Functional divergence of diacylglycerol acyltransferases in the unicellular green alga Haematococcus pluvialis.

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final committed step in triacylglycerol biosynthesis in eukaryotes. In microalgae, the copy number of DGAT genes is extraordinarily expanded, yet the functions of many DGATs remain largely unknown. This study revealed that microalgal DGAT can function as a lysophosphatidic acyltransferase (LPAAT) both in vitro and in vivo while losing its original function as DGAT. Among the five DGAT-encoding genes identified and cloned from the green microalga Haematococcus pluvialis, four encoded HpDGATs that showed triacylglycerol synthase activities in yeast functional complementation analyses; the exception was one of the type II DGAT encoding genes, HpDGTT2. The hydrophobic recombinant HpDGTT2 protein was purified in soluble form and was found to function as a LPAAT via enzymatic assay. Introducing this gene into the green microalga Chlamydomonas reinhardtii led to retarded cellular growth, enlarged cell size, and enhanced triacylglycerol accumulation, identical to the phenotypes of transgenic strains overexpressing CrLPAAT. This study provides a framework for dissecting uncharacterized DGATs, and could pave the way to decrypting the structure-function relationship of this large group of enzymes that are critical to lipid biosynthesis.

Endoplasmic reticulum acyltransferase with prokaryotic substrate preference contributes to triacylglycerol assembly in Chlamydomonas.

Understanding the unique features of triacylglycerol (TAG) metabolism in microalgae may be necessary to realize the full potential of these organisms for biofuel and biomaterial production. In the unicellular green alga Chlamydomonas reinhardtii a chloroplastic (prokaryotic) pathway has been proposed to play a major role in TAG precursor biosynthesis. However, as reported here, C. reinhardtii contains a chlorophyte-specific lysophosphatidic acid acyltransferase, CrLPAAT2, that localizes to endoplasmic reticulum (ER) membranes. Unlike canonical, ER-located LPAATs, CrLPAAT2 prefers palmitoyl-CoA over oleoyl-CoA as the acyl donor substrate. RNA-mediated suppression of CrLPAAT2 indicated that the enzyme is required for TAG accumulation under nitrogen deprivation. Our findings suggest that Chlamydomonas has a distinct glycerolipid assembly pathway that relies on CrLPAAT2 to generate prokaryotic-like TAG precursors in the ER.

Editing of phosphatidic acid and phosphatidylethanolamine by acyl-CoA: lysophospholipid acyltransferases in developing Camelina sativa seeds.

MAIN CONCLUSIONS: The main source of polyunsaturated acyl-CoA in cytoplasmic acyl-CoA pool of Camelina sativa seeds are fatty acids derived from phosphatidylcholine followed by phosphatidic acid. Contribution of phosphatidylethanolamine is negligible. While phosphatidylethanolamine (PE) is the second most abundant phospholipid, phosphatidic acid (PA) only constitutes a small fraction of C. sativa seeds' polar lipids. In spite of this, the relative contribution of PA in providing fatty acids for the synthesis of acyl-CoA, supplying cytosolic acyl-CoA pool seems to be much higher than the contribution of PE. Our data indicate that up to 5% of fatty acids present in mature C. sativa seeds are first esterified with PA, in comparison to 2% first esterified with PE, before being transferred into acyl-CoA pool via backward reactions of either acyl-CoA:lysophosphatidic acid acyltransferases (CsLPAATs) or acyl-CoA:lysophoshatidylethanolamine acyltransferases (CsLPEATs). Those acyl-CoAs are later reused for lipid biosynthesis or remodelling. In the forward reactions both aforementioned acyltransferases display the highest activity at 30 °C. The spectrum of optimal pH differs for both enzymes with CsLPAATs most active between pH 7.5-9.0 and CsLPEATs between pH 9.0 to 10.0. Whereas addition of magnesium ions stimulates CsLPAATs, calcium and potassium ions inhibit them in concentrations of 0.05-2.0 mM. All three types of ions inhibit CsLPEATs activity. Both tested acyltransferases present the highest preferences towards 16:0-CoA and unsaturated 18-carbon acyl-CoAs in forward reactions. However, CsLPAATs preferentially utilise 18:1-CoA and CsLPEATs preferentially utilise 18:2-CoA while catalysing fatty acid remodelling of PA and PE, respectively.

