Digalactosyldiacylglycerol

Rapid, Untargeted Chemical Profiling of Single Cells in Their Native Environment.[Pubmed:30955322]

Anal Chem. 2019 Apr 8.

We report a method that enables untargeted, high throughput and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture-mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into a LVC-MS sampling probe. Once exposed to an appropriate solvent the cell is lysed, extracted and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG) and Digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.

Galactolipids are essential for internal membrane transformation during etioplast-to-chloroplast differentiation.[Pubmed:30892620]

Plant Cell Physiol. 2019 Mar 20. pii: 5406926.

Etioplasts developed in angiosperm cotyledon cells in darkness rapidly differentiate into chloroplasts with illumination. This process involves dynamic transformation of internal membrane structures from the prolamellar bodies (PLBs) and prothylakoids (PTs) in etioplasts to thylakoid membranes in chloroplasts. Although two galactolipids, monogalactosyldiacylglycerol (MGDG) and Digalactosyldiacylglycerol (DGDG), are predominant lipid constituents of membranes in both etioplasts and chloroplasts, their roles in the structural and functional transformation of internal membranes during etioplast-to-chloroplast differentiation are unknown. We previously reported that a 36% loss of MGDG by an artificial microRNA targeting major MGDG synthase (amiR-MGD1) only slightly affected PLB structures but strongly impaired PT formation and protochlorophyllide biosynthesis. Meanwhile, strong DGDG deficiency in a DGDG synthase mutant (dgd1) disordered the PLB lattice structure in addition to impaired PT development and protochlorophyllide biosynthesis. In this study, thylakoid biogenesis after PLB disassembly with illumination was strongly perturbed by amiR-MGD1. The amiR-MGD1 expression impaired the accumulation of Chl and the major light-harvesting complex II protein (LHCB1), which may inhibit rapid transformation from disassembled PLBs to the thylakoid membrane. As did amiR-MGD1 expression, dgd1 mutation impaired the accumulation of Chl and LHCB1 during etioplast-to-chloroplast differentiation. Furthermore, unlike in amiR-MGD1 seedlings, in dgd1 seedlings, disassembly of PLBs after illumination was retarded. Because DGDG but not MGDG prefers to form the bilayer lipid phase in membranes, the MGDG-to-DGDG ratio may strongly affect the transformation of PLBs to the thylakoid membrane during etioplast-to-chloroplast differentiation.

Suicoccus acidiformans gen. nov., sp. nov., isolated from a sick pig.[Pubmed:30882297]

Int J Syst Evol Microbiol. 2019 Mar 18.

A Gram-stain-positive, non-spore-forming, catalase-positive and facultatively anaerobic coccus, designated ZY16052(T), was isolated from mesenteric lymph nodes of a sick piglet in Kunming, Yunnan Province, PR China and its taxonomic position was studied by following a polyphasic approach. Optimal growth was observed at 37 degrees C, pH 8.0 and 2 % NaCl (w/v) on Columbia agar. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY16052(T) formed a separated evolutionary lineage from recognized genera of the family Aerococcaceae and shared low similarity to its closest related species Facklamiasourekii (93.8 %) and Ignavigranum ruoffiae (93.4 %). Phylogenetic analysis based on the housekeeping gene recA indicated that strain ZY16052(T) represented a deep and distinct evolutionary lineage, and was well separated from all genera in the family Aerococcaceae, with very low sequence similarity(5 %) were C16 : 0, C18 : 1omega9c, C14 : 0 and summed feature 5 (C18 : 2omega6,9c and/or C18 : 0 ante). The major polar lipids were Digalactosyldiacylglycerol, phosphatidylglycerol, diacylglycerols, triacylglycerol and phosphatidic acid. The peptidoglycan contained the amino acids lysine, glycine, alanine and glutamic acid, which is characteristic of peptidoglycan type A1a. Based on the phylogenetic and phenotypic evidence, we propose that the unknown bacterium be classified as Suicoccus acidiformans gen. nov., sp. nov. The type strain of Suicoccus acidiformans is ZY16052(T) (=CCTCC AB 2017017(T)=DSM 105755(T)).

Modulation of Polar Lipid Profiles in Chlorella sp. in Response to Nutrient Limitation.[Pubmed:30823401]

Metabolites. 2019 Feb 28;9(3). pii: metabo9030039.

