Degradation Of Storage Lipids Research Articles

Dynamics of macronutrients during short-term germination of flax seeds

Abstract: Germination is an environmentally friendly and convenient approach to enhancing the biochemical potential of food plant raw materials. The nutritional value of raw materials and the functional properties of protein contained therein can be significantly improved by activatying the inherent enzyme system. Bioactivation not only increases the amount of water-soluble protein fractions, but also promotes accumulation of free amino and fatty acids and easily soluble reducing sugars. We used flax seeds as a source of essential polyunsaturated fatty acids, dietary fibres, complete protein, polypeptides and lignans to support the most important physiological functions of the human body. The aim was to study the dynamics of macronutrients in the process of short-term germination of flax seeds by chemical and spectroscopic methods. Flax seeds were germinated in laboratory conditions in special trays at a temperature of 18–20 ºС for 5 days with periodic moistening. Visual changes occurring in flax seeds at all stages of short-term germination are demonstrated. The periodicity of changes in the main macronutrients of flax seeds is shown. It is concluded that, during the studied period of germination, the principal hydrolytic decomposition of seed storage proteins was incomplete, leading to a permanent decrease, first of all, in the content of proteins and protein nitrogen. Based on the changes in the lipid content and acid number, the intensity of the peaks associated with functional groups in the lipid region, in particular, the band at 1748 cm-1 assigned to stretching vibrations of C=Ogroups of fatty acids, no degradation of storage lipids at an early stage of germination was assumed. The accumulation of soluble substances during the studied germination period is demonstrated, including watersoluble protein compounds, as well as water-soluble polysaccharides and products of their hydrolytic degradation. Sprouted flax seeds are a valuable ingredient for producing healthy foods.

Biomodification of fats and oils and scenarios of adding value on renewable fatty materials through microbial fermentations: Modelling and trials with Yarrowia lipolytica

The ex novo lipid accumulation process in the yeast Yarrowia lipolytica was systematically studied in the current investigation. Various fatty materials of plant (i.e. olive, sunflower, palm, linseed oils) or animal (i.e. cod liver oil, beef tallow) origin, the fatty acid (FA) composition of which resembled in several cases to the one of important low- or negative-cost hydrophobic resources (i.e. stickwaters, fat-rich wastes from meat-processing facilities, used-cooked oils, etc) were employed as substrates and a detailed mathematical model which included description of substrate fat hydrolysis, biomass and reserve lipid formation and cellular lipid turnover was developed. The kinetic parameter values were calculated by fitting the model on experimental data and used to estimate the ability of the yeast to grow on the various substrate fats and modify their FA composition. With few exceptions the substrate fat was efficiently converted into yeast cells (biomass) rich in lipids consisting mainly of neutral lipids (often >90% on the total lipids), indicating, therefore the potential of the yeast Y. lipolytica as cell factory amenable to ferment several types of low-cost fatty materials. The contribution of each substrate fatty acid (FA) to the synthesis of fat free biomass varied according to the theoretical FA uptake rate and typically was higher for palmitic, oleic and linoleic acid than the remaining FAs. Eicosapentaenoic and docosohexaenoic acids, typically found in several stickwaters or crude fish oils, were also rapidly taken up when cod liver oil was used as substrate. Respectively, the contribution of the substrate FAs to the synthesis of storage lipid was more important for linoleic and palmitic acids. Higher proportions of unsaturated FAs were esterified in phospholipid fraction. Upon exhaustion of the substrate fat, storage lipid degradation occurred. Unsaturated (primarily monounsaturated) FA uptake rates were higher than those of saturated FAs and therefore storage lipid was enriched in saturated FAs during lipid turnover. Storage lipid degradation can be successfully repressed (or even its biosynthesis can be restored) with the addition of a carbon source (e.g. glycerol, glucose, olive oil) in the culture medium during lipid degradation. We concluded that the mathematical approach proposed in this paper can constitute an effective tool for better understanding several key biochemical events, including substrate fat modification, during growth of Y. lipolytica on common oils. Through the proposed approach, low cost fatty materials such as stickwaters, fat-rich wastewaters from meat-processing facilities, used-cooked oils, soap stocks, stearins, etc., can be efficiently converted in higher-value cellular lipids by Y. lipolytica through completely eco-friendly and green processes.

