diacylglyceryl-N,N,N-trimethylhomoserine Research Articles

Discovering oxidized polar lipids in microalgae lipidome using liquid chromatography mass spectrometry approaches

Microalgae are rich in polar lipids, such as glycolipids, phospholipids and betaine lipids esterified with omega-3 polyunsaturated fatty acids (PUFA), which are prone to oxidation. Under stress conditions, the redox balance in microalgae is altered, leading to an abnormal production of reactive oxygen species (ROS) that can affect surrounding lipids. While few oxidized polar lipids have been described to play important roles in mammals and possess interesting bioactive properties, their occurrence in microalgae is poorly described, hindering their exploitation as novel bioactive compounds. To enhance our understanding of these lipids, we conducted a targeted analysis of oxidized polar lipids in lipid extracts obtained from five microalgae species: Chlorella vulgaris, Chlorococcum amblystomatis, Scendesmus obliquus, Nanochloropsis oceanica and Phaeodactylum ticornutum, using reverse-phase liquid chromatography mass spectrometry (RP-LC-MS). A detailed analysis of the LC-MS data identified 150 oxidized polar lipid species across different classes of phospholipids, glycolipids and betaine lipids in the five microalgae species. These modified oxidized lipids featured oxygenated species with 1–3 additional oxygen atoms in the fatty acyl chains. The predominant oxidized fatty acids esterified to these lipids were omega-3 eicosapentaenoic acid (EPA, 20:5 n-3) and α-linonelic acid (ALA, 18:3 n-3). Notably, most oxidized lipid species were identified in diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) in C. amblystomatis, S. obliquus and N. oceanica, monogalactosyldiacylglycerol (MGDG) in P. tricornutum, and phosphatidylethanolamine (PE) in C. vulgaris. Furthermore, distinct fragmentation patterns across lipid classes allowed the unequivocal identification of oxidized polar lipids. These findings reveal a diverse array of oxidized polar lipids in microalgae, predominantly enriched with oxidized omega-3 PUFA, highlighting microalgae as a natural source of oxidized polar lipids that may serve as a natural reservoir of bioactive omega-3 oxylipins.

Diacylglyceryl-N,N,N-trimethylhomoserine-dependent lipid remodeling in a green alga, Chlorella kessleri

Membrane lipid remodeling contributes to the environmental acclimation of plants. In the green lineage, a betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), is included exclusively among green algae and nonflowering plants. Here, we show that the green alga Chlorella kessleri synthesizes DGTS under phosphorus-deficient conditions through the eukaryotic pathway via the ER. Simultaneously, phosphatidylcholine and phosphatidylethanolamine, which are similar to DGTS in their zwitterionic properties, are almost completely degraded to release 18.1% cellular phosphorus, and to provide diacylglycerol moieties for a part of DGTS synthesis. This lipid remodeling system that substitutes DGTS for extrachloroplast phospholipids to lower the P-quota operates through the expression induction of the BTA1 gene. Investigation of this lipid remodeling system is necessary in a wide range of lower green plants for a comprehensive understanding of their phosphorus deficiency acclimation strategies.

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii, a well-established model organism, has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms. In previous studies, it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type, but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis. The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C. reinhardtii sta6. By utilizing an electrospray ionization/mass spectrometry (ESI/MS)-based lipidomics approach, a total of 105 membrane lipid molecules of C. reinhardtii were resolved, including 16 monogalactosyldiacylglycerol (MGDG), 16 digalactosyldiacylglycerol (DGDG), 11 phosphatidylglycerol (PG), 6 sulfoquinovosyldiacylglycerol (SQDG), 49 diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), 2 phosphatidylethanolamine (PE), and 5 phosphatidylinositol (PI) molecules. The quantitative results indicated that the membrane lipid profiles were similar between the two C. reinhardtii strains grown under both low- and high-light conditions, but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis. Under low-light conditions, sta6 accumulated more PI, MGDG, DGDG but less amounts of DGTS as compared to WT. Under high light, sta6 cells contained higher content membrane lipids than cc-124, except for PG, which is more or less similar in both strains. Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant, and thereby its physiological impact remains to be explored.

