Validation of a New Multicistronic Plasmid for the Efficient and Stable Expression of Transgenes in Microalgae.

Low stability of transgenes and high variability of their expression levels among the obtained transformants are still pending challenges in the nuclear genetic transformation of microalgae. We have generated a new multicistronic microalgal expression plasmid, called Phyco69, to make easier the large phenotypic screening usually necessary for the selection of high-expression stable clones. This plasmid contains a polylinker region (PLK) where any gene of interest (GOI) can be inserted and get linked, through a short viral self-cleaving peptide to the amino terminus of the aminoglycoside 3'-phosphotransferase (APHVIII) from Streptomyces rimosus, which confers resistance to the antibiotic paromomycin. The plasmid has been validated by expressing a second antibiotic resistance marker, the ShBLE gene, which confers resistance to phleomycin. It has been shown, by RT-PCR and by phenotypic studies, that the fusion of the GOI to the selective marker gene APHVIII provides a simple method to screen and select the transformants with the highest level of expression of both the APHVIII gene and the GOI among the obtained transformants. Immunodetection studies have shown that the multicistronic transcript generated from Phyco69 is correctly processed, producing independent gene products from a common promoter.

Evaluation and optimization of a methodology for the long-term cryogenic storage of Tetradesmus obliquus at - 80 °C.

Cryopreservation is a common methodology for long-term microalgae storage. Current cryopreservation methods are based on using diverse cryoprotectants and two-step cooling protocols, followed by sample storage at the temperature of liquid nitrogen (- 196 °C). However, the use of this methodology requires a continuous liquid N2 supply as well as facilities with dedicated equipment, which is not affordable for every laboratory. In our work, we report on the successful development of a simple and cost-effective method for the long-term cryogenic storage of Tetradesmus obliquus at temperatures (- 80 °C) used in commonly available deep freezers that are more readily accessible to laboratories. Two procedures were evaluated that were originally devised for other microalgae; this was followed by the optimization of critical parameters such as the sample's microalgal concentration and the cryoprotectant reagent's incubation time. Cell viability was monitored using the survival rates obtained by direct agar plating and the growth recovery times in liquid cultures. Viability-related variables were recorded following different storage times of up to 3 years. The main operational factors involved in the process (cell concentration, incubation time, and storage time) were statistically analyzed with regard to their influence on the survival rate. The statistical analysis showed interdependence (a two-factor interaction) between the cellular concentration and the cryoprotectant's incubation time, on the one hand, and between the incubation time and the storage time on the other. Survival rates above 70% were obtained under optimized conditions after 3 months of storage, along with 20-35% viabilities after 3 years. These results open up the possibility of extending this method to other Scenedesmaceae, or even other microalgal species, and for its use in resource-limited laboratories.

Overexpression of a flower-specific aerolysin-like protein from the dioecious plant Rumex acetosa alters flower development and induces male sterility in transgenic tobacco.

Sex determination in Rumex acetosa, a dioecious plant with a complex XY1 Y2 sex chromosome system (females are XX and males are XY1 Y2 ), is not controlled by an active Y chromosome but depends on the ratio between the number of X chromosomes and autosomes. To gain insight into the molecular mechanisms of sex determination, we generated a subtracted cDNA library enriched in genes specifically or predominantly expressed in female floral buds in early stages of development, when sex determination mechanisms come into play. In the present paper, we report the molecular and functional characterization of FEM32, a gene encoding a protein that shares a common architecture with proteins in different plants, animals, bacteria and fungi of the aerolysin superfamily; many of these function as ß pore-forming toxins. The expression analysis, assessed by northern blot, RT-PCR and in situ hybridization, demonstrates that this gene is specifically expressed in flowers in both early and late stages of development, although its transcripts accumulate much more in female flowers than in male flowers. The ectopic expression of FEM32 under both the constitutive promoter 35S and the flower-specific promoter AP3 in transgenic tobacco showed no obvious alteration in vegetative development but was able to alter floral organ growth and pollen fertility. The 35S::FEM32 and AP3::FEM32 transgenic lines showed a reduction in stamen development and pollen viability, as well as a diminution in fruit set, fruit development and seed production. Compared with other floral organs, pistil development was, however, enhanced in plants overexpressing FEM32. According to these effects, it is likely that FEM32 functions in Rumex by arresting stamen and pollen development during female flower development. The aerolysin-like pore-forming proteins of eukaryotes are mainly involved in defence mechanisms against bacteria, fungi and insects and are also involved in apoptosis and programmed cell death (PCD), a mechanism that could explain the role of FEM32 in Rumex sex determination.

DNA Sequencing Sensors: An Overview.

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS) meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years.

Asn112 in Plasmodium falciparum glutathione S-transferase is essential for induced reversible tetramerization by phosphate or pyrophosphate.

