Betaine Lipids Research Articles

Nitrogen and phosphorus limitations induce carbon partitioning and membrane lipid remodelling in the marine diatom Phaeodactylum tricornutum

ABSTRACT Nitrogen (N) and phosphorus (P) limitations induce triacylglycerol (TAG) accumulation and membrane lipid remodelling in the marine diatom Phaeodactylum tricornutum. However, a clear understanding of the metabolic reorientation is still lacking. Carbon partitioning is of great interest because this microalga produces various highly valuable molecules such as lipids and polyunsaturated fatty acids. This study compared growth, photosynthetic activity, biochemical and transcriptional responses of P. tricornutum throughout batch culture under N or P limitation. The integrated results show that the photosynthetic intensity was greatly reduced under N or P limitation. Under N limitation, the degradation and re-use of cellular N-containing compounds contributed to TAG accumulation, whilst P limitation favoured TAG accumulation due to the efficiency of carbon fixation, without massive degradation of essential compounds at cellular level. There was no difference in the partitioning of carbon to neutral lipids between N and P limitation. Substitution of phospholipids with betaine lipids appeared to be a P-specific acclimation strategy in P. tricornutum, which was largely regulated at the gene expression level. Betaine lipid synthesis was induced by P limitation. The lipid remodelling began once the medium became deficient in P. While the phospholipid biosynthesis pathway was not completely inhibited, a shift of lipid classes occurred immediately after their synthesis via phospholipid-recycling mechanisms.

Polar lipid profile of Saccharina latissima, a functional food from the sea

Increasing global demand for food has generated a need for new strategies to feed a fast-growing population. Oceans appear as a sustainable solution, providing alternative sources of food such as macroalgae. These sea vegetables have high nutritional value and provide functional and health benefits. The sugar kelp Saccharina latissima is an emerging edible seaweed used mainly for human consumption. Although much is known about its biochemical compositions its lipidome remains unexplored. The aim of the present study was to characterize the polar lipid profile of S. latissima using a lipidomic-mass spectrometry HILIC-LC-MS based analysis. This approach allowed the identification of 197 molecular species of polar lipids, including glycolipids, phospholipids and betaine lipids. Several molecular species identified are carriers of polyunsaturated fatty acids with nutritional value and have been reported with anti-inflammatory, anti-microbial and anti-proliferative activity. Saccharina latissima is an emerging candidate to promote blue biotechnology inspired by the ocean.

The PHO signaling pathway directs lipid remodeling in Cryptococcus neoformans via DGTS synthase to recycle phosphate during phosphate deficiency.

The phosphate sensing and acquisition (PHO) pathway of Cryptococcus neoformans is essential for growth in phosphate-limiting conditions and for dissemination of infection in a mouse model. Its key transcription factor, Pho4, regulates expression of genes controlling the acquisition of phosphate from both external and cellular sources. One such gene, BTA1, is highly up-regulated during phosphate starvation. Given that a significant proportion of cellular phosphate is incorporated into phospholipids, and that the Pho4-dependent BTA1 gene encodes an enzyme predicted to catalyse production of a phosphorus-free betaine lipid, we investigated whether phospholipids provide an accessible reservoir of phosphate during phosphate deficiency. By comparing lipid profiles of phosphate-starved WT C. neoformans, PHO4 (pho4Δ) and BTA1 (bta1Δ) deletion mutants using thin layer chromatography and liquid chromatography mass spectrometry, we showed that phosphatidylcholine (PC) is substituted by the phosphorus-free betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylgyceryl hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA) in a Pho4- and Bta1-dependent manner, and that BTA1 encodes a functional DGTS synthase. Synthesis of DGTA tightly correlated with that of DGTS, consistent with DGTS being the precursor of DGTA. Similar to pho4Δ, bta1Δ grew more slowly than WT in cell culture medium (RPMI) and was hypovirulent in a murine model of cryptococcosis. In contrast to pho4Δ, bta1Δ tolerated alkaline pH and disseminated to the brain. Our results demonstrate that Bta1-dependent substitution of PC by betaine lipids is tightly regulated in C. neoformans by the PHO pathway, to conserve phosphate and preserve membrane integrity and function. This phospholipid remodeling strategy may also contribute to cryptococcal virulence during host infection.

