Respective role of plastoglobules and lipid droplets in leaf neutral lipid accumulation during senescence and nitrogen deprivation.

Upon abiotic stress or senescence, the size and/or abundancy of plastid-localized plastoglobules and cytosolic lipid droplets, both compartments devoted to neutral lipid storage, increase in leaves. Meanwhile, plant lipid metabolism is also perturbed, notably with the degradation of thylakoidal monogalactosyldiacylglycerol (MGDG) and the accumulation of neutral lipids. Although these mechanisms are probably linked, they have never been jointly studied, and the respective roles of plastoglobules and lipid droplets in the plant response to stress are totally unknown. To address this question, we determined and compared the glycerolipid composition of both lipid droplets and plastoglobules, followed their formation in response to nitrogen starvation and studied the kinetics of lipid metabolism in Arabidopsis leaves. Our results demonstrated that plastoglobules preferentially store phytyl-esters, while triacylglycerols (TAGs) and steryl-esters accumulated within lipid droplets. Thanks to a pulse chase labeling approach and lipid analyses of fatty acid desaturase 2 (fad2) mutant, we showed that MGDG-derived C18:3 fatty acids were exported to lipid droplets, while MGDG-derived C16:3 fatty acids were stored within plastoglobules. The export of lipids from plastids to lipid droplets was likely facilitated by the physical contact occurring between both organelles, as demonstrated by our electron tomography study. The accumulation of lipid droplets and neutral lipids was transient, suggesting that stress-induced TAGs were remobilized during the plant recovery phase by a mechanism that remains to be explored.

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

Plastidic Δ6 Fatty-Acid Desaturases with Distinctive Substrate Specificity Regulate the Pool of C18-PUFAs in the Ancestral Picoalga Ostreococcus tauri.

Eukaryotic Δ6-desaturases are microsomal enzymes that balance the synthesis of ω-3 and ω-6 C18-polyunsaturated fatty acids (C18-PUFAs) according to their specificity. In several microalgae, including Ostreococcus tauri, plastidic C18-PUFAs are strictly regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFAs. Here, we identified two putative front-end Δ6/Δ8-desaturases from O tauri that, together with putative homologs, cluster apart from other characterized Δ6-desaturases. Both were plastid-located and unambiguously displayed a Δ6-desaturation activity when overexpressed in the heterologous hosts Nicotiana benthamiana and Synechocystis sp. PCC6803, as in the native host. Detailed lipid analyses of overexpressing lines unveiled distinctive ω-class specificities, and most interestingly pointed to the importance of the lipid head-group and the nonsubstrate acyl-chain for the desaturase efficiency. One desaturase displayed a broad specificity for plastidic lipids and a preference for ω-3 substrates, while the other was more selective for ω-6 substrates and for lipid classes including phosphatidylglycerol as well as the peculiar 16:4-galactolipid species occurring in the native host. Overexpression of both Δ6-desaturases in O tauri prevented the regulation of C18-PUFA under phosphate deprivation and triggered glycerolipid fatty-acid remodeling, without causing any obvious alteration in growth or photosynthesis. Tracking fatty-acid modifications in eukaryotic hosts further suggested the export of plastidic lipids to extraplastidic compartments.

LPIAT, a lyso-Phosphatidylinositol Acyltransferase, Modulates Seed Germination in Arabidopsis thaliana through PIP Signalling Pathways and is Involved in Hyperosmotic Response.

