Phosphatidylcholine:diacylglycerol cholinephosphotransferase's unique regulation of castor bean oil quality.

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with "common" acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

Camelina sativa phosphatidylcholine:diacylglycerol cholinephosphotransferase-catalyzed interconversion does not discriminate between substrates.

Phosphatidylcholine:diacylglycerol cholinephosphotransferases (PDCT) regulate the fatty acid composition of seed oil (triacylglycerol, TAG) by interconversion of diacylglycerols (DAG) and phosphatidylcholine (PtdCho). PtdCho is the substrate for polyunsaturated fatty acid biosynthesis, as well as for a number of unusual fatty acids. By the action of PDCT, these fatty acids can be transferred into the DAG pool to be utilized in TAG biosynthesis by the action of acyl-CoA:DAG and phospholipid:diacylglycerol acyltransferases. Despite its importance in regulating seed oil composition, biochemical characterization of PDCT enzymes has been lacking. We characterized Camelina sativa PDCT in microsomal preparations of a yeast strain expressing Camelina PDCT and lacking the capacity of producing TAG. Camelina PDCT was specific for PtdCho and the sn-1,2 enantiomer of DAG and could not utilize ceramide. The interconversion reaches equilibrium within 15 min of incubation, indicating that only distinct pools of DAG and PtdCho were available for exchange. However, the pool sizes of DAG and PtdCho involved in the exchange were not fixed but increased with the amount of exogenous DAG or PtdCho added. Camelina PDCT showed about the same selectivity for di-oleoyl, di-linoleoyl, and di-linolenoyl species in both PtdCho and DAG substrates, suggesting that no unidirectional transfer of particular unsaturated substrates occurred. Camelina PDCT had a good activity with erucoyl-DAG as a substrate despite low erucic acid levels in PtdCho in plant species accumulating a high amount of this fatty acid in the seed oil.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Acyltransferases Regulate Oil Quality in Camelina sativa Through Both Acyl Donor and Acyl Acceptor Specificities.

Camelina sativa is an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuring in vitro substrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically, C. sativa acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase in C. sativa seeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressing C. sativa seeds. Thus, the in vitro data correlate well with the changes in the overall fatty acid profile and TAG species in C. sativa seeds with altered DGAT1 and PDAT activity. Additionally, in vitro studies of C. sativa phosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previous in vivo studies regarding this enzyme.

Oil crops for the future.

Agriculture faces enormous challenges including the need to substantially increase productivity, reduce environmental footprint, and deliver renewable alternatives that are being addressed by developing new oil crops for the future. The efforts include domestication of Lepidium spp. using genomics-aided breeding as a cold hardy perennial high-yielding oil crop that provides substantial environmental benefits, expands the geography for oil crops, and improves farmers' economy. In addition, genetic engineering in Crambe abyssinica may lead to a dedicated industrial oil crop to replace fossil oil. Redirection of photosynthates from starch to oil in plant tubers and cereal endosperm also provides a path for enhancing oil production to meet the growing demands for food, fuel, and biomaterials. Insect pheromone components are produced in seed oil plants in a cost-effective and environmentally friendly pest management replacing synthetically produced pheromones. Autophagy is explored for increasing crop fitness and oil accumulation using genetic engineering in Arabidopsis.

Crambe hispanica Subsp. abyssinica Diacylglycerol Acyltransferase Specificities Towards Diacylglycerols and Acyl-CoA Reveal Combinatorial Effects That Greatly Affect Enzymatic Activity and Specificity.

