Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds

Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.     

Triacylglycerol content,productivity and fatty acid profile in Scenedesmus acutus PVUW12

A detailed lipid characterization of Scenedesmus acutus PVUW12, with emphasis on the evaluation of triacylglycerols (TAGs) as a biodiesel feedstock, is presented. When algal cells were grown in nitrogen-free medium (N stress), a lipid increase was detected that was mainly due to TAG accumulation. In situ fluorescence measurements allowed the kinetics and extent of neutral lipid accumulation to be followed. Under N stress, the productivity of total lipids and TAGs increased significantly (80.99 and 63.74 mg L^?1 day^?1, respectively) compared with controls (29.51 and 16.23 mg L^?1 day^?1, respectively). Monounsaturated fatty acids were the major fraction and increased further (49.74 %) in stressed cells, with oleic acid as the most abundant compound (46.97 %). The polyunsaturated fatty acid composition of this algal oil appears to meet the European Standard EN 14214. These results indicate that S. acutus oil meets the requirements for its use as a biodiesel feedstock. Since this strain was also proposed for wastewater bioremediation, this opens up the possibility of its use in an integrated system combined with biofuel production.     

Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars

Oil bodies were purified from mature seed of two Brassica napus crop cultivars, Reston and Westar. Purified oil body proteins were subjected to both 2-DE followed by LC-MS/MS and multidimensional protein identification technology. Besides previously known oil body proteins oleosin, putative embryo specific protein ATS1, (similar to caleosin), and 11-beta-hydroxysteroid dehydrogenase-like protein (steroleosin), several new proteins were identified in this study. One of the identified proteins, a short chain dehydrogenase/reductase, is similar to a triacylglycerol-associated factor from narrow-leafed lupin while the other, a protein annotated as a myrosinase associated protein, shows high similarity to the lipase/hydrolase family of enzymes with GDSL-motifs. These similarities suggest these two proteins could be involved in oil body degradation. Detailed analysis of the two other oil body components, polar lipids (lipid monolayer) and neutral lipids (triacylglycerol matrix) was also performed. Major differences were observed in the fatty acid composition of polar lipid fractions between the two B. napus cultivars. Neutral lipid composition confirmed erucic acid and oleic acid accumulation in Reston and Westar seed oil, respectively.     

Evaluating the usability of 19 effluents for heterotrophic cultivation of microalgal consortia as biodiesel feedstock

A key reason inhibiting commercialization of algal oil as biodiesel feedstock, is cultivation cost. For this reason, the usability of 19 readily available industrial effluents (autoclaved and non-autoclaved) to support heterotrophic growth and lipid accumulation was evaluated using six mixed algal cultures. Autoclaved whey effluent was the best with 14.32 g biomass L^?1, 13.23% lipids, resulting in a lipid production of 1.91 g lipids L^?1. Biomass production and lipid accumulation were in many cases inverse, e.g. mixed algal culture termed TUT4 accumulating 84.25% lipids on autoclaved acid mine drainage, with very little biomass produced. Biomass production was dependent on the effluent type, whereas the lipid accumulation was influenced mostly by the specific mixed algal cultures. The fatty acid composition of the algal oil (fish cannery and whey effluents) showed high saturation, leading to acceptable cetane numbers, kinematic viscosity, good oxidative stability, but poor cold flow properties.     

Fatty acid composition of the seed lipids of Chamaerops humilis L. natural populations and its relation with the environment

Seed lipids composition is a tool to discriminate among plant taxa and is related to phylogeny and biogeographic distribution. Aim of the present study was to evaluate the fatty acid (FA) composition of the seed lipids from nine natural Chamaerops humilis L. population and its relation with the climatic traits of the collection sites. The average seed lipids content was 54.8 g kg^? 1 and the most represented FAs were oleic (478 g kg^? 1 oil) and linoleic (230 g kg^? 1 oil), with significant differences among the accessions. Most of the lipid traits significantly correlated with climatic traits. In particular, oleic acid negatively correlated with year potential evapotranspiration. These relations could emerge from a genotypic adaptation to the environment. The seed of C. humilis showed high content of lipids, which implies an importance of the species for feeding wild life during winter, and a high degree of unsaturation. Considering the importance of the unsaturated FAs in human and animal nutrition, the present results suggest that European fan palm could be introduced in breeding programmes and its seed extract used as FA in diet supplementation. Further studies are needed to identify the content of anti-nutritional or nutraceutical compounds.     