The deterioration of Moringa oleifera Lam. seeds in the course of storage involves reserve degradation

Seed deterioration in the course of storage may involve hydrolytic reactions. Hence, we aimed to evaluate viability, vigour, contents of reserves and metabolites, and activities of hydrolytic enzymes in Moringa oleifera Lam. seeds during storage under controlled conditions. Seeds were packaged in semipermeable plastic and maintained in a growth chamber (27 ± 2 °C and RH 60–65%) and under refrigeration (4 ± 2 °C and RH 20–25%) for 18 months. Samples were taken at the start of the experiment and every 3 months. During the first 12 months, water content, viability, and vigour remained almost unaffected, while the content of neutral lipids, starch, soluble sugars and free amino acids did not reduce in the seeds kept under refrigeration. After this period, the loss of viability and vigour was accompanied by the degradation of storage lipids, storage proteins, and non-reducing sugars associated with the increase of lipase and acid protease activity in both environmental conditions. As the seed water content remained below 8% in the course of the experiment, we suggest that non-enzymatic hydrolysis might play a role in the deterioration of M. oleifera seeds during storage. At least for planting, we recommend that M. oleifera seeds be kept at low relative humidity under refrigeration for up to 12 months.

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

BackgroundAcetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts.ResultsIn order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media.ConclusionOur results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.

Regulation of reserve mobilisation in sunflower during late seedling establishment in continuous darkness.

Reserve mobilisation, metabolite partitioning and reserve-degrading enzyme activity were studied in sunflower seedlings cultivated invitro under a 12-h photoperiod or in the dark to investigate the involvement of source-sink relation and carbon starvation in the regulation of reserve mobilisation under continuous darkness. Reserves, metabolites and enzyme activity were determined with standard spectrophotometric methods. At the first 24h of treatment (acclimation phase), darkness did not affect growth, but restricted carbon and nitrogen use, as indicated by sugar and amino acid accumulation in the different seedling parts. After 5days of treatment (survival phase), extended darkness limited growth and retarded storage lipid mobilisation due to carbon starvation, as evidenced by the depletion of carbohydrates in cotyledons and hypocotyl, as well as the consumption of amino acids in hypocotyls and roots. Alterations in the source-sink relationship might have been a response to prolonged darkness, instead of a mechanism used to regulate reserve mobilisation, as these alterations cannot be associated with negative feedback mediated by metabolite accumulation. Storage lipid degradation depends, at least in part, on mechanisms that co-ordinately regulate the activities of lipases and isocitrate lyase. Taking these results together, it is possible that reserve mobilisation in sunflower seedlings cultivated in the dark might be regulated by mechanisms that perceive the absence of light and predict carbon starvation, adjusting reserve use according to future energy demands to allow, at least in the short term, seedling survival.

Use of a lipid rich strain reveals mechanisms of nitrogen limitation and carbon partitioning in the haptophyte Tisochrysis lutea

Haptophytes are a diverse monophyletic group with a worldwide distribution, known to be significantly involved in global climate regulation in their role as a carbon sink. Because nitrogen is a major limiting macronutrient for phytoplankton in oceans and for cultures of microalgae, understanding the involvement of nitrogen availability in haptophyte carbon partitioning is of global and biotechnological importance. Here, we made an ecophysiological study coupled with comprehensive large scale proteomic analysis to examine differences of behavior in reaction to nitrogen availability changes between a wild type strain of Tisochrysis lutea (WTc1) and a mutant strain (2Xc1) known to accumulate more storage lipids. Strains were grown in chemostats and studied under different ecophysiological conditions including N limitation, N repletion and N depletion. Whereas short time N repletion triggered consumption of carbohydrates in both strains, storage lipid degradation and accumulation during changes of ecophysiological status were recorded in 2Xc1 but not in WTc1. After 3months of continuous culture, 2Xc1 exhibited an unexpected increase of carbon sequestration ability (+50%) by producing twofold more carbohydrates for the same nitrogen availability. Deep proteomic analysis by LC-MS/MS identified and compared the abundance of 4332 proteins, i.e. the deepest coverage of a microalgal proteome obtained to date. Results revealed that storage lipid accumulation is favored by an overall reorganization of carbon partitioning in 2Xc1 cells that increases the metabolism of carbon and energy acquisition, and decreases mitochondrial activity and metabolic conversion of storage lipids to phosphoenolpyruvate before gluconeogenesis.

Identification, duplication, evolution and expression analyses of caleosins in Brassica plants and Arabidopsis subspecies.