The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previously validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl‑β‑alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of β‑carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function includes protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.

Intact polar lipids in the water column of the eastern tropical North Pacific: abundance and structural variety of non-phosphorus lipids

Abstract. Intact polar lipids (IPLs) are the main building blocks of cellular membranes and contain chemotaxonomic, ecophysiological and metabolic information, making them valuable biomarkers in microbial ecology and biogeochemistry. This study investigates IPLs in suspended particulate matter (SPM) in the water column of the eastern tropical North Pacific Ocean (ETNP), one of the most extensive open-ocean oxygen minimum zones (OMZs) in the world, with strong gradients of nutrients, temperature and redox conditions. A wide structural variety in polar lipid head-group composition and core structures exists along physical and geochemical gradients within the water column, from the oxygenated photic zone to the aphotic OMZ. We use this structural diversity in IPLs to evaluate the ecology and ecophysiological adaptations that affect organisms inhabiting the water column, especially the mid-depth OMZ in the context of biogeochemical cycles. Diacylglycerol phospholipids are present at all depths, but exhibit the highest relative abundance and compositional variety (including mixed acyl/ether core structures) in the upper and core OMZ where prokaryotic biomass was enriched. Surface ocean SPM is dominated by diacylglycerol glycolipids that are found in photosynthetic membranes. These and other glycolipids with varying core structures composed of ceramides and hydroxylated fatty acids are also detected with varying relative abundances in the OMZ and deep oxycline, signifying additional non-phototrophic bacterial sources for these lipids. Betaine lipids (with zero or multiple hydroxylations in the core structures) that are typically assigned to microalgae are found throughout the water column down to the deep oxycline but do not show a depth-related trend in relative abundance. Archaeal IPLs comprised of glycosidic and mixed glycosidic-phosphatidic glycerol dibiphytanyl glycerol tetraethers (GDGTs) are most abundant in the upper OMZ, where nitrate maxima point to ammonium oxidation but increase in relative abundance in the core OMZ and deep oxycline. The presence of non-phosphorus “substitute” lipids within the OMZ suggest that the indigenous microbes might be phosphorus limited (P starved) at ambient phosphate concentrations of 1 to 3.5 µM, although specific microbial sources for many of these lipids still remain unknown.

Does silica solid-phase extraction of soil lipids isolate a pure phospholipid fraction?

Phospholipids are isolated from crude lipid extracts by silica solid phase extraction (SPE), but for soils we don't know if phospholipids are the only fatty acid-based lipids present in the polar lipid fraction. Lipids extracted from three soils were fractionated with a silica SPE protocol commonly used for soils, with “neutrals” eluted by chloroform, “glycolipids” eluted by acetone, and “phospholipids” eluted by methanol. Fatty acid-based lipids were identified and quantified by liquid chromatography-mass spectrometry. Phospholipids were recovered in the methanol fraction, but this fraction also included betaine lipids. In two soils the methanol fraction was 3–6% betaine lipid while in one soil betaine lipids accounted for 48% of lipids in the methanol fraction. Clearly the fraction obtained by eluting lipids from silica with methanol is not purely phospholipid but can contain significant amounts of other polar lipids.

Lipidomic signature of the green macroalgae Ulva rigida farmed in a sustainable integrated multi-trophic aquaculture

Ulva species, green macroalgae, are widely distributed across the globe, being one of the most heavily traded edible seaweeds. Nonetheless, although this genus has been largely used in scientific studies, its lipidome remains rather unexplored. The present study sheds light over the lipid profile of Ulva rigida produced in a land-based integrated multi-trophic aquaculture (IMTA) system using liquid chromatography coupled to high-resolution mass spectrometry for molecular lipid species identification. The lipidome of U. rigida revealed the presence of distinct beneficial n-3 fatty acids for human health, namely alpha-linoleic acid (ALA) and docosapentaenoic acid (DPA). A total of 87 molecular species of glycolipids, 58 molecular species of betaine lipids, and 57 molecular species of phospholipids were identified in the lipidome of U. rigida including some species bearing PUFA and with described bioactive properties. Overall, the present study contributes to the valorization and quality validation of sustainably farmed U. rigida.