We evaluate the effects of nutrient limitation on cellular composition of polar lipid classes/species in Chlorella sp. using modern polar lipidomic profiling methods (liquid chromatography(-)tandem mass spectrometry; LC-MS/MS). Total polar lipid concentration was highest in nutrient-replete (HN) cultures with a significant reduction in monogalactosyldiacylglycerol (MGDG), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) class concentrations for nutrient-deplete (LN) cultures. Moreover, reductions in the abundance of MGDG relative to total polar lipids versus an increase in the relative abundance of Digalactosyldiacylglycerol (DGDG) were recorded in LN cultures. In HN cultures, polar lipid species composition remained relatively constant throughout culture with high degrees of unsaturation associated with acyl moieties. Conversely, in LN cultures lipid species composition shifted towards greater saturation of acyl moieties. Multivariate analyses revealed that changes in the abundance of a number of species contributed to the dissimilarity between LN and HN cultures but with dominant effects from certain species, e.g., reduction in MGDG 34:7 (18:3/16:4). Results demonstrate that Chlorella sp. significantly alters its polar lipidome in response to nutrient limitation, and this is discussed in terms of physiological significance and polar lipids production for applied microalgal production systems.

Measurement of Lipid Transport in Mitochondria by the MTL Complex.[Pubmed:30790250]

Methods Mol Biol. 2019;1949:69-93.

Membrane biogenesis requires an extensive traffic of lipids between different cell compartments. Two main pathways, the vesicular and non-vesicular pathways, are involved in such a process. Whereas the mechanisms involved in vesicular trafficking are well understood, fewer is known about non-vesicular lipid trafficking, particularly in plants. This pathway involves the direct exchange of lipids at membrane contact sites (MCSs) between organelles. In plants, an extensive traffic of the chloroplast-synthesized Digalactosyldiacylglycerol (DGDG) to mitochondria occurs during phosphate starvation. This lipid exchange occurs by non-vesicular trafficking pathways at MCSs between mitochondria and plastids. By a biochemical approach, a mitochondrial lipoprotein super-complex called MTL (Mitochondrial Transmembrane Lipoprotein complex) involved in mitochondria lipid trafficking has been identified in Arabidopsis thaliana. This protocol describes the method to isolate the MTL complex and to study the implication of a component of this complex (AtMic60) in mitochondria lipid trafficking.

Chloroplast Lipids and Their Biosynthesis.[Pubmed:30786236]

Annu Rev Plant Biol. 2019 Feb 20.

Chloroplasts contain high amounts of monogalactosyldiacylglycerol (MGDG) and Digalactosyldiacylglycerol (DGDG) and low levels of the anionic lipids sulfoquinovosyldiacylglycerol (SQDG), phosphatidylglycerol (PG), and glucuronosyldiacylglycerol (GlcADG). The mostly extraplastidial lipid phosphatidylcholine is found only in the outer envelope. Chloroplasts are the major site for fatty acid synthesis. In Arabidopsis, a certain proportion of glycerolipids is entirely synthesized in the chloroplast (prokaryotic lipids). Fatty acids are also exported to the endoplasmic reticulum and incorporated into lipids that are redistributed to the chloroplast (eukaryotic lipids). MGDG, DGDG, SQDG, and PG establish the thylakoid membranes and are integral constituents of the photosynthetic complexes. Phosphate deprivation induces phospholipid degradation accompanied by the increase in DGDG, SQDG, and GlcADG. During freezing and drought stress, envelope membranes are stabilized by the conversion of MGDG into oligogalactolipids. Senescence and chlorotic stress lead to lipid and chlorophyll degradation and the deposition of acyl and phytyl moieties as fatty acid phytyl esters. Expected final online publication date for the Annual Review of Plant Biology Volume 70 is April 29, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Interplay between Jasmonic Acid, Phosphate Signaling and the Regulation of Glycerolipid Homeostasis in Arabidopsis.[Pubmed:30753691]

Plant Cell Physiol. 2019 Feb 8. pii: 5310097.

Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of glycerolipid remodeling in response to Pi-deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and glycerolipid remodeling in wild type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine (PC), phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and Digalactosyldiacylglycerol (DGDG). JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi-deprivation, JA seems rather associated with a specific MGDG increase. Following Pi-resupply, whereas the expression of glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, i.e. downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.

Glycerolipid Composition of the Red Macroalga Agarophyton Chilensis and Comparison to the Closely Related Agarophyton Vermiculophyllum Producing Different Types of Eicosanoids.[Pubmed:30717350]

Mar Drugs. 2019 Feb 2;17(2). pii: md17020096.