Biosynthetic Enzymes of Membrane Glycerophospholipid Diversity as Therapeutic Targets for Drug Development.

Biophysical properties of membranes are dependent on their glycerophospholipid compositions. Lysophospholipid acyltransferases (LPLATs) selectively incorporate fatty chains into lysophospholipids to affect the fatty acid composition of membrane glycerophospholipids. Lysophosphatidic acid acyltransferases (LPAATs) of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family incorporate fatty chains into phosphatidic acid during the de novo glycerophospholipid synthesis in the Kennedy pathway. Other LPLATs of both the AGPAT and the membrane bound O-acyltransferase (MBOAT) families further modify the fatty chain compositions of membrane glycerophospholipids in the remodeling pathway known as the Lands' cycle. The LPLATs functioning in these pathways possess unique characteristics in terms of their biochemical activities, regulation of expressions, and functions in various biological contexts. Essential physiological functions for LPLATs have been revealed in studies using gene-deficient mice, and important roles for several enzymes are also indicated in human diseases where their mutation or dysregulation causes or contributes to the pathological condition. Now several LPLATs are emerging as attractive therapeutic targets, and further understanding of the mechanisms underlying their physiological and pathological roles will aid in the development of novel therapies to treat several diseases that involve altered glycerophospholipid metabolism.

Increased triacylglycerol production in oleaginous microalga Neochloris oleoabundans by overexpression of plastidial lysophosphatidic acid acyltransferase.

BACKGROUND: Microalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesis RESULTS: NeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years. CONCLUSIONS: Our results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.

Docosahexaenoic acid preserves visual function by maintaining correct disc morphology in retinal photoreceptor cells.

Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction.

Expression of AGPAT2, an enzyme involved in the glycerophospholipid/triacylglycerol biosynthesis pathway, is directly regulated by HIF-1 and promotes survival and etoposide resistance of cancer cells under hypoxia.

Hypoxia inducible factor-1 (HIF-1) supports survival of normal cells under low oxygen concentration and cancer cells in the hypoxic tumor microenvironment. This involves metabolic reprogramming via upregulation of glycolysis, downregulation of oxidative phosphorylation and, less well documented, effects on lipid metabolism. To investigate the latter, we examined expression of relevant enzymes in cancer cells grown under hypoxia. We show that expression of acylglycerol-3-phosphate acyltransferase 2 (AGPAT2), also known as lysophosphatidic acid acyltransferase ß (LPAATß), was upregulated under hypoxia and this was impaired by siRNA-mediated knockdown of HIF-1α. Moreover, a sequence of the AGPAT2 gene promoter region, containing 6 putative Hypoxia Response Elements (HREs), activated transcription of a reporter gene under hypoxic conditions or in normoxic cells over-expressing HIF-1α. Chromatin immunoprecipitation experiments confirmed binding of HIF-1α to one of these HREs, mutation of which abolished hypoxic activation of the AGPAT2 promoter. Knockdown of AGPAT2 by siRNA reduced lipid droplet accumulation and cell viability under hypoxia and increased cancer cell sensitivity to the chemotherapeutic etoposide. In conclusion, our findings demonstrate that AGPAT2, which is mutated in patients with congenital generalized lipodystrophy and over-expressed in different types of cancer, is a direct transcriptional target of HIF-1, suggesting that upregulation of lipid storage by HIF-1 plays an important role in adaptation and survival of cancer cells under low oxygen conditions.

Lpaatδ/Agpat4 deficiency impairs maximal force contractility in soleus and alters fibre type in extensor digitorum longus muscle.

