Extraction and Quantification of Lipids from Plant or Algae.

In plants and algae, the glycerolipidome changes in response to environmental modifications. For instance, in phosphate starvation, phospholipids are degraded and replaced by non-phosphorus lipids, and in nitrogen starvation, storage lipids accumulate. In addition to the well-known applications of oil crops for food, algae lipids are becoming a model for potential applications in health, biofuel, and green chemistry and are used as a platform for genetic engineering. It is therefore important to measure accurately and quickly the glycerolipid content in plants and algae. Here we describe the methods to extract the lipid and quantify the fatty acid amount of the lipid extract and the different lipid classes that are present in these samples.

Phosphatidylinositol-4-phosphate joins the dance of plant autophagosome formation.

In plants, macroautophagy/autophagy is a key mechanism that contributes to their ability to cope with a wide range of environmental constraints such as drought, nutrient starvation or pathogen resistance. Nevertheless, the molecular mechanisms of plant autophagy, and notably that of autophagosome formation, remain poorly understood. As the starting point of our recent paper, we considered the potential functional contribution of lipids in the numerous membrane-remodeling steps involved in this process. By combining biochemistry, genetics, cell biology and high-resolution 3D imaging, we unraveled the function of the lipid phosphatidylinositol-4-phosphate (PtdIns4P) in autophagy in Arabidopsis thaliana, thus providing novel insights into the assembly of autophagosomes in plant cells.

Quantitative proteomic analyses reveal the impact of nitrogen starvation on the proteome of the model diatom Phaeodactylum tricornutum.

Diatoms are one of the largest groups in phytoplankton biodiversity. Understanding their response to nitrogen variations, present from micromolar to near-zero levels in oceans and fresh waters, is essential to comprehend their ecological success. Nitrogen starvation is used in biotechnological processes, to trigger the remodeling of carbon metabolism in the direction of fatty acids and triacylglycerol synthesis. We evaluated whole proteome changes in Phaeodactylum tricornutum after 7 days of cultivation with 5.5-mM nitrate (+N) or without any nitrogen source (-N). On a total of 3768 proteins detected in biological replicates, our analysis pointed to 384 differentially abundant proteins (DAP). Analysis of proteins of lower abundance in -N revealed an arrest of amino acid and protein syntheses, a remodeling of nitrogen metabolism, and a decrease of the proteasome abundance suggesting a decline in unselective whole-proteome decay. Analysis of proteins of higher abundance revealed the setting up of a general nitrogen scavenging system dependent on deaminases. The increase of a plastid palmitoyl-ACP desaturase appeared as a hallmark of carbon metabolism rewiring in the direction of fatty acid and triacylglycerol synthesis. This dataset is also valuable to select gene candidates for improved biotechnological properties.

Phosphatidylinositol-4-phosphate controls autophagosome formation in Arabidopsis thaliana.

Autophagy is an intracellular degradation mechanism critical for plant acclimation to environmental stresses. Central to autophagy is the formation of specialized vesicles, the autophagosomes, which target and deliver cargo to the lytic vacuole. How autophagosomes form in plant cells remains poorly understood. Here, we uncover the importance of the lipid phosphatidylinositol-4-phosphate in autophagy using pharmacological and genetical approaches. Combining biochemical and live-microscopy analyses, we show that PI4K activity is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis.

Features of the Opportunistic Behaviour of the Marine Bacterium Marinobacter algicola in the Microalga Ostreococcus tauri Phycosphere.