Caleosins are a class of Ca(2+) binding proteins that appear to be ubiquitous in plants. Some of the main proteins embedded in the lipid monolayer of lipid droplets, caleosins, play critical roles in the degradation of storage lipids during germination and in lipid trafficking. Some of them have been shown to have histidine-dependent peroxygenase activity, which is believed to participate in stress responses in Arabidopsis. In the model plant Arabidopsis thaliana, caleosins have been examined extensively. However, little is known on a genome-wide scale about these proteins in other members of the Brassicaceae. In this study, 51 caleosins in Brassica plants and Arabidopsis lyrata were investigated and analyzed in silico. Among them, 31 caleosins, including 7 in A. lyrata, 11 in Brassica oleracea and 13 in Brassica napus, are herein identified for the first time. Segmental duplication was the main form of gene expansion. Alignment, motif and phylogenetic analyses showed that Brassica caleosins belong to either the H-family or the L-family with different motif structures and physicochemical properties. Our findings strongly suggest that L-caleosins are evolved from H-caleosins. Predicted phosphorylation sites were differentially conserved in H-caleosin and L-caleosins, respectively. 'RY-repeat' elements and phytohormone-related cis-elements were identified in different caleosins, which suggest diverse physiological functions. Gene structure analysis indicated that most caleosins (38 out of 44) contained six exons and five introns and their intron phases were highly conserved. Structurally integrated caleosins, such as BrCLO3-3 and BrCLO4-2, showed high expression levels and may have important roles. Some caleosins, such as BrCLO2 and BoCLO8-2, lost motifs of the calcium binding domain, proline knot, potential phosphorylation sites and haem-binding sites. Combined with their low expression, it is suggested that these caleosins may have lost function.

OsLOX2, a rice type I lipoxygenase, confers opposite effects on seed germination and longevity.

Rice production and seed storage are confronted with grain deterioration and loss of seed viability. Some members of the lipoxygenase (LOX) family function in degradation of storage lipids during the seed germination, but little is known about their influence on seed longevity during storage. We characterized the role of rice OsLOX2 gene in seed germination and longevity via over-expression and knock-down approaches. Abundant expression of OsLOX2 was detected in panicles, roots, and stems, but not in leaves. Moreover, OsLOX2 was highly induced during germination. OsLOX2 protein, located in the cytoplasm, showed a wide range of temperature adaptation (20-50°C) and a substrate preference to linoleic acid. Lines over-expressing OsLOX2 showed accelerated seed germination under normal condition and lower seed viability after accelerated aging. RNA interference (RNAi) of OsLOX2 caused delayed germination and enhanced seed longevity. RNAi lines with strongly repressed OsLOX2 activity completely lost the capability of germination after accelerated aging. More lipid hydroperoxide were found in OE15 than the control, but less in RNAi lines than in the WT Nipponbare. Therefore, OsLOX2 acts in opposite directions during seed germination and longevity during storage. Appropriate repression of the OsLOX2 gene may delay the aging process during the storage without compromising germination under normal conditions.

Degradation of lipoxygenase-derived oxylipins by glyoxysomes from sunflower and cucumber cotyledons

BackgroundOilseed germination is characterized by the degradation of storage lipids. It may proceed either via the direct action of a triacylglycerol lipase, or in certain plant species via a specific lipid body 13-lipoxygenase. For the involvement of a lipoxygenase previous results suggested that the hydroxy- or oxo-group that is being introduced into the fatty acid backbone by this lipoxygenase forms a barrier to continuous β-oxidation.ResultsThis study shows however that a complete degradation of oxygenated fatty acids is possible by isolated cucumber and sunflower glyoxysomes. Interestingly, degradation is accompanied by the formation of saturated short chain acyl-CoAs with chain length between 4 and 12 carbon atoms lacking the hydroxy- or oxo-diene system of the oxygenated fatty acid substrate. The presence of these CoA esters suggests the involvement of a specific reduction of the diene system at a chain length of 12 carbon atoms including conversion of the hydroxy-group at C7.ConclusionsTo our knowledge this metabolic pathway has not been described for the degradation of polyunsaturated fatty acids so far. It may represent a new principle to degrade oxygenated fatty acid derivatives formed by lipoxygenases or chemical oxidation initiated by reactive oxygen species.