Lysophosphatidic acid acyltransferase (LPAAT) δ/acylglycerophosphate acyltransferase 4 is a mitochondrial enzyme and one of five homologues that catalyze the acyl-CoA-dependent synthesis of phosphatidic acid (PA) from lysophosphatidic acid. We studied skeletal muscle LPAATδ and found highest levels in soleus, a red oxidative fibre-type that is rich in mitochondria, and lower levels in extensor digitorum longus (EDL) (white glycolytic) and gastrocnemius (mixed fibre-type). Using Lpaatδ-deficient mice, we found no change in soleus or EDL mass, or in treadmill time-to-exhaustion compared to wildtype littermates. There was, however, a significant reduction in the proportion of type I and type IIA fibres in EDL but, surprisingly, not soleus, where these fibre-types predominate. Also unexpectedly, there was no impairment in force generation by EDL, but a significant reduction by soleus. Oxidative phosphorylation and activity of complexes I, I + II, III, and IV in soleus mitochondria was unchanged and therefore could not explain this effect. However, pyruvate dehydrogenase activity was significantly reduced in Lpaatδ-/- soleus and EDL. Analysis of cellular lipids indicated no difference in soleus triacylglycerol, but specific elevations in soleus PA and phosphatidylethanolamine levels, likely due to a compensatory upregulation of Lpaatß and Lpaatε in Lpaatδ-/- mice. An anabolic effect for PA as an activator of skeletal muscle mTOR has been reported, but we found no change in serine 2448 phosphorylation, indicating reduced soleus force generation is unlikely due to the loss of mTOR activation by a specific pool of LPAATδ-derived PA. Our results identify an important role for LPAATδ in soleus and EDL.

A genome-wide analysis of the lysophosphatidate acyltransferase (LPAAT) gene family in cotton: organization, expression, sequence variation, and association with seed oil content and fiber quality.

BACKGROUND: Lysophosphatidic acid acyltransferase (LPAAT) encoded by a multigene family is a rate-limiting enzyme in the Kennedy pathway in higher plants. Cotton is the most important natural fiber crop and one of the most important oilseed crops. However, little is known on genes coding for LPAATs involved in oil biosynthesis with regard to its genome organization, diversity, expression, natural genetic variation, and association with fiber development and oil content in cotton. RESULTS: In this study, a comprehensive genome-wide analysis in four Gossypium species with genome sequences, i.e., tetraploid G. hirsutum- AD1 and G. barbadense- AD2 and its possible ancestral diploids G. raimondii- D5 and G. arboreum- A2, identified 13, 10, 8, and 9 LPAAT genes, respectively, that were divided into four subfamilies. RNA-seq analyses of the LPAAT genes in the widely grown G. hirsutum suggest their differential expression at the transcriptional level in developing cottonseeds and fibers. Although 10 LPAAT genes were co-localised with quantitative trait loci (QTL) for cottonseed oil or protein content within a 25-cM region, only one single strand conformation polymorphic (SSCP) marker developed from a synonymous single nucleotide polymorphism (SNP) of the At-Gh13LPAAT5 gene was significantly correlated with cottonseed oil and protein contents in one of the three field tests. Moreover, transformed yeasts using the At-Gh13LPAAT5 gene with the two sequences for the SNP led to similar results, i.e., a 25-31% increase in palmitic acid and oleic acid, and a 16-29% increase in total triacylglycerol (TAG). CONCLUSIONS: The results in this study demonstrated that the natural variation in the LPAAT genes to improving cottonseed oil content and fiber quality is limited; therefore, traditional cross breeding should not expect much progress in improving cottonseed oil content or fiber quality through a marker-assisted selection for the LPAAT genes. However, enhancing the expression of one of the LPAAT genes such as At-Gh13LPAAT5 can significantly increase the production of total TAG and other fatty acids, providing an incentive for further studies into the use of LPAAT genes to increase cottonseed oil content through biotechnology.

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels.

The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.

Molecular evolution of the lysophosphatidic acid acyltransferase (LPAAT) gene family.