The red macroalga Agarophyton chilensis is a well-known producer of eicosanoids such as hydroxyeicosatetraenoic acids, but the alga produces almost no prostaglandins, unlike the closely related A. vermiculophyllum. This indicates that the related two algae would have different enzyme systems or substrate composition. To carry out more in-depth discussions on the metabolic pathway of eicosanoids between the two algae, we investigated the characteristics of glycerolipids, which are the substrates of eicosanoids production, of A. chilensis and compared them to the reported values of A. vermiculophyllum. In A. chilensis, monogalactosyldiacylglycerol (MGDG), Digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylcholine (PC) were the major lipid classes and accounted for 44.4% of the total lipid extract. The predominant fatty acids were arachidonic acid (20:4n-6), an eicosanoids precursor, and palmitic acid (16:0). The 20:4n-6 content was extremely high in MGDG and PC (>70%), and the 16:0 content was extremely high in DGDG and SQDG (>40%). A chiral-phase HPLC analysis showed that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The glycerolipid molecular species were determined by reversed-phase HPLC(-)ESI(-)MS analysis. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (63.8%) and PC (48.2%), 20:4n-6/16:0 for DGDG (71.1%) and SQDG (29.4%). These lipid characteristics of A. chilensis were almost the same as those of A. vermiculophyllum. Hence, the differences of the eicosanoids producing ability between the two algae would not be due to the difference of substrate composition but the difference of enzyme system.

The effect of carotenoids on the concentration of singlet oxygen in lipid membranes.[Pubmed:30689980]

Biochim Biophys Acta Biomembr. 2019 Apr 1;1861(4):845-851.

An effect of beta-carotene and its polar derivative, zeaxanthin, on a concentration of singlet oxygen in lipid membranes was studied in a model system. The carotenoids were incorporated into the membranes of small unilamellar liposomes at a concentration of 0.15mol% with respect to lipid. Singlet oxygen was generated in a liposome suspension via photosensitization of toluidine blue, and its concentration in a membrane was detected with application of a specific fluorescence probe (singlet oxygen sensor green reagent) located in the lipid bilayer. The results show the carotenoid-dependent decrease in the concentration of singlet oxygen in the membranes formed with unsaturated lipids (egg yolk phosphatidylcholine and Digalactosyldiacylglycerol) but not in the case of the membranes formed with a saturated lipid (dimyristoylphosphatidylcholine). The effect of carotenoids was about twice as high as in the case of cholesterol present in liposomes at the same concentration. The results suggest that carotenoids protect membranes formed with unsaturated lipids against singlet oxygen through combined activity of different mechanisms: modification of structural properties of the lipid bilayers, physical quenching of singlet oxygen and chemical reactions leading to the pigment oxidation. The latter conclusion is based on the analysis of the absorption spectra of liposomes before and after light exposure. An importance of the different modes of protection by carotenoids against single oxygen toxicity towards biomembranes is discussed.

Suboptimal Temperature Acclimation Affects Kennedy Pathway Gene Expression, Lipidome and Metabolite Profile of Nannochloropsis salina during PUFA Enriched TAG Synthesis.[Pubmed:30388843]

Mar Drugs. 2018 Nov 1;16(11). pii: md16110425.

In humans, dietary polyunsaturated fatty acids (PUFAs) are involved in therapeutic processes such as prevention and treatment of cardiovascular diseases, neuropsychiatric disorders, and dementia. We examined the physiology, PUFA accumulation and glycerol lipid biosynthesis in the marine microalga Nannochloropsis salina in response to constant suboptimal temperature (<20 degrees C). As expected, N. salina exhibited significantly reduced growth rate and photosynthetic activity compared to optimal cultivation temperature. Total fatty acid contents were not significantly elevated at reduced temperatures. Cultures grown at 5 degrees C had the highest quantity of eicosapentanoic acid (EPA) (C20:5n3) and the lowest growth rate. Additionally, we monitored broadband lipid composition to model the occurrence of metabolic alteration and remodeling for various lipid pools. We focused on triacylglycerol (TAG) with elevated PUFA content. TAGs with EPA at all three acyl positions were higher at a cultivation temperature of 15 degrees C. Furthermore, monogalactosyldiacylglycerol and Digalactosyldiacylglycerol, which are polar lipids associated with chloroplast membranes, decreased with reduced cultivation temperatures. Moreover, gene expression analysis of key genes involved in Kennedy pathway for de novo TAG biosynthesis revealed bimodal variations in transcript level amongst the temperature treatments. Collectively, these results show that Nannochloropsis salina is a promising source of PUFA containing lipids.

Uptake and toxic effects of triphenyl phosphate on freshwater microalgae Chlorella vulgaris and Scenedesmus obliquus: Insights from untargeted metabolomics.[Pubmed:30308812]

Sci Total Environ. 2019 Feb 10;650(Pt 1):1239-1249.