Lyso-lipid acyltransferases are enzymes involved in various processes such as lipid synthesis and remodelling. Here, we characterized the activity of an acyltransferase from Arabidopsis thaliana (LPIAT). In vitro, this protein, expressed in Escherichia coli membrane, displayed a 2-lyso-phosphatidylinositol acyltransferase activity with a specificity towards saturated long chain acyl CoAs (C16:0- and C18:0-CoAs), allowing the remodelling of phosphatidylinositol. In planta, LPIAT gene was expressed in mature seeds and very transiently during seed imbibition, mostly in aleurone-like layer cells. Whereas the disruption of this gene did not alter the lipid composition of seed, its overexpression in leaves promoted a strong increase in the phosphatidylinositol phosphates (PIP) level without affecting the PIP2 content. The spatial and temporal narrow expression of this gene as well as the modification of PIP metabolism led us to investigate its role in the control of seed germination. Seeds from the lpiat mutant germinated faster and were less sensitive to abscisic acid (ABA) than wild-type or overexpressing lines. We also showed that the protective effect of ABA on young seedlings against dryness was reduced for lpiat line. In addition, germination of lpiat mutant seeds was more sensitive to hyperosmotic stress. All these results suggest a link between phosphoinositides and ABA signalling in the control of seed germination.

Handgrip strength measured by a dynamometer connected to a smartphone: a new applied health technology solution for the self-assessment of rheumatoid arthritis disease activity.

OBJECTIVE: The aim was to analyse the accuracy of a hand dynamometer connected to a smartphone to assess RA disease activity through the measurement of handgrip strength (HGS). METHODS: Eighty-two RA patients participated in this prospective study. Three types of HGS were assessed: power (Po), pinch (Pi) and tripod (T). An interactive mobile application was developed to capture grip measures. A unilinear regression analysis between HGS and DAS28 was performed. A multivariate regression analysis to identify independent variables related to HGS was also conducted. RESULTS: Sixty-three patients (76.8%) were female. Mean age was 61.3 years. At baseline, a negative correlation between the three HGS measures and DAS28 score was found, as follows: Po, r = -0.65 (95% CI: -0.76, -0.51, P < 0.001); Pi, r= -0.42 (95% CI: -0.59, -0.23, P < 0.001); and T, r = -0.47 (95% CI: -0.63, -0.29, P < 0.001). In a longitudinal analysis of 32 patients, a negative correlation between ΔPo grip and ΔDAS28 was found (r = -0.76, 95% CI: -0.88, -0.56). Po grip was independently correlated with male sex (95% CI: 1.49, 4.14, P = 0.002), whereas variables inversely correlated with Po grip were disease duration (95% CI: -2.71, -1.34, P = 0.03), patient global assessment (95% CI: -2.41, -1.1, P < 0.001) and CRP level (95% CI: -3.56, -1.08, P < 0.001). CONCLUSION: HGS assessed by a hand dynamometer connected to a smartphone represents an innovative health technology solution that could prompt the self-assessment of RA disease activity in an outpatient setting.

Lipid droplets are versatile organelles involved in plant development and plant response to environmental changes.

Since decades plant lipid droplets (LDs) are described as storage organelles accumulated in seeds to provide energy for seedling growth after germination. Indeed, LDs are the site of accumulation for neutral lipids, predominantly triacylglycerols (TAGs), one of the most energy-dense molecules, and sterol esters. Such organelles are present in the whole plant kingdom, from microalgae to perennial trees, and can probably be found in all plant tissues. Several studies over the past decade have revealed that LDs are not merely simple energy storage compartments, but also dynamic structures involved in diverse cellular processes like membrane remodeling, regulation of energy homeostasis and stress responses. In this review, we aim to highlight the functions of LDs in plant development and response to environmental changes. In particular, we tackle the fate and roles of LDs during the plant post-stress recovery phase.

Isolation of Plastoglobules for Lipid Analyses.

Plastoglobules are plastid compartments designed for the storage of neutral lipids. They share physical and structural characteristics with cytosolic lipid droplets. Hence, special care must be taken to avoid contamination by cytosolic lipid droplets during plastoglobule purification. We describe the isolation of pure plastoglobules from Arabidopsis thaliana leaves, and the methods we use to determine their lipid composition. After preparation of a crude chloroplast fraction, plastoglobules are isolated from plastid membranes by two steps of ultracentrifugation on discontinuous sucrose gradients. For lipid analyses, total lipids are then extracted by a standard chloroform-methanol protocol, and polar lipids are separated from neutral lipids by liquid-liquid extraction. While polar lipid classes are subsequently separated by thin-layer chromatography (TLC) with the classical Vitiello solvent mix, a double TLC development has to be performed for neutral lipids, to separate phytyl and steryl esters. Lipids are quantified by gas chromatography after conversion of the fatty acids into methyl esters.

