pH and color and enhancing oxidative and bacterial stability without unpleased flavor for consumers.

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography-electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds.

Cyclopropane fatty acids, which can be simply converted to methylated fatty acids, are good unusual fatty acid candidates for long-term resistance to oxidization and low-temperature fluidity useful for oleochemistry and biofuels. Cyclopropane fatty acids are present in low amounts in plants or bacteria. In order to develop a process for large-scale biolipid production, we expressed 10 cyclopropane fatty acid synthases from various organisms in the oleaginous yeast Yarrowia lipolytica, a model yeast for lipid metabolism and naturally capable of producing large amounts of lipids. The Escherichia coli cyclopropane fatty acid synthase expression in Y. lipolytica allows the production of two classes of cyclopropane fatty acids, a C17:0 cyclopropanated form and a C19:0 cyclopropanated form, whereas others produce only the C17:0 form. Expression optimization and fed-batch fermentation set-up enable us to reach a specific productivity of 0.032 g·L-1 ·hr-1 with a genetically modified strain containing cyclopropane fatty acid up to 45% of the total lipid content corresponding to a titre of 2.3 ± 0.2 g/L and a yield of 56.2 ± 4.4 mg/g.

RationaleWhile the GC-Orbitrap, marketed in 2015, represents a technological breakthrough in terms of sensitivity, resolution and mass stability, many studies have reported ion ratio modification in mass spectra using the standard 70 eV electron ionisation.MethodsWe studied the influence of the acquisition and sample parameters leading to these modifications on fatty acid methyl esters (FAMEs).ResultsFAMEs showed that these variations in relative intensities of ions were related to the acquisition parameters such as the mass range and the offset values of the C-TRAP, but also directly related to the column concentration of the sample, and especially that it was molecule-dependent. Advantageously, it is possible to use this feature to promote the molecular ions of FAMEs sometimes not present in a spectrum under electron ionisation at 70 eV.ConclusionsThe 70 eV electron ionisation mass spectra from the GC-Orbitrap were clearly molecule-dependent and could be due to metastable ions during storage states in the C-TRAP.

BackgroundThe mucilage is a model to study the polysaccharide biosynthesis since it is produced in large amounts and composed of complex polymers. In addition, it is of great economic interest for its technical and nutritional value. A fast method for phenotyping the released mucilage and the seed morphometric parameters will be useful for fundamental, food, pharmaceutical and breeding researches. Current strategies to phenotype soluble mucilage are restricted to visual evaluations or are highly time-consuming.ResultsHere, we developed a high-throughput phenotyping method for the simultaneous measurement of the soluble mucilage content released on a gel and the seed morphometric parameters. Within this context, we combined a biochemical assay and an open-source computer-aided image analysis tool, MuSeeQ. The biochemical assay consists in sowing seeds on an agarose medium containing the dye toluidine blue O, which specifically stains the mucilage once it is released on the gel. The second part of MuSeeQ is a macro developed in ImageJ allowing to quickly extract and analyse 11 morphometric data of seeds and their respective released mucilages. As an example, MuSeeQ was applied on a flax recombinant inbred lines population (previously screened for fatty acids content.) and revealed significant correlations between the soluble mucilage shape and the concentration of some fatty acids, e.g. C16:0 and C18:2. Other fatty acids were also found to correlate with the seed shape parameters, e.g. C18:0 and C18:2. MuSeeQ was then showed to be used for the analysis of other myxospermous species, including Arabidopsis thaliana and Camelina sativa.ConclusionsMuSeeQ is a low-cost and user-friendly method which may be used by breeders and researchers for phenotyping simultaneously seeds of specific cultivars, natural variants or mutants and their respective soluble mucilage area released on a gel. The script of MuSeeQ and video tutorials are freely available at http://MuSeeQ.free.fr.

Fatty acid methyl esters (FAMES), which are commonly used to characterize lipids, have several limitations to conclude on many structures. 3-Pyridylcarbinol esters (3-PCE) are used to characterize fatty acid structures [1], in particular, to identify ring and double bond positions on the carbon chain. Chromatographic separation of these esters is complex due to their polarity and high boiling points. In this study, we used a column with high resolutive power based on ionic liquids to increase the separation quality in gas chromatography (GC). In addition, we used a high-resolution detector (Orbitrap) to limit non-specific signals and improve the detection limits. This detector could be used with a mass filter at 5 ppm for the rapid determination of 3-PCE from its characteristic ions (m/z = 108.0441 and 92.0495). This filter allowed the identification of derivative fatty acids with good sensibility. Thus, it was possible to characterize 3-PCE by measuring the exact fragment masses to confirm structures such as C19:2n12cyclo Delta 9. (C) 2018 Elsevier B.V. All rights reserved.

In the context of the growing demand for α-linolenic acid due to its high nutritional value as a polyunsaturated fatty acid, we have investigated the contribution of 2-lysophosphatidic acid acyltransferase (LPAAT) enzymes from flax (Linum usitatissimum) in the accumulation of α-linolenic acid into the oil fraction of flax seed. We have isolated the cDNAs encoding three class A microsomal LPAAT2 isoforms from developing flax seeds. The three isoforms, denominated LPAAT2A, LPAAT2A2 and LPAAT2B, are able to complement the LPAAT deficient JC201 E. coli mutant, confirming their functionality. We have performed enzymatic assays showing that the specific activity of the LPAAT2A isoform is significantly higher than that of the LPAAT2A2 and LPAAT2B toward the unsaturated oleic, linoleic and linolenic acids. Moreover, LPAAT2A presents in vitro a high specificity and selectivity for linoleic and linolenic acids as compared to saturated fatty acids. The three isoforms are expressed during all the stages of seed development and in stem and leaf tissues, as shown by an analysis of the transcription level of the corresponding genes. The heterologous expression of LPAAT2A in Arabidopsis seeds leads to an increase in the accumulation of linoleic and linolenic acids in the oil fraction of the seeds from two transgenic lines.

In context of fluxomic studies, 13C labeling analysis of amino acids are very important for solving the carbon flow calculation, because they are synthesized in various biosynthesis pathways and cellular compartments in plant cells. Traditionally, 13C labeling analysis are performed using low resolution mass spectrometry detector by GC-MS. We compared a method using capillary electrophoresis-high resolution mass spectrometry without derivatization and with better accuracy assessment of labeling measurements comparing to classical GC-MS. Our method allowed us to show that valine, leucine, alanine are not synthesized from the same pyruvate pool during the period of reserves accumulation in flax seeds.