Soil microbial populations substitute phospholipids with betaine lipids in response to low P availability

Soil microbes can be limited by phosphorus availability. A significant proportion of cellular P is accounted for by the phospholipids that comprise membranes. Experiments with aquatic microbial communities and isolated microbes have shown one way microbes can reduce their cellular P requirements is by substituting phospholipids with P-free polar lipids. We do not know if soil microbial communities can also spare P by using P-free lipids instead of phospholipids.The aims of this study were to examine a range of contrasting soils and a) determine the profile of polar fatty-acid-based lipids; and b) examine if P-free polar lipids are more abundant in P-poor soils. To obtain a broad range in P availability we examined soils from 16 sites that encompassed a range of geologies and vegetation types. Intact lipids were identified and quantified using liquid-chromatography-mass spectrometry. An independent estimate of polar lipid classes was obtained by hydrolysing lipids and quantifying headgroups with capillary electrophoresis-mass spectrometry.Among sites soil P status varied from supra-optimal (N:P = 2.4 g g−1) through to severely deficient (N:P = 80 g g−1). The dominant polar lipids among sites were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Among sites as P availability declined a larger proportion of polar lipids was accounted for by betaine lipids such that in the most severely P-deficient soils phospholipids were only 61% of total polar lipids. If we assume that phospholipids account for one-third of cellular P, the measured substitution of phospholipids with betaine lipids would reduce the amount of P required for growth by around 10%. The substantial P saving due to lipid substitution could contribute to the selection of taxa under P deficiency.

Glycerolipid remodeling triggered by phosphorous starvation and recovery in Nannochloropsis oceanica

To understand the function of DGTS (diacylglyceryl-N,N,N-trimethyl-homoserine) as substitution of PC (phosphatidylcholine), Nannochloropsis oceanica has been chosen as the model system because it has DGTS and PC simultaneously. Phosphorus is a regulator for DGTS and PC. Generally, DGTS increases and correspondingly PC decreases under phosphorous starvation. However, the function of DGTS and its fatty acid origin under phosphorous starvation, and the resurgence of PC triggered by phosphorus recovery remain not clarified completely. In the present study, we investigated the effect of phosphorus starvation and recovery on polar glycerolipid remodeling in N. oceanica based on quantitative lipidomic data. Through targeted lipidomics analysis, we proposed that, under phosphorus deprivation, DGTS serves as the central intermediate in glycerolipid remodeling via acting as the carrier of the C18 fatty acids desaturation by replacement of PC, maintaining the biosynthesis of EPA (eicosapentaenoic acid) and providing the EPA acyl as a precursor for formation of chloroplast glycolipids. The prokaryotic molecular species of DGTS are mainly from de novo biosynthesis pathway and make the major contribution for the DGTS increase. In the process of resupplying phosphorus, PC increased attributing to hydrolysis of TAG, generating diacylglycerol for PC biosynthesis. These new information demonstrated that DGTS and PC varied conversely in different metabolic pathways as revealed by targeted lipidomic data.

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.

Metabolomic and transcriptomic analyses reveal the effects of ultraviolet radiation deprivation on Isochrysis galbana at high temperature

The microalga Isochrysis galbana exhibits a seasonal fluctuation of biomass referred to as ‘decline’ during summer, leading to a great economic loss in the mariculture industry. In the present study, we aimed to explore the role of ultraviolet radiation (UVR) in I. galbana decline at high temperature by integrated analysis of metabolomic and transcriptomic data. The positive effects of UVR deprivation on the growth and photosynthetic activity of I. galbana were preliminarily evidenced by the increase in biomass concentration, the effective photochemical efficiency of photosystem II (PSII) (Fv′/Fm′), and oxygen evolution rate. Furthermore, we compared the contents of eight organic acids, total lipids and gene expression of I. galbana grown under natural sunlight versus UVR-deprived conditions, as well as. The contents of some tricarboxylic acid (TCA) cycle-related organic acids were higher in the UVR-deprived group. Lipidomic analysis indicated that UVR deprivation resulted in the accumulation of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC), diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), and lyso-lipids, while triacylglycerol (TAG) was decreased. Consistent with these results, the transcriptomic data showed that the expressions of some key genes involved in the TCA cycle and the photosynthesis pathways were clearly induced by UVR deprivation. Besides, the down-regulation of diacylglycerol O-acyltransferases (DGAT) and the up-regulation of TAG lipase collectively resulted in the lower accumulation of TAG, which was consistent with the metabolomic data. Taken together, the decline of I. galbana in summer could be explained by the disorder of a series of biological processes caused by UVR, especially the damage to photosynthesis, the slowdown of TCA cycle, and the disturbance of lipid metabolism. This study allowed us to better understand the responses of I. galbana to UVR stress and provided useful information for the cultivation of I. galbana in summer, greatly expanding our current knowledge on the genetic information of I. galbana.