The flame retardant triphenyl phosphate (TPhP) has been widely detected in surface waters. Yet, little information is known regarding its impact on microalgae. We investigated the uptake and toxicity of TPhP on two freshwater microalgae Chlorella vulgaris (CV) and Scenedesmus obliquus (SO) after exposure to 10mug/l-10mg/l for 5days. The presence of microalgae significantly enhanced TPhP degradation, with the final concentrations dropped to 5.5-35.1% of the original concentrations. Most of the medium TPhP were sorbed and transformed by microalgae in just one day. Growth of CV was inhibited in a concentration-dependent manner, whereas growth of SO were only inhibited significantly at 10mg/l TPhP exposure. Mass spectrometry-based untargeted metabolomics revealed concentration- and species-dependent metabolic responses. Exposure to TPhP in CV resulted in enhanced respiration (increase of fumarate and malate) and osmoregulation (increase of sucrose and myo-inositol), synthesis of membrane lipids (accumulation of monogalactosyldiacylglycerol (MGDG) and Digalactosyldiacylglycerol (DGDG), decrease of lysoglycerolipids, fatty acids, and glyceryl-glucoside). Exposure to TPhP in SO resulted in enhanced osmoregulation (increase of valine, proline, and raffinose) and lipolysis (decrease of MGDG, accumulation of fatty acids, lysophospholipids, and glycerol phosphate). Although chlorophyll a and b contents did not change significantly, decrease of chlorophyll derivatives was observed in both CV and SO at high exposure concentrations. Further bioassays confirmed that CV exhibited enhanced membrane integrity and decreased cellular reactive oxygen species (ROS) possibly as a defense strategy, whereas SO showed disruption of membrane integrity and induction of ROS at 10mg/l exposure. This study demonstrated the potential of microalgae to remove TPhP in water, and offered new insights for the risk assessment of TPhP on freshwater microalgae using metabolomics.

Triacylglycerol accumulates exclusively outside the chloroplast in short-term nitrogen-deprived Chlamydomonas reinhardtii.[Pubmed:30266428]

Biochim Biophys Acta Mol Cell Biol Lipids. 2018 Dec;1863(12):1478-1487.

In microalgae, triacylglycerol (TAG) biosynthesis occurs by parallel pathways involving both the chloroplast and endoplasmic reticulum. A better understanding of contribution of each pathway to TAG assembly facilitates enhanced TAG production via rational genetic engineering of microalgae. Here, using a UPLC-MS(/MS) coupled with TLC-GC-based lipidomic platform, the early response of the major glycerolipids to nitrogen stress was analyzed at both the cellular and chloroplastidic levels in the model green alga Chlamydomonas reinhardtii. Subcellular lipidomic analysis demonstrated that TAG was accumulated exclusively outside the chloroplast, and remained unaltered inside the chloroplast after 4h of nitrogen starvation. This study ascertained the existence of the glycolipid, Digalactosyldiacylglycerol (DGDG), outside the chloroplast and the betaine lipid, diacylglycerol-N,N,N-trimethylhomoserine (DGTS), inside the chloroplast. The newly synthesized DGDG and DGTS prominently increased at the extra-chloroplastidic compartments and served as the major precursors for TAG biosynthesis. In particular, DGDG contributed to the extra-chloroplastidic TAG assembly in form of diacylglycerol (DAG) and DGTS in form of acyl groups. The chloroplastidic membrane lipid, monogalactosyldiacylglycerol (MGDG), was proposed to primarily offer DAG for TAG formation outside the chloroplast. This study provides valuable insights into the subcellular glycerolipidomics and unveils the acyl flux into the extra-chloroplastidic TAG in microalgae.

Quantitative Assessment of the Chloroplast Lipidome.[Pubmed:29987726]

Methods Mol Biol. 2018;1829:241-252.

In plants and algae, photosynthetic membranes have a unique lipid composition. They differ from all other cellular membranes by their very low amount of phospholipids, besides some phosphatidylglycerol (PG), and high proportion of glycolipids. These glycolipids are the uncharged galactolipids, i.e., monogalactosyldiacylglycerol and Digalactosyldiacylglycerol (MGDG and DGDG), and an anionic sulfolipid, i.e., sulfoquinovosyldiacylglycerol (SQDG). In all photosynthetic membranes analyzed to date, from cyanobacteria to algae, protists, and plants, the lipid quartet constituted by MGDG, DGDG, SQDG, and PG has been highly conserved but the composition in fatty acids of these lipids can vary a lot from an organism to another. To better understand chloroplast biogenesis, it is therefore essential to know their lipid content. Establishing chloroplast lipidome requires first to purify chloroplast from plant or algae tissue. Here we describe the methods to extract lipids, quantify the lipids of the chloroplast, and qualify and quantify the different lipid classes that might be present in these fractions.