Comparative Proteomic Analysis of Streptomyces lividans Wild-Type andppkMutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

Effect of Carbon and Nitrogen Availability on Metabolism of Amino Acids in Germinating Spores of Arbuscular Mycorrhizal Fungi

The effects of carbon (C) and nitrogen (N) sources on N utilization and biosynthesis of amino acids were examined in the germinating spores of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith after exposure to various N substrates, CO 2, glucose, and/or root exudates. The N uptake and de novo biosynthesis of amino acids were analyzed using stable isotopic labeling with mass spectrometric detection. High-performance liquid chromatography-based analysis was used to measure amino acid levels. In the absence of exogenous N sources and in the presence of 25 mL L −1 CO 2, the germinating AM fungal spores utilized internal N storage as well as C skeletons derived from the degradation of storage lipids to biosynthesize the free amino acids, in which serine and glycine were produced predominantly. The concentrations of internal amino acids increased gradually as the germination time increased from 0 to 1 or 2 weeks. However, asparagine and glutamine declined to the low levels; both degraded to provide the biosynthesis of other amino acids with C and N donors. The availability of exogenous inorganic N (ammonium and nitrate) and organic N (urea, arginine, and glutamine) to the AM fungal spores using only CO 2 for germination generated more than 5 times more internal free amino acids than those in the absence of exogenous N. A supply of exogenous nitrate to the AM fungal spores with only CO 2 gave rise to more than 10 times more asparagine than that without exogenous N. In contrast, the extra supply of exogenous glucose to the AM fungal spores generated a significant enhancement in the uptake of exogenous N sources, with more than 3 times more free amino acids being produced than those supplied with only exogenous CO 2. Meanwhile, arginine was the most abundant free amino acid produced and it was incorporated into the proteins of AM fungal spores to serve as an N storage compound.

Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) catalyzes oxygenation of polyenoic fatty acids, which precedes the degradation of storage lipids during seed germination in sunflower. In the present work, it has been confirmed that 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) produces fluorescence in presence of lipid hydroperoxides (LOX reaction products). This work provides new information on spatial localization of transiently enhanced LOX activity in protoplasts from 5d old seedling cotyledons of sunflower (Helianthus annuus L. cv. Morden) by exploiting H(2)DCFDA as a probe for fluorescence detection from LOX activity sites. Use of LOX inhibitors [nordihydroguaiaretic acid (NDGA) and propyl gallate (PG)] confirms oil bodies as LOX activity sites. Oil body surface has been shown to possess LOX activity in 5d old seedling cotyledons.

Autophagy-related lipase FgATG15 of Fusarium graminearum is important for lipid turnover and plant infection

Autophagy is a non-selective degradation pathway in eukaryotic cells that is conserved from yeasts to humans. Autophagy is involved in the virulence of several pathogenic fungi such as Magnaporthe grisea or Colletotrichum orbiculare. In the current study, we identified and disrupted an autophagy-like lipase FgATG15 in Fusarium graminearum. We showed that FgATG15 exhibits lipase activity when heterologously expressed in P. pastoris. We used a gene deletion approach to characterize the function of the enzyme. We demonstrate that FgATG15 is involved in fungal growth and aerial hyphae production. FgATG15 is also involved in conidia production and germination, and disruption of FgATG15 led to aberrant conidia shapes. FgATG15 disruptants were reduced in storage lipid degradation under starvation conditions, implicating FgATG15’s involvement in lipid turnover. Moreover, wheat head infection by the disruptants was severely attenuated, indicating the involvement of FgATG15 in pathogenesis. Additionally, we found that the deoxynivalenol levels of FgATG15 disruptants were significantly decreased compared with the wild type strain. Taken together, we show that FgATG15 is involved in numerous developmental processes and could be exploited as an antifungal target.

The lipoxygenase-dependent oxygenation of lipid body membranes is promoted by a patatin-type phospholipase in cucumber cotyledons.

Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal β-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.

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.

Proteomic Identification and Characterization of a Novel Peroxisomal Adenine Nucleotide Transporter Supplying ATP for Fatty Acid β-Oxidation in Soybean and Arabidopsis

We have identified the novel protein Glycine max PEROXISOMAL ADENINE NUCLEOTIDE CARRIER (Gm PNC1) by proteomic analyses of peroxisomal membrane proteins using a blue native/SDS-PAGE technique combined with peptide mass fingerprinting. Gm PNC1, and the Arabidopsis thaliana orthologs At PNC1 and At PNC2, were targeted to peroxisomes. Functional integration of Gm PNC1 and At PNC2 into the cytoplasmic membranes of intact Escherichia coli cells revealed ATP and ADP import activities. The amount of Gm PNC1 in cotyledons increased until 5 d after germination under constant darkness and then decreased very rapidly in response to illumination. We investigated the physiological functions of PNC1 in peroxisomal metabolism by analyzing a transgenic Arabidopsis plant in which At PNC1 and At PNC2 expression was suppressed using RNA interference. The pnc1/2i mutant required sucrose for germination and suppressed the degradation of storage lipids during postgerminative growth. These results suggest that PNC1 contributes to the transport of adenine nucleotides that are consumed by reactions that generate acyl-CoA for peroxisomal fatty acid beta-oxidation during postgerminative growth.