Lysophosphatidic acid acyltransferases (LPAATs) perform an essential cellular function by controlling the production of phosphatidic acid (PA), a key intermediate in the synthesis of membrane, signaling and storage lipids. Although LPAATs have been extensively explored by functional and biotechnological studies, little is known about their molecular evolution and diversification. We performed a genome-wide analysis using data from several plants and animals, as well as other eukaryotic and prokaryotic species, to identify LPAAT genes and analyze their evolutionary history. We used phylogenetic and molecular evolution analysis to test the hypothesis of distinct origins for these genes. The reconstructed phylogeny supported the ancient origin of some isoforms (plant LPAAT1 and LPAATB; animal AGPAAT1/2), while others emerged more recently (plant LPAAT2/3/4/5; AGPAAT3/4/5/8). Additionally, the hypothesis of endosymbiotic origin of the plastidic isoform LPAAT1 was confirmed. LPAAT genes from plants and animals mainly experienced strong purifying selection pressures with limited functional divergence after the species-specific duplications. Gene expression analyses of LPAAT isoforms in model plants demonstrated distinct LPAAT expression patterns in these organisms. The results showed that distinct origins followed by diversification of the LPAAT genes shaped the evolution of TAG biosynthesis. The expression pattern of individual genes may be responsible for adaptation into multiple ecological niches.

A novel lysophosphatidic acid acyltransferase enzyme (LPAAT4) with a possible role for incorporating docosahexaenoic acid into brain glycerophospholipids.

Glycerophospholipids are important components of cellular membranes, required for constructing structural barriers, and for providing precursors of bioactive lipid mediators. Lysophosphatidic acid acyltransferases (LPAATs) are enzymes known to function in the de novo glycerophospholipid biosynthetic pathway (Kennedy pathway), using lysophosphatidic acid (LPA) and acyl-CoA to form phosphatidic acid (PA). Until now, three LPAATs (LPAAT1, 2, and 3) have been reported from the 1-acyl-glycerol-3-phosphate O-acyltransferase (AGPAT) family. In this study, we identified a fourth LPAAT enzyme, LPAAT4, previously known as an uncharacterized enzyme AGPAT4 (LPAATδ), from the AGPAT family. Although LPAAT4 was known to contain AGPAT motifs essential for acyltransferase activities, detailed biochemical properties were unknown. Here, we found that mouse LPAAT4 (mLPAAT4) possesses LPAAT activity with high acyl-CoA specificity for polyunsaturated fatty acyl-CoA, especially docosahexaenoyl-CoA (22:6-CoA, DHA-CoA). mLPAAT4 was distributed in many tissues, with relatively high expression in the brain, rich in docosahexaenoic acid (DHA, 22:6). mLPAAT4 siRNA in a neuronal cell line, Neuro 2A, caused a decrease in LPAAT activity with 22:6-CoA, suggesting that mLPAAT4 functions endogenously. siRNA in Neuro 2A cells caused a decrease in 18:0-22:6 PC, whereas mLPAAT4 overexpression in Chinese hamster ovary (CHO)-K1 cells caused an increase in this species. Although DHA is considered to have many important functions for the brain, the mechanism of its incorporation into glycerophospholipids is unknown. LPAAT4 might have a significant role for maintaining DHA in neural membranes. Identification of LPAAT4 will possibly contribute to understanding the regulation and the biological roles of DHA-containing glycerophospholipids in the brain.

Phosphatidylethanolamines link ferroptosis and autophagy during appressorium formation of rice blast fungus.

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.

Lipid Metabolism in Cancer: The Role of Acylglycerolphosphate Acyltransferases (AGPATs).

Altered lipid metabolism is an emerging hallmark of aggressive tumors, as rapidly proliferating cancer cells reprogram fatty acid (FA) uptake, synthesis, storage, and usage to meet their increased energy demands. Central to these adaptive changes, is the conversion of excess FA to neutral triacylglycerides (TAG) and their storage in lipid droplets (LDs). Acylglycerolphosphate acyltransferases (AGPATs), also known as lysophosphatidic acid acyltransferases (LPAATs), are a family of five enzymes that catalyze the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA), the second step of the TAG biosynthesis pathway. PA, apart from its role as an intermediate in TAG synthesis, is also a precursor of glycerophospholipids and a cell signaling molecule. Although the different AGPAT isoforms catalyze the same reaction, they appear to have unique non-overlapping roles possibly determined by their distinct tissue expression and substrate specificity. This is best exemplified by the role of AGPAT2 in the development of type 1 congenital generalized lipodystrophy (CGL) and is also manifested by recent studies highlighting the involvement of AGPATs in the physiology and pathology of various tissues and organs. Importantly, AGPAT isoform expression has been shown to enhance proliferation and chemoresistance of cancer cells and correlates with increased risk of tumor development or aggressive phenotypes of several types of tumors.