Triacylglycerol accumulates exclusively outside the chloroplast in short-term nitrogen-deprived Chlamydomonas reinhardtii

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 4 h 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.

A liquid chromatography–mass spectrometry method for analysis of intact fatty‐acid‐based lipids extracted from soil

Fatty‐acid‐based lipids are considered to be indicators of the structure and function of soil microbial biomass. Identifying and quantifying intact fatty‐acid‐based lipids in soil extracts is challenging because lipids range from polar (e.g. phospholipids and other membrane lipids) to non‐polar (e.g. triacylglycerols involved in energy storage). The aim of this study was to develop a liquid chromatography–mass spectrometry (LC–MS) method to identify and quantify intact fatty‐acid‐based lipids in soil extracts. Reversed‐phase LC separated fatty‐acid‐based lipids ranging from polar to non‐polar in one chromatographic analysis. Within classes, individual lipid species were separated predictably from one another on the basis of carbon numbers and degree of unsaturation, such that chromatographic retention time could be used to aid identification. Lipid species were identified based on intact mass of the lipid and by fragmentation into structure‐specific fragments that enabled compound classes and fatty acyl chains to be determined. Detection limits in positive and negative mode were better than 10 nmol l−1 for most lipid standards. In an extract of soil, 74 lipids from 10 classes were identified. The main non‐polar lipids were 12 diacylglyerol species and 16 triacylglycerol species. Polar lipids included phosphatidylcholines, phosphatidylethanolamines, monomethyl‐phosphatidylethanolamines, phosphatidylinositols and three classes of betaine lipid (diacylgycerol‐N‐trimethylhomoserine; diacylglycerylhydroxymethy‐N,N,N‐trimethyl‐β‐alanine; and diacylglycerylcarboxyhydroxymethylcholine). The LC–MS method developed provides a powerful analytical tool for analysis of fatty‐acid‐based lipids ranging from polar to non‐polar and should be useful for the culture‐ and gene‐independent assay of soil function.HighlightsA novel LC–MS method is described for identification of soil lipids. Polar and non‐polar lipids were separated in one chromatographic analysis 74 lipids from 10 classes were identified Lipids identified included 25 species of betaine lipid

Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis

Diacylglyceryl-N,N,N-trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica, DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica.

High-Resolution Lipidomics of the Early Life Stages of the Red Seaweed Porphyra dioica.

Porphyra dioica is a commercial seaweed consumed all over the world, mostly in the shape of nori sheets used for “sushi” preparation. It is a well-known part of the Asian diet with health benefits, which have been associated, among others, to the high levels of n-3 and n-6 fatty acids in this red alga. However, other highly valued lipids of Porphyra are polar lipids that remain largely undescribed and can have both nutritional value and bioactivity, thus could contribute to the valorization of this seaweed. In this context, the present work aims to identify the lipidome of two life cycle stages of the Atlantic species Porphyra dioica: the early life stage conchocelis produced in an indoor-nursery, and young blades produced outdoors using an integrated multitrophic aquaculture (IMTA) framework. Both the blades (gametophyte) and conchocelis (sporophyte) are commercialized in the food and cosmetics sectors. Liquid chromatography coupled to Q–Exactive high resolution-mass spectrometry (MS) platform was used to gain insight into the lipidome of these species. Our results allowed the identification of 110 and 100 lipid molecular species in the lipidome of the blade and conchocelis, respectively. These lipid molecular species were distributed as follows (blade/conchocelis): 14/15 glycolipids (GLs), 93/79 phospholipids (PLs), and 3/6 betaine lipids. Both life stages displayed a similar profile of GLs and comprised 20:4(n-6) and 20:5(n-3) fatty acids that contribute to n-3 and n-6 fatty acid pool recorded and rank among the molecular species with higher potential bioactivity. PLs’ profile was different between the two life stages surveyed, mainly due to the number and relative abundance of molecular species. This finding suggests that differences between both life stages were more likely related with shifts in the lipids of extraplastidial membranes rather than in plastidial membranes. PLs contained n-6 and n-3 precursors and in both life stages of Porphyra dioica the n-6/n-3 ratio recorded was less than 2, highlighting the potential benefits of using these life stages in human diet to prevent chronic diseases. Atherogenic and thrombogenic indexes of blades (0.85 and 0.49, respectively) and conchocelis (0.34 and 0.30, respectively) are much lower than those reported for other Rhodophyta, which highlights their potential application as food or as functional ingredients. Overall, MS-based platforms represent a powerful tool to characterize lipid metabolism and target lipids along different life stages of algal species displaying complex life cycles (such as Porphyra dioica), contributing to their biotechnological application.