Polyphasic approach to a characteristic Ulva population from a limno-rheocrenic, mineral (chloride, sodium, sulphate) spring in the Siwa Oasis (Western Desert of Egypt)

An interesting population of ulvacean green algae was collected from a limno-rheocrenic, thermal, mineral (chloride, sodium, sulphate) spring known as Ain Abu Sherouf in the Siwa Oasis, the Western Desert of Egypt. A detailed combined investigation on its morphotaxonomy, autecology, and a multilocus sequence data set including the chloroplast-encoded rbcL gene and the nuclear-encoded nrDNA SSU and ITS allowed us to identify this population as Ulva flexuosa subsp. paradoxa (syn. Ulva paradoxa, Ulvales, Chlorophyta), although the algal thalli were in average narrower than usual in this taxon. Analysis of the molecular rbcL-ITS sequencing data demonstrated a close phylogenetic relationship of the studied population to two Ulva isolates from Japan and China and their taxonomic status was further discussed. The pigment profiling confirmed chlorophylls and carotenoids typical of the Ulvaceae (lutein, α/β-carotenes, violaxanthin, and neoxanthin). Lipidomic analysis revealed the presence of the monogalactosyl diacylglycerols (MGDG), digalactosyl diacylglycerols (DGDG), sulfoquinovosyl diacylglycerols (SQDG), and diacylglyceryl N,N,N-trimethylhomoserine (DGTS) lipid classes. Presence of the plasma membrane DGTS lipids in remarkable proportion might be an adaptation to this nutrient-poor mineral spring habitat, with relatively low phosphorus values. The lower unsaturation index (UI) values of the plastidial DGDG and SQDG lipids are likely to reflect the need to maintain adequate membrane fluidity in this thermal mineral spring system. This polyphasic study not only extended our little knowledge on the distribution, autecology, and adaptive mechanisms of U. flexuosa subsp. paradoxa in the Saharan habitats of Africa, but also re-assessed and refined the taxonomic and phylogenetic affiliation of previously sampled close relatives from East Asia.

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.

Low-molecular-mass organic acid and lipid responses of Isochrysis galbana Parke to high temperature stress during the entire growth stage

Isochrysis galbana is an important food source in aquaculture especially in the rearing of bivalve mollusks and usually cultured in summer, when water temperatures in breeding pools may reach 35°C. However, the impact of high temperature stress on the growth and metabolism of I. galbana is poorly understood. In this study, the changes of total superoxide dismutase (T-SOD) activity, organic acid, and lipid levels were analyzed at different growth stages when I. galbana Parke was cultured under normal temperature (20°C) and extreme high temperature (35°C), respectively. The result showed that T-SOD activity reached the highest level at exponential phase at both temperatures but exhibited lower level during growth at 35°C. The lower content of citric acid and α-ketoglutaric acid during late stationary phase indicated that tricarboxylic acid (TCA) cycle, amino acid metabolism, and the synthesis of fatty acids may severely be damaged under high temperature stress. The upregulation of monogalactosyldiacylglycerol (MGDG), diacylglycerylcarboxyhydroxymethylcholine (DGCC), diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), triacylglycerol (TAG), and lyso-lipids was observed from adaptation phase to early stationary phase. But downregulation of glyceroglycolipids (MGDG, DGDG, and SQDG), DGCC, and DGTS were found between early stationary phase and late stationary phase at high temperature. Besides, the level of DHA decreased under high temperature stress, revealing that the nutritional value of I. galbana Parke had been reduced.This is the first report addressing metabolic responses of I. galbana Parke to high temperature stress combining T-SOD activity, organic acid, and lipid analysis, and the results demonstrated that the extreme high temperature (35°C) will not only suppress the activity of T-SOD and the formation of organic acids in the TCA cycle and photorespiration, but also influence the lipid metabolism in I. galbana Parke. This study provided a useful reference value for the cultivation of I. galbana under high temperature condition.

Metabolic transformation of microalgae due to light acclimation and genetic modifications followed by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

Metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and lipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid productions were monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72 hour time period under high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5 times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgal population. These capabilities are expected to facilitate the exploration of genetically altered microalgal strains for biofuel production.