The synthesis and accumulation of stearidonic acid in transgenic plants: a novel source of 'heart-healthy' omega-3 fatty acids

Dietary omega-3 polyunsaturated fatty acids have a proven role in reducing the risk of cardiovascular disease and precursor disease states such as metabolic syndrome. Although most studies have focussed on the predominant omega-3 fatty acids found in fish oils (eicosapentaenoic acid and docosahexaenoic acid), recent evidence suggests similar health benefits from their common precursor, stearidonic acid. Stearidonic acid is a Δ6-unsaturated C18 omega-3 fatty acid present in a few plant species (mainly the Boraginaceae and Primulaceae ) reflecting the general absence of Δ6-desaturation from higher plants. Using a Δ6-desaturase from Primula vialii , we generated transgenic Arabidopsis and linseed lines accumulating stearidonic acid in their seed lipids. Significantly, the P. vialii Δ6-desaturase specifically only utilises α-linolenic acid as a substrate, resulting in the accumulation of stearidonic acid but not omega-6 γ-linolenic acid. Detailed lipid analysis revealed the accumulation of stearidonic acid in neutral lipids such as triacylglycerol but an absence from the acyl-CoA pool. In the case of linseed, the achieved levels of stearidonic acid (13.4% of triacylglycerols) are very similar to those found in the sole natural commercial plant source ( Echium spp.) or transgenic soybean oil. However, both those latter oils contain γ-linolenic acid, which is not normally present in fish oils and considered undesirable for heart-healthy applications. By contrast, the stearidonic acid-enriched linseed oil is essentially devoid of this fatty acid. Moreover, the overall omega-3/omega-6 ratio for this modified linseed oil is also significantly higher. Thus, this nutritionally enhanced linseed oil may have superior health-beneficial properties.     

Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20–30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20–30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.     

From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula

? The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. ? We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. ? Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. ? Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.     

EVALUATION OF BIOTECHNOLOGICAL POTENTIALS OF SOME INDUSTRIAL FUNGI IN ECONOMICAL LIPID ACCUMULATION AND BIOFUEL PRODUCTION AS A FIELD OF USE

Considering the vast number of scientific reports on various potential uses of fungi, there was an attempt to select the best lipid producer of some fungi at optimized conditions (Aspergillus versicolor, Rhizopus oryzae, Rhizopus arrhizus, Tramates versicolor). The aim was to offer new fields of use to the industries already culturing and using such materials. Aspergillus versicolor mycelia were found to be accumulating the highest amount of lipids. Experiments to improve lipid accumulation and transesterification properties were performed in molasses medium; the first steps were testing the effects of different pH values and different nitrogen sources on lipid accumulation. Various concentrations of KNO₃ (0.5, 1.0, 1.5 gL^?1) and molasses (6%, 8%, 10%) were tried in order to find the optimum carbon and nitrogen requirements. Maximum lipid content was 22.8% in the samples containing 6% molasses solution and 1.0 gL^?1 KNO₃ at pH 4 after 10 days of incubation. The highest fatty acid ethyl ester yield of these samples was 77% (5.0 ethanol:oil, 0.4 sulfuric acid:oil at 30°C for 6 hr). Since the crude lipids were rich in C16 and C18 fatty acids, this was considered as suitable feedstock for biodiesel production.     

Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans

It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues.     

Effect of pH on neutral lipid and biomass accumulation in microalgal strains native to the Canadian prairies and the Athabasca oil sands

Algal biodiesel has been a subject of growing importance in the realm of renewable energy due to carbon capture properties and its potential for photosynthetic efficiency with high lipid output. This study identified five isolates of freshwater green algae, belonging to the Chlorellaceae, and measured the lipid classes and fatty acid profiles of these species to determine suitability for biodiesel production. To induce the greater accumulation of lipids, especially in the form of triacylglycerols (TAGs) desired for biodiesel, we examined the lipid accumulation in cells stressed by nitrogen limitation, sulfur deficiency, or pH stress. Increases in biomass were monitored in order to determine if adjusting pH incrementally over the course of the experiment had any effect on growth and lipid accumulation of several isolates. TAG accumulation was visually screened by Nile Red fluorescence and further assessed by gas chromatography. Lipid amounts were comparably equal or better for pH stress treatments than for standard nutrient-deprivation treatments. Incrementally adjusted pH over the course of growth triggered lipid accumulation comparable to constant pH stress treatments, yet biomass accumulation was equivalent to unstressed growth. One isolate obtained from the Athabasca oil-sands region of Alberta, OS4-2, is a good candidate for biodiesel production, having accumulated over 45 % of its dry weight as lipid, with over 80 % of the lipid as triacylglycerols, and contains an abundance of 18:1 fatty acids. This class of fatty acids improves the cold flow and oxidative stability of biodiesel and is ideal for biofuel used in a Canadian climate.     