Crambe is an oil crop suitable for industrial purposes due to the high content of erucic acid (22:1) in the seed oil. The final acylation of diacylglycerols (DAG) with acyl-CoA in the production of triacylglycerols (oil) is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. We identified eight forms of DGATs in crambe and characterized them in microsomal preparations of yeast expressing the enzymes using various acyl-CoAs and both di-6:0-DAG and long-chain DAG species as acyl acceptors. All DGATs accepted 22:1-CoA when using di-6:0-DAG as acyl acceptor. When di-22:1-DAG was the acyl acceptor, the DGAT1 type of enzyme utilized 22:1-CoA at a much-reduced rate compared to assays with sn-1-22:1-sn-2-18:1(oleoyl)-DAG, the most frequently available DAG precursor in crambe seeds. None of the DGAT2 enzymes was able to acylate di-22:1-DAG. Our results indicate that formation of trierucin by crambe DGATs is a limiting step for further increasing the levels of 22:1 in the previously developed transgenic crambe lines due to their poor abilities to acylate di-22:1-DAG. We also show that the acyl-CoA specificities and the enzymatic activities are highly influenced by the fatty acid composition of the DAG acyl acceptor. This finding implies that the use of artificial acyl acceptors (e.g. di-6:0-DAG) may not always reflect the actual acyl-CoA specificities of DGATs in planta. The relevance of the here reported pronounced specificities for specific DAG species exerted by DGAT enzymes is discussed in the context of the findings of DAG pools of distinct catalytic origin in triacylglycerol biosynthesis in the seed oil.

Isoforms of Acyl-CoA:Diacylglycerol Acyltransferase2 Differ Substantially in Their Specificities toward Erucic Acid.

In most oilseeds, two evolutionarily unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, are the main contributors to the acylation of diacylglycerols in the synthesis of triacylglycerol. DGAT1 and DGAT2 are both present in the important crop oilseed rape (Brassica napus), with each type having four isoforms. We studied the activities of DGAT isoforms during seed development in microsomal fractions from two oilseed rape cultivars: edible, low-erucic acid (22:1) MONOLIT and nonedible high-erucic acid MAPLUS. Whereas the specific activities of DGATs were similar with most of the tested acyl-CoA substrates in both cultivars, MAPLUS had 6- to 14-fold higher activity with 22:1-CoA than did MONOLIT. Thus, DGAT isoforms with different acyl-CoA specificities are differentially active in the two cultivars. We characterized the acyl-CoA specificities of all DGAT isoforms in oilseed rape in the microsomal fractions of yeast cells heterologously expressing these enzymes. All four DGAT1 isoforms showed similar and broad acyl-CoA specificities. However, DGAT2 isoforms had much narrower acyl-CoA specificities: two DGAT2 isoforms were highly active with 22:1-CoA, while the ability of the other two isoforms to use this substrate was impaired. These findings elucidate the importance, which a DGAT isoform with suitable acyl-CoA specificity may have, when aiming for high content of a particular fatty acid in plant triacylglycerol reservoirs.

Specialized lysophosphatidic acid acyltransferases contribute to unusual fatty acid accumulation in exotic Euphorbiaceae seed oils.

MAIN CONCLUSION: In vivo and in vitro analyses of Euphorbiaceae species' triacylglycerol assembly enzymes substrate selectivity are consistent with the co-evolution of seed-specific unusual fatty acid production and suggest that many of these genes will be useful for biotechnological production of designer oils. Many exotic Euphorbiaceae species, including tung tree (Vernicia fordii), castor bean (Ricinus communis), Bernardia pulchella, and Euphorbia lagascae, accumulate unusual fatty acids in their seed oils, many of which have valuable properties for the chemical industry. However, various adverse plant characteristics including low seed yields, production of toxic compounds, limited growth range, and poor resistance to abiotic stresses have limited full agronomic exploitation of these plants. Biotechnological production of these unusual fatty acids (UFA) in high yielding non-food oil crops would provide new robust sources for these valuable bio-chemicals. Previous research has shown that expression of the primary UFA biosynthetic gene alone is not enough for high-level accumulation in transgenic seed oils; other genes must be included to drive selective UFA incorporation into oils. Here, we use a series of in planta molecular genetic studies and in vitro biochemical measurements to demonstrate that lysophosphatidic acid acyltransferases from two Euphorbiaceae species have high selectivity for incorporation of their respective unusual fatty acids into the phosphatidic acid intermediate of oil biosynthesis. These results are consistent with the hypothesis that unusual fatty acid accumulation arose in part via co-evolution of multiple oil biosynthesis and assembly enzymes that cooperate to enhance selective fatty acid incorporation into seed oils over that of the common fatty acids found in membrane lipids.

PlantFAdb: a resource for exploring hundreds of plant fatty acid structures synthesized by thousands of plants and their phylogenetic relationships.