Detection and characterization of phosphatidylcholine in various strains of the genus Chlamydomonas (Volvocales, Chlorophyceae).

The laboratory strains of Chlamydomonas reinhardtii have been reported to contain no phosphatidylcholine (PC), which is considered to be replaced by another zwitterionic lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). According to the recently published classification, the strains belonged to the subgroup Reinhardtinia. Screening for PC in 13 selected strains of Chlamydomonas in the NIES Algal Collection, which are different in habitats and belong to different phylogenetic subgroups in the genus, revealed the presence of PC in four strains: a strain in the subgroup Polytominia, and three strains in Reinhardtinia. PC was not detected in three other strains of Reinhardtinia analyzed. The presence/absence of PC was not related to the phylogenetic relationship based on 18S rRNA. DGTS was detected in all the strains analyzed. The rare isomer of linolenic acid, 18:3(5,9,12), which has been found in the DGTS of C. reinhardtii, was found in the PC of the two strains and in the DGTS of the five strains. The occurrence of this fatty acid seems limited to a branch of Reinhardtinia. Acquisition and loss of PC in various strains of Chlamydomonas are discussed from the viewpoint of evolution of PC biosynthetic pathway.

Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus‐free betaine‐lipid diacylglyceryl‐N,N,N‐trimethylhomoserine (605.22)

Diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS) is a phosphorus‐free betaine‐lipid analog of phosphatidylcholine (PtdCho) synthesized by many soil bacteria, algae, and non‐vascular plants. Synthesis of DGTS and other phosphorus‐free lipids in bacteria occurs in response to phosphorus (P) deprivation, and results in the replacement of phospholipids by non‐phosphorous lipids. The genes encoding DGTS biosynthetic enzymes have previously been identified and characterized in bacteria and the alga Chlamydomonas reinhardtii. We now report that many fungal genomes, including those of plant and animal pathogens, encode the enzymatic machinery for DGTS biosynthesis, and that fungi synthesize DGTS during P‐limitation. This finding demonstrates that replacement of phospholipids by non‐phosphorous lipids is a strategy used in divergent eukaryotic lineages for the conservation of P under P‐limiting conditions. Mutants of Neurospora crassa were used to show that DGTS synthase encoded by the BTA1 locus is solely responsible for DGTS biosynthesis, and is under the control of the fungal phosphorus‐deprivation regulon, mediated by the NUC‐1/Pho4p transcription factor. Furthermore, we describe the rational re‐engineering of lipid metabolism in the yeast S. cerevisiae such that PtdCho is completely replaced by DGTS, and show that membrane and organelle biogenesis are largely unaffected in the engineered strain.Grant Funding Source: National Institutes of Health

The major lipid changes of some important diet microalgae during the entire growth phase

Abstract In the present study, we investigated the major lipid changes of six important diet microalgae (Chlorella sp., Nannochloropsis oculata, Nannochloropsis sp., Isochrysis galbana, Phaeodactylum tricornutum Bohlin and Chaetoceros calcitrans) during the entire growth phase using UPLC-Q-TOF MS system. Our results suggested that EPA/AA-rich lipids were largely detected in both Nannochloropsis sp. and N. oculata as a component of diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Moreover, docosahexaenoic acids (DHAs) were detected only in I. galbana mainly as a component of monogalactosyldiacylglycerol (MGDG) and sulphoquinovosyldiacylglycerol (SQDG). Besides, the growth phase of microalgae could be a useful variable for the content enrichment of EFA-rich lipids. A higher content EPA/AA-rich DGTS could be obtained at the end of stationary phase for Nannochloropsis sp. and N. oculata, and a higher content of DHA-rich lipids could be obtained at the end of stationary phase for I. galbana. However, EPA/AA-rich lipids were mainly detected in both P. tricornutum and C. calcitrans mainly as a component of TAG and MGDG, respectively. In addition, a higher content of EPA-rich TAG could be obtained at the end of the stationary phase for P. tricornutum. A higher content of EPA-rich MGDG could be obtained at the end of stationary phase for C. calcitrans. Taken together, considering the high EFA requirement for artificial rearing of marine organisms, Nannochloropsis sp., N. oculata, P. tricornutum, C. calcitrans and I. galbana would be highly recommended to be harvested at the end of the stationary phase. The distribution characteristics of EFA-rich lipids related to 6 microalgae are described in detail.