Intact polar lipid export in the temperate western North Atlantic and Sargasso Sea

Intact polar diacylglycerolipids (IP-DAGs), abundant membrane components in phytoplankton and bacterioplankton, comprised 5.9±0.5% of suspended particulate organic carbon (POC) in the euphotic zone at two stations in the western North Atlantic Ocean. The IP-DAG assemblage included 9 phospholipid, glycolipid and betaine lipid classes that are routinely found in marine samples. We also report the pelagic occurrence of two recently discovered IP-DAGs, phosphatidyl-S,S-dimethylpropanethiol (PDPT) and a betaine-like lipid, both originally identified in Emiliania huxleyi. At the mesotrophic station north of the Gulf Stream, IP-DAGs comprised 0.62±0.38% of sinking POC collected in sediment traps, and at the oligotrophic station in the Sargasso Sea they comprised 0.31±0.05% of sinking POC.Phospholipids and glycolipids with polyunsaturated fatty acid (PUFA) moieties were relatively abundant in sinking particles at 150m compared with suspended particles in the euphotic zone at the mesotrophic station north of the Gulf Stream. The sinking particles were probably associated with the demise of a dinoflagellate or diatom bloom that resulted in a maximum total IP-DAG export flux of 1.8μmol/m2/day. At the oligotrophic station in the Sargasso Sea, sinking particles included microalgal glycolipids, phospholipids and betaine lipids with PUFAs; picocyanobacterial glycolipids with saturated and monounsaturated FAs; and bacterial phospholipids with saturated and monounsaturated FAs. The microalgal and bacterial IP-DAGs were relatively abundant in sinking particles compared with suspended particles. The IP-DAG export flux at the station in the Sargasso Sea was similar in three sediment traps deployed on consecutive days, with an average of 0.46±0.08μmol/m2/day.A diel cycle was observed in the IP-DAG euphotic zone inventory in the Sargasso Sea, with higher concentration in samples taken at dusk than at dawn, and we propose a model using this dynamic to calculate IP-DAG residence time. Export efficiency was calculated from the residence time and the ratio of IP-DAG export flux to euphotic zone inventory, with values ranging from 0.02% for PDPT to 6.9% for monogalactosyldiacylglycerol (MGDG) with PUFAs. The greater export flux and export efficiency of IP-DAGs with PUFAs contrasts with previous results for total FAs in sinking particles, which showed preferential export of saturated and monounsaturated FAs. The difference likely resulted from IP-DAGs with PUFAs being membrane components of rapidly sinking organic matter from microalgae. Alternatively, PUFA moieties cause steric hindrance of microbial lipase activity, potentially decreasing their hydrolysis rate and increasing the relative concentration of IP-DAGs with PUFAs in sinking particles.

Kleptoplasty does not promote major shifts in the lipidome of macroalgal chloroplasts sequestered by the sacoglossan sea slug Elysia viridis

Sacoglossan sea slugs, also known as crawling leaves due to their photosynthetic activity, are highly selective feeders that incorporate chloroplasts from specific macroalgae. These “stolen” plastids - kleptoplasts - are kept functional inside animal cells and likely provide an alternative source of energy to their host. The mechanisms supporting the retention and functionality of kleptoplasts remain unknown. A lipidomic mass spectrometry-based analysis was performed to study kleptoplasty of the sacoglossan sea slug Elysia viridis fed with Codium tomentosum. Total lipid extract of both organisms was fractionated. The fraction rich in glycolipids, exclusive lipids from chloroplasts, and the fraction rich in betaine lipids, characteristic of algae, were analysed using hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-LC-MS). This approach allowed the identification of 81 molecular species, namely galactolipids (8 in both organisms), sulfolipids (17 in C. tomentosum and 13 in E. viridis) and betaine lipids (51 in C. tomentosum and 41 in E. viridis). These lipid classes presented similar lipidomic profiles in C. tomentosum and E. viridis, indicating that the necessary mechanisms to perform photosynthesis are preserved during the process of endosymbiosis. The present study shows that there are no major shifts in the lipidome of C. tomentosum chloroplasts sequestered by E. viridis.