Over 450 structurally distinct fatty acids are synthesized by plants. We have developed PlantFAdb.org, an internet-based database that allows users to search and display fatty acid composition data for over 9000 plants. PlantFAdb includes more than 17 000 data tables from >3000 publications and hundreds of unpublished analyses. This unique feature allows users to easily explore chemotaxonomic relationships between fatty acid structures and plant species by displaying these relationships on dynamic phylogenetic trees. Users can navigate between order, family, genus and species by clicking on nodes in the tree. The weight percentage of a selected fatty acid is indicated on phylogenetic trees and clicking in the graph leads to underlying data tables and publications. The display of chemotaxonomy allows users to quickly explore the diversity of plant species that produce each fatty acid and that can provide insights into the evolution of biosynthetic pathways. Fatty acid compositions and other parameters from each plant species have also been compiled from multiple publications on a single page in graphical form. Links provide simple and intuitive navigation between fatty acid structures, plant species, data tables and the publications that underlie the datasets. In addition to providing an introduction to this resource, this report illustrates examples of insights that can be derived from PlantFAdb. Based on the number of plant families and orders that have not yet been surveyed we estimate that a large number of novel fatty acid structures are still to be discovered in plants.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis.

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells.

Determination of Substrate Preferences for Desaturases and Elongases for Production of Docosahexaenoic Acid from Oleic Acid in Engineered Canola.

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola (Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants.

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil.

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS.

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).

Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols.

Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids.

Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.

RNAi targeting putative genes in phosphatidylcholine turnover results in significant change in fatty acid composition in Crambe abyssinica seed oil.

The aim of this study was to evaluate the importance of three enzymes, LPCAT, PDCT and PDAT, involved in acyl turnover in phosphatidylcholine in order to explore the possibility of further increasing erucic acid (22:1) content in Crambe seed oil. The complete coding sequences of LPCAT1-1 and LPCAT1-2 encoding lysophosphatidylcholine acyltransferase (LPCAT), PDCT1 and PDCT2 encoding phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), and PDAT encoding phospholipid:diacylglycerol acyltransferase (PDAT) were cloned from developing Crambe seeds. The alignment of deduced amino acid sequences displayed a high similarity to the Arabidopsis homologs. Transgenic lines expressing RNA interference (RNAi) targeting either single or double genes showed significant changes in the fatty acid composition of seed oil. An increase in oleic acid (18:1) was observed, to varying degrees, in all of the transgenic lines, and a cumulative effect of increased 18:1 was shown in the LPCAT-PDCT double-gene RNAi. However, LPCAT single-gene RNAi led to a decrease in 22:1 accumulation, while PDCT or PDAT single-gene RNAi had no obvious effect on the level of 22:1. In agreement with the abovementioned oil phenotypes, the transcript levels of the target genes in these transgenic lines were generally reduced compared to wild-type levels. In this paper, we discuss the potential to further increase the 22:1 content in Crambe seed oil through downregulation of these genes in combination with fatty acid elongase and desaturases.

Novel reactions in acyl editing of phosphatidylcholine by lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities in microsomal preparations of plant tissues.

MAIN CONCLUSION: Plants have lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities. The combined action of LPCT and GPCAT provides a novel route of PC re-synthesis after its deacylation. Phosphatidylcholine (PC) is the major lipid in eukaryotic membranes and has a central role in overall plant lipid metabolism. It is also the site of production of polyunsaturated fatty acids in plants. The recently discovered acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activity in yeast provides a novel route of re-synthesising PC via lysophosphatidylcholine (LPC) after its deacylation. This route does not require the degradation of the glycerophosphocholine (GPC) into free choline, the activation of choline to CDP-choline, nor the utilization of CDP-choline by the CDP-choline:diacylglycerol cholinephosphotransferase. We show here that GPCAT activities also are present in membrane preparations from developing oil seeds of safflower and other species as well as in membrane preparations of roots and leaves of Arabidopsis, indicating that GPCAT activity plays a ubiquitous role in plant lipid metabolism. The last step in formation of GPC, the substrate for GPCAT, is the deacylation of LPC. Microsomal membranes of developing safflower seeds utilized LPC in LPC:LPC transacylation reactions (LPCT activities) creating PC and GPC. The results demonstrate that safflower membranes have LPCT and GPCAT activities that represent novel reactions for PC acyl editing. The physiological relevance of these reactions probably has to await identification of the enzymes catalysing these reactions.