Although interactions between microalgae and bacteria are observed in both natural environment and the laboratory, the modalities of coexistence of bacteria inside microalgae phycospheres in laboratory cultures are mostly unknown. Here, we focused on well-controlled cultures of the model green picoalga Ostreococcus tauri and the most abundant member of its phycosphere, Marinobacter algicola. The prevalence of M. algicola in O. tauri cultures raises questions about how this bacterium maintains itself under laboratory conditions in the microalga culture. The results showed that M. algicola did not promote O. tauri growth in the absence of vitamin B12 while M. algicola depended on O. tauri to grow in synthetic medium, most likely to obtain organic carbon sources provided by the microalgae. M. algicola grew on a range of lipids, including triacylglycerols that are known to be produced by O. tauri in culture during abiotic stress. Genomic screening revealed the absence of genes of two particular modes of quorum-sensing in Marinobacter genomes which refutes the idea that these bacterial communication systems operate in this genus. To date, the 'opportunistic' behaviour of M. algicola in the laboratory is limited to several phytoplanktonic species including Chlorophyta such as O. tauri. This would indicate a preferential occurrence of M. algicola in association with these specific microalgae under optimum laboratory conditions.

How Lipids Contribute to Autophagosome Biogenesis, a Critical Process in Plant Responses to Stresses.

Throughout their life cycle, plants face a tremendous number of environmental and developmental stresses. To respond to these different constraints, they have developed a set of refined intracellular systems including autophagy. This pathway, highly conserved among eukaryotes, is induced by a wide range of biotic and abiotic stresses upon which it mediates the degradation and recycling of cytoplasmic material. Central to autophagy is the formation of highly specialized double membrane vesicles called autophagosomes which select, engulf, and traffic cargo to the lytic vacuole for degradation. The biogenesis of these structures requires a series of membrane remodeling events during which both the quantity and quality of lipids are critical to sustain autophagy activity. This review highlights our knowledge, and raises current questions, regarding the mechanism of autophagy, and its induction and regulation upon environmental stresses with a particular focus on the fundamental contribution of lipids. How autophagy regulates metabolism and the recycling of resources, including lipids, to promote plant acclimation and resistance to stresses is further discussed.

The Role of Chloroplast Membrane Lipid Metabolism in Plant Environmental Responses.

Plants are nonmotile life forms that are constantly exposed to changing environmental conditions during the course of their life cycle. Fluctuations in environmental conditions can be drastic during both day-night and seasonal cycles, as well as in the long term as the climate changes. Plants are naturally adapted to face these environmental challenges, and it has become increasingly apparent that membranes and their lipid composition are an important component of this adaptive response. Plants can remodel their membranes to change the abundance of different lipid classes, and they can release fatty acids that give rise to signaling compounds in response to environmental cues. Chloroplasts harbor the photosynthetic apparatus of plants embedded into one of the most extensive membrane systems found in nature. In part one of this review, we focus on changes in chloroplast membrane lipid class composition in response to environmental changes, and in part two, we will detail chloroplast lipid-derived signals.

The Puzzling Conservation and Diversification of Lipid Droplets from Bacteria to Eukaryotes.

Membrane compartments are amongst the most fascinating markers of cell evolution from prokaryotes to eukaryotes, some being conserved and the others having emerged via a series of primary and secondary endosymbiosis events. Membrane compartments comprise the system limiting cells (one or two membranes in bacteria, a unique plasma membrane in eukaryotes) and a variety of internal vesicular, subspherical, tubular, or reticulated organelles. In eukaryotes, the internal membranes comprise on the one hand the general endomembrane system, a dynamic network including organelles like the endoplasmic reticulum, the Golgi apparatus, the nuclear envelope, etc. and also the plasma membrane, which are linked via direct lateral connectivity (e.g. between the endoplasmic reticulum and the nuclear outer envelope membrane) or indirectly via vesicular trafficking. On the other hand, semi-autonomous organelles, i.e. mitochondria and chloroplasts, are disconnected from the endomembrane system and request vertical transmission following cell division. Membranes are organized as lipid bilayers in which proteins are embedded. The budding of some of these membranes, leading to the formation of the so-called lipid droplets (LDs) loaded with hydrophobic molecules, most notably triacylglycerol, is conserved in all clades. The evolution of eukaryotes is marked by the acquisition of mitochondria and simple plastids from Gram-positive bacteria by primary endosymbiosis events and the emergence of extremely complex plastids, collectively called secondary plastids, bounded by three to four membranes, following multiple and independent secondary endosymbiosis events. There is currently no consensus view of the evolution of LDs in the Tree of Life. Some features are conserved; others show a striking level of diversification. Here, we summarize the current knowledge on the architecture, dynamics, and multitude of functions of the lipid droplets in prokaryotes and in eukaryotes deriving from primary and secondary endosymbiosis events.