Lipid turnover between membrane lipids and neutral lipids via inhibition of diacylglyceryl N,N,N-trimethylhomoserine synthesis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii of the microalgal model species lacks phosphatidylcholine (PC), and PC is replaced by diacylglyceryl N,N,N-trimethylhomoserine (DGTS). DGTS is a betaine lipid that is placed in the endoplasmic reticulum (ER) and is synthesized by a single gene, BTA1. In this study, we aimed to ascertain the turnover between membrane and neutral lipids via BTA1 knockdown transformants. Transgenic lines CrBta-hm13 and CrBta-hm31 were 80% and 60% downregulated in BTA1 gene expression levels, respectively. Both transformants had half the amount of DGTS, which coincided with decreased monogalactosyldiacylglycerol (MGDG), which was increased approximately threefold in neutral lipids. While the reduction of DGTS was shown to arise from inhibition of DGTS synthesis, decreased MGDG was affected by internal stress, such as ER stress, which was induced to have a decreased amount of DGTS in the ER. However, galactolipids, except for MGDG, and phospholipids in the transformants were maintained at similar levels. In the transformants, the molar proportion of C16:4 and C18:3(9,12,15), which are the major fatty acids of MGDG, was significantly increased in triacylglycerol (TAG) because of MGDG degradation. Thus, lipid turnover arising from the downregulation of DGTS and induction of ER stress caused a decrease in DGTS and MGDG, which generated a synergy effect on the accumulation of TAG. This study implies that genetic modification of a membrane lipid synthesis pathway could not only be a suitable approach to target accumulation of TAG, but could also suggest a mechanism for the lipid turnover between membrane lipids and neutral lipids.

Seaweed Competition: Ulva Sp. has the Potential to Produce the Betaine Lipid Diacylglyceryl-O-4'-(N,N,N,-Trimethyl) Homoserine (DGTS) in Order to Replace Phosphatidylcholine (PC) Under Phosphate-Limiting Conditions in the P-Limited Dutch Wadden Sea and Outcompete an Aggressive Non-Indigenous Gracilaria vermiculophylla Red Drift Algae Out of this Unique Unesco World Heritage Coastal Area

The present study tested in the Western Dutch Wadden Sea (WDW) UNESCO World Heritage Site why an on a global scale the aggressive non-indigenous red drift alga Gracilaria vermiculophylla didn’t succeed to overgrow the WDC. In such a multifaceted complex ecosystem like the dynamic WDC it seems like unraveling a Gordian knob in order to describe the inextricable relationship between this seaweed invader suppression and its (a) biotic environment. However, we succeeded at the molecular level to give a convincing reasoning at first grounded in the awareness of a since 1987 river Rhine-North-Sea-WDC severely Phosphorus (P) restricted ecosystem. Our ecological datasets gave via final DCA (Detrended Correspondence Analysis) awareness of the very compelling interaction between Ulva sp. and G.vermiculophylla. Based on LCMS-techniques we discovered that Ulva sp. have the advantage to use the biochemical pathway solely rarely observed in some euckaryotes- to have the potential to produce the betaine lipid diacylglyceryl-O-4’-(N,N,N,-trimethyl)homoserine (DGTS) which replaces the plant/seaweed cell wall structure phosphatidylcholine (PC) “lecithine” under phosphate-limiting growth conditions. Also we hope this lipidomics based compound DGTS can serve as an ecological biomarker in order to protect vulnerable ecosystems like the Wadden Sea (UNESCO World Heritage).

Aspects of phosphorus physiology associated with phosphate-induced polar lipid remodelling in marine microalgae.