Arabidopsis LDIP protein locates at a confined area within the lipid droplet surface and favors lipid droplet formation.

Lipid droplets (LDs) are cell organelles specialized in neutral lipid storage. Extendedly studied in seeds, LDs also accumulate in leaves during senescence or in response to abiotic stresses. However the mechanisms underlying their biogenesis remain relatively unknown. Here, we deciphered the distinct roles of two proteins during LD biogenesis: LD-associated protein 1 (AtLDAP1) and LDAP-interacting protein (AtLDIP). We demonstrated that AtLDIP overexpression favors the neo-formation of small LDs under growing conditions where LD accumulation is usually not observed. In addition, atldip knock-out mutant displayed fewer but larger LDs, confirming a role of AtLDIP in LD biogenesis. Interestingly, a synergistic effect of the overexpression of both AtLDIP and AtLDAP1 was observed, resulting in an increase of LD cluster occurrence and LD abundance within the clusters and the cells. AtLDIP overexpression has no significant impact on triacylglycerol and steryl ester accumulation but AtLDIP inactivation is associated with an increase of neutral lipid content, that is probably a consequence of the enlarged but less abundant LDs present in this line. Our localization study demonstrated that AtLDIP is localized at specific dotted sites within the LD in contrast to AtLDAP1 that covers the whole LD. In addition, AtLDIP sometimes localized away from the LD marker, but always associated with the ER network, suggesting a location at LD nascent sites within the ER. Taken together, our results suggested that AtLDIP promotes the formation of new LDs from ER localized TAG lenses.

Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions.

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data suggest that leaf LDs are functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER.

Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis.

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity.

Metabolic disorders, including obesity, diabetes, and cardiovascular disease, are widespread in Westernized nations. Gut microbiota composition is a contributing factor to the susceptibility of an individual to the development of these disorders; therefore, altering a person's microbiota may ameliorate disease. One potential microbiome-altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N-acylphosphatidylethanolamines (NAPEs) are precursors to the N-acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. Here, we demonstrated that administration of engineered NAPE-expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the levels of obesity in mice fed a high-fat diet. Mice that received modified bacteria had dramatically lower food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria. The protective effects conferred by NAPE-expressing bacteria persisted for at least 4 weeks after their removal from the drinking water. Moreover, administration of NAPE-expressing bacteria to TallyHo mice, a polygenic mouse model of obesity, inhibited weight gain. Our results demonstrate that incorporation of appropriately modified bacteria into the gut microbiota has potential as an effective strategy to inhibit the development of metabolic disorders.

Prefrontal cortex activity during motor tasks with additional mental load requiring attentional demand: a near-infrared spectroscopy study.

Functional near-infrared spectroscopy (fNIRS) is suitable for investigating cerebral oxygenation changes during motor and/or mental tasks. In the present study, we investigated how an additional mental load during a motor task at two submaximal loadings affects the fNIRS-measured brain activation over the right prefrontal cortex (PFC). Fifteen healthy males performed isometric grasping contractions at 15% and 30% of the maximal voluntary contraction (MVC) with or without an additional mental (i.e., arithmetic) task. Mental performance, force variability, fNIRS and subjective perception responses were measured in each condition. The performance of the mental task decreased significantly while the force variability increased significantly at 30% MVC as compared to 15% MVC, suggesting that performance of dual-task required more attentional resources. PFC activity increased significantly as the effort increased from 15% to 30% MVC (p<.001). Although a larger change in the deoxyhemoglobin was observed in dual-task conditions (p=.051), PFC activity did not change significantly as compared to the motor tasks alone. In summary, participants were unable to invest more attention and effort in performing the more difficult levels in order to maintain adequate mental performance.

Occurrence, biosynthesis and functions of N-acylphosphatidylethanolamines (NAPE): not just precursors of N-acylethanolamines (NAE).