Longer Duration of Active Oil Biosynthesis during Seed Development Is Crucial for High Oil Yield-Lessons from Genome-Wide In Silico Mining and RNA-Seq Validation in Sesame.

Sesame, one of the ancient oil crops, is an important oilseed due to its nutritionally rich seeds with high protein content. Genomic scale information for sesame has become available in the public databases in recent years. The genes and their families involved in oil biosynthesis in sesame are less studied than in other oilseed crops. Therefore, we retrieved a total of 69 genes and their translated amino acid sequences, associated with gene families linked to the oil biosynthetic pathway. Genome-wide in silico mining helped identify key regulatory genes for oil biosynthesis, though the findings require functional validation. Comparing sequences of the SiSAD (stearoyl-acyl carrier protein (ACP)-desaturase) coding genes with known SADs helped identify two SiSAD family members that may be palmitoyl-ACP-specific. Based on homology with lysophosphatidic acid acyltransferase (LPAAT) sequences, an uncharacterized gene has been identified as SiLPAAT1. Identified key regulatory genes associated with high oil content were also validated using publicly available transcriptome datasets of genotypes contrasting for oil content at different developmental stages. Our study provides evidence that a longer duration of active oil biosynthesis is crucial for high oil accumulation during seed development. This underscores the importance of early onset of oil biosynthesis in developing seeds. Up-regulating, identified key regulatory genes of oil biosynthesis during early onset of seed development, should help increase oil yields.

A Review of Erucic Acid Production in Brassicaceae Oilseeds: Progress and Prospects for the Genetic Engineering of High and Low-Erucic Acid Rapeseeds (Brassica napus).

Erucic acid (C22:1, ω-9, EA) is a very-long-chain monounsaturated fatty acid (FA) that is an important oleochemical product with a wide range of uses in metallurgy, machinery, rubber, the chemical industry, and other fields because of its hydrophobicity and water resistance. EA is not easily digested and absorbed in the human body, and high-EA rapeseed (HEAR) oil often contains glucosinolates. Both glucosinolates and EA are detrimental to health and can lead to disease, which has resulted in strict guidelines by regulatory bodies on maximum EA contents in oils. Increasingly, researchers have attempted to enhance the EA content in Brassicaceae oilseeds to serve industrial applications while conversely reducing the EA content to ensure food safety. For the production of both LEAR and HEAR, biotechnology is likely to play a fundamental role. Elucidating the metabolic pathways of EA can help inform the improvement of Brassicaceae oilseeds through transgenic technology. In this paper, we introduce the industrial applications of HEAR oil and health benefits of low-EA rapeseed (LEAR) oil first, following which we review the biosynthetic pathways of EA, introduce the EA resources from plants, and focus on research related to the genetic engineering of EA in Brassicaceae oilseeds. In addition, the effects of the environment on EA production are addressed, and the safe cultivation of HEAR and LEAR is discussed. This paper supports further research into improving FAs in Brassicaceae oilseeds through transgenic technologies and molecular breeding techniques, thereby advancing the commercialization of transgenic products for better application in various fields.

Cellular phosphatidic acid sensor, α-synuclein N-terminal domain, detects endogenous phosphatidic acid in macrophagic phagosomes and neuronal growth cones.

Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation.

Enzymes of glycerol-3-phosphate pathway in triacylglycerol synthesis in plants: Function, biotechnological application and evolution.