Marine microalgae exhibit a diversified phosphorus physiology and have also been recently found to show high inter-taxa variability in their phosphate induced-polar lipids’ remodelling. Identification of phosphorus physiology aspects that are more related to lipid remodelling can contribute to better understanding of such intricate phytoplankton lipid metabolism. Therefore, some aspects of phosphorus physiology related to its uptake, storage and use were evaluated in a taxonomically diversified group of nine marine microalgae that was arranged into three subgroups, each of them including species showing similar polar lipid responses to phosphate.Luxury phosphate uptake (PU) was the physiological aspect best associated to microalgal polar lipid metabolism as it was maximal in species (Picochlorum atomus, Tetraselmis suecica and Nannochloropsis gaditana) that were able to counterbalance between phospholipids (PL) and betaine lipids (BL). Cryptophytes (Rhodomonas baltica, Chroomonas placoidea), characterized by their constitutive BL and flexible PL contents in response to phosphate, had almost no luxury PU and showed higher phosphorus cell quota (QP) under phosphate deprivation. Haptophyes (Isochrysis galbana, Diacronema vlkianum), with constitutive BL contents and permanently minimal PL contents, showed the lowest QP when deprived of phosphate while their luxury PU was below that for green microalgae. Induction of alkaline phosphatase activity following phosphate depletion was maximal in diatoms (Phaeodactylum tricornutum, Chaetoceros gracilis) and I. galbana but it was unrelated to lipid remodelling. Despite strong influence of taxonomy, polar lipid remodelling accounted for 38.8% of total variation when microalgae were ordinated using their physiological responses to phosphorus as descriptive variables.

Valorization of Lipids from Gracilaria sp. through Lipidomics and Decoding of Antiproliferative and Anti-Inflammatory Activity.

The lipidome of the red seaweed Gracilaria sp., cultivated on land-based integrated multitrophic aquaculture (IMTA) system, was assessed for the first time using hydrophilic interaction liquid chromatography-mass spectrometry and tandem mass spectrometry (HILIC–MS and MS/MS). One hundred and forty-seven molecular species were identified in the lipidome of the Gracilaria genus and distributed between the glycolipids classes monogalactosyl diacylglyceride (MGDG), digalactosyl diacylglyceride (DGDG), sulfoquinovosyl monoacylglyceride (SQMG), sulfoquinovosyl diacylglyceride (SQDG), the phospholipids phosphatidylcholine (PC), lyso-PC, phosphatidylglycerol (PG), lyso-PG, phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatic acid (PA), inositolphosphoceramide (IPC), and betaine lipids monoacylglyceryl- and diacylglyceryl-N,N,N-trimethyl homoserine (MGTS and DGTS). Antiproliferative and anti-inflammatory effects promoted by lipid extract of Gracilaria sp. were evaluated by monitoring cell viability in human cancer lines and by using murine macrophages, respectively. The lipid extract decreased cell viability of human T-47D breast cancer cells and of 5637 human bladder cancer cells (estimated half-maximal inhibitory concentration (IC50) of 12.2 μg/mL and 12.9 μg/mL, respectively) and inhibited the production of nitric oxide (NO) evoked by the Toll-like receptor 4 agonist lipopolysaccharide (LPS) on the macrophage cell line RAW 264.7 (35% inhibition at a concentration of 100 μg/mL). These findings contribute to increase the ranking in the value-chain of Gracilaria sp. biomass cultivated under controlled conditions on IMTA systems.

Glycerolipid Characterization and Nutrient Deprivation-Associated Changes in the Green Picoalga Ostreococcus tauri.

The picoalga Ostreococcus tauri is a minimal photosynthetic eukaryote that has been used as a model system. O. tauri is known to efficiently produce docosahexaenoic acid (DHA). We provide a comprehensive study of the glycerolipidome of O. tauri and validate this species as model for related picoeukaryotes. O. tauri lipids displayed unique features that combined traits from the green and the chromalveolate lineages. The betaine lipid diacylglyceryl-hydroxymethyl-trimethyl-β-alanine and phosphatidyldimethylpropanethiol, both hallmarks of chromalveolates, were identified as presumed extraplastidial lipids. DHA was confined to these lipids, while plastidial lipids of prokaryotic type were characterized by the overwhelming presence of ω-3 C18 polyunsaturated fatty acids (FAs), 18:5 being restricted to galactolipids. C16:4, an FA typical of green microalgae galactolipids, also was a major component of O. tauri extraplastidial lipids, while the 16:4-coenzyme A (CoA) species was not detected. Triacylglycerols (TAGs) displayed the complete panel of FAs, and many species exhibited combinations of FAs diagnostic for plastidial and extraplastidial lipids. Importantly, under nutrient deprivation, 16:4 and ω-3 C18 polyunsaturated FAs accumulated into de novo synthesized TAGs while DHA-TAG species remained rather stable, indicating an increased contribution of FAs of plastidial origin to TAG synthesis. Nutrient deprivation further severely down-regulated the conversion of 18:3 to 18:4, resulting in obvious inversion of the 18:3/18:4 ratio in plastidial lipids, TAGs, as well as acyl-CoAs. The fine-tuned and dynamic regulation of the 18:3/18:4 ratio suggested an important physiological role of these FAs in photosynthetic membranes. Acyl position in structural and storage lipids together with acyl-CoA analysis further help to determine mechanisms possibly involved in glycerolipid synthesis.