Human health benefits of very-long-chain polyunsaturated fatty acids from microalgae.

Microalgae are single-cell, photosynthetic organisms whose biodiversity places them at the forefront of biological producers of high-value molecules including lipids and pigments. Some of these organisms particular are capable of synthesizing n-3 very long chain polyunsaturated fatty acids (VLC-PUFAs), known to have beneficial effects on human health. Indeed, VLC-PUFAs are the precursors of many signaling molecules in humans involved in the complexities of inflammatory processes. This mini-review provides an inventory of knowledge on the synthesis of VLC-PUFAs in microalgae and on the diversity of signaling molecules (prostanoids, leukotrienes, SPMs, EFOX, isoprostanoids) that arise in humans from VLC-PUFAs.

Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells.

Diatoms are unicellular heterokonts, living in oceans and freshwaters, exposed to frequent environmental variations. They have a sophisticated membrane compartmentalization and are bounded by a siliceous cell-wall. Formation of lipid droplets (LDs), filled with triacylglycerol (TAG), is a common response to stress. The proteome of mature-LDs from Phaeodactylum tricornutum highlighted the lack of proteins involved in early-LD formation, TAG biosynthesis or LD-to-LD connections. These features suggest that cytosolic LDs might reach a size limit. We analyzed the dynamics of LD formation in P. tricornutum (Pt1 8.6; CCAP 1055/1) during 7 days of nitrogen starvation, by monitoring TAG by mass spectrometry-based lipidomics, and LD radius using epifluorescence microscopy and pulse field gradient nuclear magnetic resonance. We confirmed that mature LDs reach a maximal size. Based on pulse field gradient nuclear magnetic resonance, we did not detect any LD-LD fusion. Three LD subpopulations were produced, each with a different maximal size, larger-sized LDs (radius 0.675 ± 0.125 µm) being generated first. Mathematical modeling showed how smaller LDs are produced once larger LDs have reached their maximum radius. In a mutant line having larger cells, the maximal size of the first LD subpopulation was higher (0.941 ± 0.169 µm), while the principle of stepwise formation of distinct LD populations was maintained. Results suggest that LD size is determined by available cytosolic space and sensing of an optimal size reached in the previous LD subpopulation. Future perspectives include the unraveling of LD-size control mechanisms upon nitrogen shortage. This study also provides novel prospects for the optimization of oleaginous microalgae for biotechnological applications.

Relationship between acyl-lipid and sterol metabolisms in diatoms.

Diatoms are a phylum of unicellular photosynthetic eukaryotes living in oceans and fresh waters, characterized by the complexity of their plastid, resulting from a secondary endosymbiosis event. In the model diatom Phaeodactylum tricornutum, fatty acids (FAs) are synthesized from acetyl-CoA in the stroma of the plastid, producing palmitic acid. FAs are elongated and desaturated to form very-long chain polyunsaturated fatty acids (VLC-PUFAs) in domains of the endomembrane system that need to be identified. Synthesis of VLC-PUFAs is coupled with their import to the core of the plastid via the so-called "omega" pathway. The biosynthesis of sterols in diatoms is likely to be localized in the endoplasmic reticulum as well as using precursors deriving from the mevalonate pathway, using acetyl-CoA as initial substrate. These metabolic modules can be characterized functionally by genetic analyzes or chemical treatments with appropriate inhibitors. Some 'metabolic modules' are characterized by a very low level of metabolic intermediates. Since some chemical treatments or genetic perturbation of lipid metabolism induce the accumulation of these intermediates, channeling processes are possibly involved, suggesting that protein-protein interactions might occur between enzymes within large size complexes or metabolons. At the junction of these modules, metabolic intermediates might therefore play dramatic roles in directing carbon fluxes from one direction to another. Here, acetyl-CoA seems determinant in the balance between TAGs and sterols. Future lines of research and potential utilization for biotechnological applications are discussed.