Triacylglycerols (TAG) are the major form of energy storage in plants. TAG are primarily stored in seeds and fruits, but vegetative tissues also possess a high capacity for their synthesis and storage. These storage lipids are essential to plant development, being used in seedling growth during germination, pollen development, and sexual reproduction, for example. TAG are also an important source of edible oils for animal and human consumption, and are used for fuel and industrial feedstocks. The canonical pathway leading to TAG synthesis is the glycerol-3-phosphate, or Kennedy, pathway, which is an evolutionarily conserved process in most living organisms. The enzymatic machinery for synthesizing TAG is well known in several plant species, and the genes encoding these enzymes have been the focus of many studies. Here, we review recent progress on the understanding of evolutionary, functional and biotechnological aspects of the glycerol-3-phosphate pathway enzymes that produce TAG. We discuss current knowledge about their functional aspects, and summarize valuable insights into genetically engineered plants for enhancing TAG accumulation. Also, we highlight the evolutionary history of these genes and present a meta-analysis linking positive selection to gene family and plant diversification, and also to the domestication processes in oilseed crops.

The Structure and Function of Acylglycerophosphate Acyltransferase 4/ Lysophosphatidic Acid Acyltransferase Delta (AGPAT4/LPAATδ).

Lipid-modifying enzymes serve crucial roles in cellular processes such as signal transduction (producing lipid-derived second messengers), intracellular membrane transport (facilitating membrane remodeling needed for membrane fusion/fission), and protein clustering (organizing lipid domains as anchoring platforms). The lipid products crucial in these processes can derive from different metabolic pathways, thus it is essential to know the localization, substrate specificity, deriving products (and their function) of all lipid-modifying enzymes. Here we discuss an emerging family of these enzymes, the lysophosphatidic acid acyltransferases (LPAATs), also known as acylglycerophosphate acyltransferases (AGPATs), that produce phosphatidic acid (PA) having as substrates lysophosphatidic acid (LPA) and acyl-CoA. Eleven LPAAT/AGPAT enzymes have been identified in mice and humans based on sequence homologies, and their localization, specific substrates and functions explored. We focus on one member of the family, LPAATδ, a protein expressed mainly in brain and in muscle (though to a lesser extent in other tissues); while at the cellular level it is localized at the trans-Golgi network membranes and at the mitochondrial outer membranes. LPAATδ is a physiologically essential enzyme since mice knocked-out for Lpaatδ show severe dysfunctions including cognitive impairment, impaired force contractility and altered white adipose tissue. The LPAATδ physiological roles are related to the formation of its product PA. PA is a multifunctional lipid involved in cell signaling as well as in membrane remodeling. In particular, the LPAATδ-catalyzed conversion of LPA (inverted-cone-shaped lipid) to PA (cone-shaped lipid) is considered a mechanism of deformation of the bilayer that favors membrane fission. Indeed, LPAATδ is an essential component of the fission-inducing machinery driven by the protein BARS. In this process, a protein-tripartite complex (BARS/14-3-3γ/phosphoinositide kinase PI4KIIIß) is recruited at the trans-Golgi network, at the sites where membrane fission is to occur; there, LPAATδ directly interacts with BARS and is activated by BARS. The resulting formation of PA is essential for membrane fission occurring at those spots. Also in mitochondria PA formation has been related to fusion/fission events. Since PA is formed by various enzymatic pathways in different cell compartments, the BARS-LPAATδ interaction indicates the relevance of lipid-modifying enzymes acting exactly where their products are needed (i.e., PA at the Golgi membranes).

Liquid chromatography-mass spectrometry based approach for rapid comparison of lysophosphatidic acid acyltransferase activity on multiple substrates.

Lysophosphatidic acid acyltransferases (LPAAT) play an essential role in generating phosphatidic acid (PA), a key intermediate for phospholipids and triacylglycerol synthesis. The individual members have a diversity of localisation, and a strong fatty acid substrate preference. In vitro LPAAT enzymatic activity assays are necessary for understanding the physiological function of these enzymes. In this work, we have developed a liquid chromatography-mass spectrometry (LC-MS) based rapid enzymatic assay without using radioactive labelling. We show that this approach is comparable to radioactive labelling assays, using either native or non-native lysophosphatidic acid receiver molecules. Most importantly, this approach can be applied to the comparison of multiple substrates in a single assay. The approach is also adaptable for other lipid enzymatic assays.