Simultaneous structural identification of diacylglyceryl-N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) in microalgae using dual Li+ /H+ adduct ion mode by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Diacylgycerol-N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) are structural isomers that are the most commonly described betaine lipids in microalgae. The structural differentiation and precise identification of DGTS and DGTA in microalgae need to be established during mass spectrometry analysis. Total lipid was extracted from Amphora spp. with CHCl3 /CH3 OH (1:1, v/v). The qualitative analysis of DGTS and DGTA in Amphora spp. was carried out using Li+ /H+ dual mode by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry operating in MSE mode (UPLC/QTOF MSE ). Characteristic fragment ions [C10 H22 O5 N]+ at m/z 236.15 and [C7 H14 O2 N]+ at m/z 144.10 from the [M+H]+ precursor ion can be used for the qualitative analysis of both DGTA and DGTS, whereas the loss of m/z 87 and 74 from the [M+Li]+ precursor ion are specific for DGTS, and the loss of m/z 103 from the [M+Li]+ precursor ion is only for DGTA. As a result, 9 DGTSs and 16 DGTAs with different fatty acids were identified simultaneously in Amphora spp. Semi-quantitative analysis of DGTS and DGTA in Amphora spp. showed that the contents of DGTS ranged from 0.003 to 0.438nmol mg-1 dw, and that of DGTA from 0.004 to 0.414nmol mg-1 dw. This is the first report to achieve the ambiguous structural identification of DGTS and DGTA by UPLC/QTOF MSE using dual Li+ /H+ adduct ion mode, which has remained a challenge in the past. It could provide new insights into their phylogeny and be helpful to characterize the natural phytoplankton communities as intact polar lipid biomarkers. Copyright © 2017 John Wiley & Sons, Ltd.

Polar Lipids Analysis of Cultured Phytoplankton Reveals Significant Inter-taxa Changes, Low Influence of Growth Stage, and Usefulness in Chemotaxonomy.

The high lipid diversity of microalgae has been used to taxonomically differentiate phytoplankton taxa at the class level. However, important lipids such as phospholipids (PL) and betaine lipids (BL) with potential chemotaxonomy application in phytoplankton ecology have been scarcely studied. The chemotaxonomy value of PL and BL depends on their intraspecific extent of variation as microalgae respond to external changing factors. To determine such effects, lipid class changes occurring at different growth stages in 15 microalgae from ten different classes were analyzed. BL occurred in 14 species and were the less affected lipids by growth stage with diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGTA) showing the highest stability. PL were more influenced by growth stage with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidyletanolamine (PE) declining towards older culture stages in some species. Glycolipids were the more common lipids, and no evident age-related variability pattern could be associated to taxonomic diversity. Selecting BL and PL as descriptor variables optimally distinguished microalgae taxonomic variability at all growth stages. Principal coordinate analysis arranged species through a main tendency from diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGCC) containing species (mainly dinoflagellates and haptophytes) to DGTA or PC containing species (mainly cryptophytes). Two diatom classes with similar fatty acid profiles could be distinguished from their respective content in DGTA (Bacillariophyceae) or DGCC (Mediophyceae). In green lineage classes (Trebouxiophyceae, Porphyridophyceae, and Chlorodendrophyceae), PC was a better descriptor than BL. BL and PL explained a higher proportion of microalgae taxonomic variation than did fatty acids and played a complementary role as lipid markers.