Biosynthesis of Long Chain Alkyl Diols and Long Chain Alkenols in Nannochloropsis spp. (Eustigmatophyceae).

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana, by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14-18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes.

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis.

Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

Correction: LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation.

[This corrects the article DOI: 10.1371/journal.pone.0182423.].

Non-Enzymatic Synthesis of Bioactive Isoprostanoids in the Diatom Phaeodactylum following Oxidative Stress.

The ecological success of diatoms requires a remarkable ability to survive many types of stress, including variations in temperature, light, salinity, and nutrient availability. On exposure to these stresses, diatoms exhibit common responses, including growth arrest, impairment of photosynthesis, production of reactive oxygen species, and accumulation of triacylglycerol (TAG). We studied the production of cyclopentane oxylipins derived from fatty acids in the diatom Phaeodactylum tricornutum in response to oxidative stress. P. tricornutum lacks the enzymatic pathway for producing cyclopentane-oxylipins, such as jasmonate, prostaglandins, or thromboxanes. In cells subjected to increasing doses of hydrogen peroxide (H2O2), we detected nonenzymatic production of isoprostanoids, including six phytoprostanes, three F2t-isoprostanes, two F3t-isoprostanes, and three F4t-neuroprostanes, by radical peroxidation of α-linolenic, arachidonic, eicosapentaenoic, and docosahexanoic acids, respectively. H2O2 also triggered photosynthesis impairment and TAG accumulation. F1t-phytoprostanes constitute the major class detected (300 pmol per 1 million cells; intracellular concentration, ∼4 µm). Only two glycerolipids, phosphatidylcholine and diacylglycerylhydroxymethyl-trimethyl-alanine, could provide all substrates for these isoprostanoids. Treatment of P. tricornutum with nine synthetic isoprostanoids produced an effect in the micromolar range, marked by the accumulation of TAG and reduced growth, without affecting photosynthesis. Therefore, the emission of H2O2 and free radicals upon exposure to stresses can lead to glycerolipid peroxidation and nonenzymatic synthesis of isoprostanoids, inhibiting growth and contributing to the induction of TAG accumulation via unknown processes. This characterization of nonenzymatic oxylipins in P. tricornutum opens a field of research on the study of processes controlled by isoprostanoid signaling in various physiological and environmental contexts in diatoms.

Extraction and Quantification of Lipids from Plant or Algae.

In plants and algae, the glycerolipidome changes in response to environmental modifications. For instance, in phosphate starvation, phospholipids are degraded and replaced by nonphosphorus lipids and in nitrogen starvation, storage lipids accumulate. In addition to the well-known applications of oil crops for food, algae lipids are becoming a model for potential applications in health, biofuel, and green chemistry and are used as a platform for genetic engineering. It is therefore important to measure accurately and quickly the glycerolipid content in plants and algae. Here we describe the methods to extract the lipid, quantify the fatty acid amount of the lipid extract and to quantify the different lipid classes that are present in these samples.

Ecophysiology and lipid dynamics of a eukaryotic mangrove decomposer.