Bottlenecks in erucic acid accumulation in genetically engineered ultrahigh erucic acid Crambe abyssinica

Erucic acid is a valuable industrial fatty acid with many applications. The main producers of this acid are today high erucic rapeseed (Brassica napus) and mustard (Brassica juncea), which have 45%–50% of erucic acid in their seed oils. Crambe abyssinica is an alternative promising producer of this acid as it has 55%–60% of erucic acid in its oil. Through genetic modification (GM) of three genes, we have previously increased the level of erucic acid to 71% (68 mol%) in Crambe seed oil. In this study, we further investigated different aspects of oil biosynthesis in the developing GM Crambe seeds in comparison with wild‐type (Wt) Crambe, rapeseed and safflower (Carthamus tinctorius). We show that Crambe seeds have very low phosphatidylcholine‐diacylglycerol interconversion, suggesting it to be the main reason why erucic acid is limited in the membrane lipids during oil biosynthesis. We further show that GM Crambe seeds have slower seed development than Wt, accompanied by slower oil accumulation during the first 20 days after flowering (DAF). Despite low accumulation of erucic acid during early stages of GM seed development, nearly 86 mol% of all fatty acids accumulated between 27 and 50 DAF was erucic acid, when 40% of the total oil is laid down. Likely bottlenecks in the accumulation of erucic acid during early stages of GM Crambe seed development are discussed.     

Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast

Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [ Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.     

Changes in lipid status and glass properties in cotyledons of developing sunflower seeds

Biochemical events involved in the acquisition of germinability and storability during orthodox seed development are well documented; however, the roles played by the physical organization of lipids and water are poorly characterized. The aim of this work was to determine, using a thermodynamic approach, whether changes in thermal properties of lipid reserves, and intracellular glasses might play a role in sunflower (Helianthus annuus L.) seed development. Triacyglycerols (TAGs) accumulated in cotyledons until the end of seed filling, which occurred 42 days after anthesis (DAA). Further seed development, leading to mature seed at 58 DAA, was mainly associated with an enlargement of lipid bodies without significant changes either in the lipid content or in their composition. When cooled to -100 degrees C, lipid reserves from cotyledons of mature seeds displayed alpha and beta' polymorphic crystalline structures; however, the ability to form alpha crystals, which was an indicator of lipid purity, progressively appeared during seed development. Characteristics of lipid melting confirmed that seed maturation drying was associated with changes in TAG physical organization. Cotyledon development was associated with an increase in the temperature of glass to rubber transition (Tg), thus suggesting a decrease in molecular mobility during maturation drying. This phenomenon was concomitant with an increase in raffinose content. Our results demonstrate that physical characteristics of lipid reserves and glasses of sunflower cotyledons are developmentally regulated and might play a role in acquisition of seed germinability and storability.     

Composition, accumulation and utilization of yolk lipids in teleost fish

Lipid reserves in teleost eggs are stored in lipoprotein yolk and, in some species, a discrete oil globule. Lipoprotein yolk lipids are primarily polar lipids, especially phosphatidylcholine and phosphatidylethanolamine (PE), and are rich in (n–3) polyunsaturated fatty acids, especially 22:6(n–3) (docosahexaenoic acid, DHA). Oil consists of neutral lipids and is rich in monounsaturated fatty acids (MUFA). Egg lipids are derived from dietary fatty acid, fatty acid mobilized from reserves and possibly fatty acid synthesized de novo. There is selective incorporation of essential fatty acids, particularly DHA, into yolk lipids and discrimination against incorporation of 22:1(n–11). Lipid is delivered to the oocyte by vitellogenin, which is rich in polar lipids, and likely also by other lipoproteins, especially very low density lipoprotein, which is rich in triacylglycerol (TAG). All classes of lipid may be used as fuel during embryonic and larval development and MUFA are preferred fatty acids for catabolism by embryos. Catabolism of oil globules is frequently delayed until latter stages of development. In some species, DHA derived from hydrolysis of phospholipid may be conserved by transfer to the neutral lipid. Recent work has expanded knowledge of the role of DHA in membrane structure, especially in neural tissue, and molecular species analysis has indicated that PE containing sn-1 oleic acid is a prime contributor to membrane fluidity. The results of this type of study provide an explanation for the selection pressures that influence yolk lipid composition. Future work ought to expand knowledge of specific roles of individual fatty acids in embryos along with knowledge of the ecological physiology of ovarian recrudescence, environmental influences on vitellogenin and yolk lipid composition, and the control of yolk lipid accumulation and utilization.     

Detection and imaging of lipids of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> based on confocal Raman microspectroscopy

In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm^?1) measured by Raman spectroscopy was analyzed. There was significant correlation (R ² = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content.     

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa

Main conclusion

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.