Rapid Induction of Lipid Droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A

Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.

Remodeling of Membrane Lipids in Iron-starved Chlamydomonas

Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

Characterization of the Betaine Lipids, Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and Diaclyglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), in Brown- and Green-Pigmented Raphidophytes

Characterization of the Betaine Lipids, Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and Diaclyglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA), in Brown- and Green-Pigmented Raphidophytes

Betaine lipids in chlorarachniophytes

SUMMARY Evolving from the endosymbiosis of a green algal cell by a filose amoeba or amoeboflagellate, the chimearic chlorarachniophytes combine unique features retained from both of their ancestral units. They have preserved from the endosymbiont only the nucleomorph and chloroplast. Four strains from three genera of this algal class were studied to identify a set of non-phosphorous-containing polar lipids and their associated fatty acids using the techniques of positive-ion electrospray ionization/mass spectrometry (ESI/MS) and electrospray ionization/mass spectrometry/mass spectrometry (ESI/MS/MS). Fourteen non-phosphorous-containing polar lipids, classified as betaine lipids were primarily identified as forms of diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and its structural isomer diaclyglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA). Though the number of forms of DGTA and DGTA were roughly equal, DGTS composed more of the polar lipid portion present in three of the strains tested, while the fourth, Lotharella globosa, was dominated by forms of DGTA. In addition, a lipid tentatively identified as diacylglycerylcarboxyhydroxymethylcholine (DGCC) was observed twice in minor amounts. The polar lipid-associated fatty acids of the aforementioned algal strains generally included dodecanoic acid (12:0), tetradecanoic acid (14:0), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), and eicosapentaenoic acid [20:5(n-3)]. The differences in betaine lipid content among the species studied may allow for further conclusions to be drawn regarding the taxonomy of chlorarachniophytes.

Annotation of Genes Involved in Glycerolipid Biosynthesis in Chlamydomonas reinhardtii : Discovery of the Betaine Lipid Synthase BTA1 Cr

Lipid metabolism in flowering plants has been intensely studied, and knowledge regarding the identities of genes encoding components of the major fatty acid and membrane lipid biosynthetic pathways is very extensive. We now present an in silico analysis of fatty acid and glycerolipid metabolism in an algal model, enabled by the recent availability of expressed sequence tag and genomic sequences of Chlamydomonas reinhardtii. Genes encoding proteins involved in membrane biogenesis were predicted on the basis of similarity to proteins with confirmed functions and were organized so as to reconstruct the major pathways of glycerolipid synthesis in Chlamydomonas. This analysis accounts for the majority of genes predicted to encode enzymes involved in anabolic reactions of membrane lipid biosynthesis and compares and contrasts these pathways in Chlamydomonas and flowering plants. As an important result of the bioinformatics analysis, we identified and isolated the C. reinhardtii BTA1 (BTA1Cr) gene and analyzed the bifunctional protein that it encodes; we predicted this protein to be sufficient for the synthesis of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), a major membrane component in Chlamydomonas. Heterologous expression of BTA1Cr led to DGTS accumulation in Escherichia coli, which normally lacks this lipid, and allowed in vitro analysis of the enzymatic properties of BTA1Cr. In contrast, in the bacterium Rhodobacter sphaeroides, two separate proteins, BtaARs and BtaBRs, are required for the biosynthesis of DGTS. Site-directed mutagenesis of the active sites of the two domains of BTA1Cr allowed us to study their activities separately, demonstrating directly their functional homology to the bacterial orthologs BtaARs and BtaBRs.

Seasonal changes of lipid content in the leaves of some ferns

Seasonal distribution of total lipids, of total and individual phospholipids as well as of one betaine lipid, diacylglyceryl‐N,N,N‐trimethylhomoserine (DGTS), found in the leaves of Dryopteris carthusiana, Dryopteris filix‐mas, Matteuccia struthiopteris and Pteridium aquilinum was studied. Using thin‐layer chromatography it was shown that lipid composition was different in these species and changed substantially throughout the development of scrolls (unrolled young leaves) to vegetatively mature leaves. The maximum concentration of DGTS for all the examined species was found in scrolls. DGTS then decreased to its minimum level or entirely disappeared to reappear at later stages of leaf development. This may indicate a specific role of DGTS in the plant membrane.