Aurantiochytrium limacinum is an osmo-heterotrophic Stramenopile and a pioneering mangrove decomposer which is taxonomically assigned to the family of Thraustochytriaceae (class: Labyrinthulomycetes). The life cycle of A. limacinum involves different cell types including mono- and multi-nucleated cells as well as flagellated zoospores which colonize new fallen leaves. The ecological relevance of thraustochytrids is underestimated and eclipsed by their biotechnological importance, due to their ability to accumulate large amount of lipids, mainly triacylglycerols (TAGs). In this study, we aimed to understand the ecophysiological parameters that trigger zoospore production and the interplay between the life cycle of A. limacinum and its lipid metabolism. When grown in a rich medium, cells accumulated large amounts of TAGs at the end of their growth period, but no zoospores were produced. In poor media such as artificial sea water, zoospores were produced in massive quantities. In the absence of organic carbon, the zoospores remained swimming for at least 6 days, consuming their TAGs in the process. Addition of glucose rapidly triggered the maturation of the zoospores. On the basis of these data, we propose a life cycle for A. limacinum integrating the potential perturbations/changes in the environment surrounding a mangrove leaf that could lead to the production of zoospores and colonization of new areas.

Screening for Biologically Annotated Drugs That Trigger Triacylglycerol Accumulation in the Diatom Phaeodactylum.

Microalgae are a promising feedstock for the production of triacylglycerol (TAG) for a variety of potential applications, ranging from food and human health to biofuels and green chemistry. However, obtaining high TAG yields is challenging. A phenotypic assay for the accumulation of oil droplets was developed to screen a library of 1,200 drugs, annotated with pharmacology information, to select compounds that trigger TAG accumulation in the diatom Phaeodactylum tricornutum Using this screen, we identified 34 molecules acting in a dose-dependent manner. Previously characterized targets of these compounds include cell division and cell signaling effectors, membrane receptors and transporters, and sterol metabolism. Among the five compounds possibly acting on sterol metabolism, we focused our study on ethynylestradiol, a synthetic form of estrogen that is used in contraceptive pills and known for its ecological impact as an endocrine disruptor. Ethynylestradiol impaired the production of very-long-chain polyunsaturated fatty acids, destabilized the galactolipid versus phospholipid balance, and triggered the recycling of fatty acids from membrane lipids to TAG. The P. tricornutum transcriptomic response to treatment with ethynylestradiol was consistent with the reallocation of carbon from sterols to acetyl-coenzyme A and TAG. The mode of action and catabolism of ethynylestradiol are unknown but might involve several up-regulated cytochrome P450 proteins. A fatty acid elongase, Δ6-ELO-B1, might be involved in the impairment of very-long-chain polyunsaturated fatty acids and fatty acid turnover. This phenotypic screen opens new perspectives for the exploration of novel bioactive molecules, potential target genes, and pathways controlling TAG biosynthesis. It also unraveled the sensitivity of diatoms to endocrine disruptors, highlighting an impact of anthropogenic pollution on phytoplankton.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation.

Methods to analyze lipidomes have considerably evolved, more and more based on mass spectrometry technics (LC-MS/MS). However, accurate quantifications using these methods require 13C-labeled standards for each lipid, which is not feasible because of the very large number of molecules. Thus, quantifications rely on standard molecules representative of a whole class of lipids, which might lead to false estimations of some molecular species. Here, we determined and compared glycerolipid distributions from three different types of cells, two microalgae (Phaeodactylum tricornutum, Nannochloropsis gaditana) and one higher plant (Arabidopsis thaliana), using either LC-MS/MS or Thin Layer Chromatography coupled with Gas Chromatography (TLC-GC), this last approach relying on the precise quantification of the fatty acids present in each glycerolipid class. Our results showed that the glycerolipid distribution was significantly different depending on the method used. How can one reconcile these two analytical methods? Here we propose that the possible bias with MS data can be circumvented by systematically running in tandem with the sample to be analyzed a lipid extract from a qualified control (QC) of each type of cells, previously analyzed by TLC-GC, and used as an external standard to quantify the MS results. As a case study, we applied this method to compare the impact of a nitrogen deficiency on the three types of cells.