N-acylphosphatidylethanolamine (NAPE) is a minor phospholipid resulting from the transfer of an acyl chain from an acyl donor to the primary amine of the ethanolamine moiety of phosphatidylethanolamine (PE). Occurring in plant and animal kingdoms as well as in prokaryotic cells, it is synthesized in higher amounts in membranes during cellular stresses and tissue damage, and it is widely thought to be the precursor of the lipid mediator, N-acylethanolamine (NAE), which modulates the endocannabinoid signaling pathway and therefore regulates various physiological processes. However, recent studies have shown that NAPE is also a bioactive molecule that is involved in several physiological functions. The present paper reviews the occurrence of NAPE in animals and plants and focuses on the various properties of NAPE observed in vitro and in vivo. The different metabolic pathways promoting the synthesis and degradation of NAPE are also discussed and the differences between animals and plants are underlined.

N-Acylethanolamines and related compounds: aspects of metabolism and functions.

N-Acylethanolamines (NAE) are fatty acid derivates that are linked with an ethanolamine group via an amide bond. NAE can be characterized as lipid mediators in the plant and animal kingdoms owing to the diverse functions throughout the eukaryotic domain. The functions of NAE have been widely investigated in animal tissues in part due to their abilities to interact with the cannabinoid receptors, vanilloid receptors or peroxisome proliferator activated receptors. However, the interest of studying the functions of these lipids in plants is progressively becoming more apparent. The number of publications about the functions related to NAE and to structural analogs (homoserine lactone and alkamides) is greatly increasing, showing the importance of these lipids in various plant physiological processes. This review sheds light on their role in different processes such as seedling development, plant pathogen interaction, phospholipase D alpha inhibition and senescence of cut flowers, and underlines the interaction between NAE and NAE-related molecules with plant hormone signaling. The different metabolic pathways promoting the synthesis and degradation of NAE are also discussed, in particular the oxygenation of polyunsaturated N-acylethanolamines, which leads to NAE-oxylipins, a new family of bioactive lipids.

Discovery and characterization of an Arabidopsis thaliana N-acylphosphatidylethanolamine synthase.

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D beta/gamma isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. beta-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.

PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast.

In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either phosphatidylinositol is synthesized from distinct cytidine diphosphate-diacylglycerol molecules, or that, after its synthesis, it is modified by a hypothetical acyltransferase that incorporates saturated fatty acid into neo-synthesized molecules of phosphatidylinositol. We used database search methods to identify an acyltransferase that could catalyze such an activity. Among the various proteins that we studied, we found that Psi1p (phosphatidylinositol stearoyl incorporating 1 protein) is required for the incorporation of stearate into phosphatidylinositol because GC and MS analyses of psi1Delta lipids revealed an almost complete disappearance of stearic (but not of palmitic acid) at the sn-1 position of this phospholipid. Moreover, it was found that, whereas glycerol 3-phosphate, lysophosphatidic acid and 1-acyl lysophosphatidylinositol acyltransferase activities were similar in microsomal membranes isolated from wild-type and psi1Delta cells, microsomal membranes isolated from psi1Delta cells are devoid of the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity that is present in microsomal membranes isolated from wild-type cells. Moreover, after the expression of PSI1 in transgenic psi1Delta cells, the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity was recovered, and was accompanied by a strong increase in the stearic acid content of lysophosphatidylinositol. As previously suggested for phosphatidylinositol from animal cells (which contains almost exclusively stearic acid as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol.

Regioselective acylation of flavonoids catalyzed by immobilized Candida antarctica lipase under reduced pressure.

A single-step acylation of rutin and naringin, catalyzed by immobilized Candida antarctica lipase B in 2-methyl-2-butanol, occurred preferentially on the primary hydroxyl group. Using palmitic methyl ester as acyl donor, the acylation rate of naringin was 10-fold higher than that of rutin. Under optimal conditions, i.e. a molar ratio acyl donor/naringin of 7:1 and 200 mbar, 92% naringin was acylated.