The glutathione S-transferase from Plasmodium falciparum presents distinct features which are absent from mammalian GST isoenzyme counterparts. Most apparent among these are the ability to tetramerize and the presence of a flexible loop. The loop, situated between the 113-119 residues, has been reported necessary for the tetramerization process. In this article, we report that a residue outside of this loop, Asn112, is a key to the process - to the point where the single Asn112Leu mutation prevents tetramerization altogether. We propose that a structural pattern involving the interaction of the Asn112 and Lys117 residues from two neighboring subunits plays a role in keeping the tetramer structure stable. We also report that, for the tetramerization of the wild-type PfGST to occur, phosphate or pyrophosphate anions must be present. In other words, tetramerization is a phosphate- or pyrophosphate-induced process. Furthermore, the presence of magnesium reinforces this induction. We present experimental evidence for these claims as well as a preliminary calorimetric and kinetic study of the dimeric Asn112Leu PfGST mutant. We also propose a putative binding site for phosphate or pyrophosphate anions through a comparative structural analysis of PfGST and pyrophosphatases from several organisms. Our results highlight the differences between PfGST and the human isoenzymes, which make the parasite enzyme a suitable antimalarial target.

ß-Cyclodextrin-bearing gold glyconanoparticles for the development of site specific drug delivery systems.

Three novel gold nanoparticles containing multiple long, flexible linkers decorated with lactose, ß-cyclodextrin, and both simultaneously have been prepared. The interaction of such nanoparticles with ß-d-galactose-recognizing lectins peanut agglutinin (PNA) and human galectin-3 (Gal-3) was demonstrated by UV-vis studies. Gal-3 is well-known to be overexpressed in several human tumors and can act as a biorecognizable target. This technique also allowed us to estimate their loading capability toward the anticancer drug methotrexate (MTX). Both results make these glyconanoparticles potential site-specific delivery systems for anticancer drugs.

Development of genetic transformation methodologies for an industrially-promising microalga: Scenedesmus almeriensis.

The development of the microalgal industry requires advances in every aspect of microalgal biotechnology. In this regard, the availability of genetic engineering tools for industrially-promising species is key. As Scenedesmus almeriensis has promise for industrial use, we describe here an Agrobacterium-based methodology that allows stable genetic transformation of it for the first time, thus opening the way to its genetic manipulation. Transformation was accomplished using two different antibiotic resistance genes [hygromicine phophotransferase (hpt) and Shble] and it is credited by PCR amplification of both hpt/Shble and GUS genes and by the ß-glucuronidase activity of transformed cells. Nevertheless, the single 35S promoter seems unable to direct gene expression to a convenient level in S. almeriensis as suggested by the low GUS enzymatic activity. Temperature was critical for the transformation efficiency.

Calorimetric studies of ligands binding to glutathione S-transferase from the malarial parasite Plasmodium falciparum.

Glutathione S-transferase, from the malarial parasite Plasmodium falciparum (PfGST), exerts a protective role in the organism and is thus considered an interesting target for antimalarial drug development. In contrast to other GSTs, it is present in solution as a tetramer and a dimer in equilibrium, which is induced by glutathione (GSH). These properties prevent a calorimetric titration from being conducted upon binding of ligands to this protein's G-site. Thermodynamic characterization can be an optimal strategy for antimalarial drug development, and isothermal titration calorimetry (ITC) is the only technique that allows the separation of the binding energy into both enthalpic and entropic contributions. This information facilitates an understanding of the changes in the drugs' substituents, improving their affinity and specificity. In this study, we have applied a nontypical ITC procedure, based on the dissociation of the ligand-protein complex, to calorimetrically study the binding of the GSH substrate, and the glutathione sulfonate competitive inhibitor, to dimeric PfGST over a temperature range of 15-37 °C. The optimal experimental conditions for applying this procedure have been optimized by studying the dimer to tetramer conversion using size exclusion chromatography. The binding of these ligands to dimeric PfGST is noncooperative, the affinity of glutathione sulfonate being approximately 2 orders of magnitude higher than that of its natural substrate GSH. The binding of both ligands is enthalpically favorable and entropically unfavorable at all the studied temperatures. These results demonstrate that, although PfGST presents differences when compared to other known GSTs, these ligands bind to its dimeric form with a similar affinity and energetic balance. However, in contrast to that of other GSTs, the binding of GSH to protein, in the absence of the ligand, is slow.

Study of the biological activity of photoactive bipyridyl-Ru(II) complexes containing 1,3,5-triaza-7-phosphaadamantane (PTA).

The water-soluble ruthenium complex cis-[Ru(dcbpyH)2(PTAH)2]Cl2·3H2O (1) (dcbpy = 4,4'-dicarboxy-2,2'-bipyridine; PTA = 1,3,5-triaza-7-phosphaadamantane) has been synthesized and characterised by NMR, IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction. The optical properties of 1 were studied, including photoactivation under visible light, as well as its biological properties, together with those of the previously published Ru complexes cis-[Ru(bpy)2(PTA)2]Cl2 (2), trans-[Ru(bpy)2(PTA)2](CF3SO3)2 (3) and cis-[Ru(bpy)2(H2O)(PTA)](CF3SO3)2 (4) (bpy = 2,2'-bipyridine). Anticancer activities of the complexes against human lung (A549), cervical (HeLa) and prostate (PC3) carcinoma cells were evaluated under dark conditions and upon photoactivation with visible light. None of the complexes exhibited cytotoxic activity in the absence of light irradiation (IC50 > 100 µM). However, after photoactivation, the cytotoxicity of complexes 1, 2 and 3 against the three cell lines markedly increased, resulting in IC50 values between 25.3 µM and 9.3 µM. Notably, these complexes did not show toxicity against red blood cells. These findings show the potential of complexes 1, 2 and, particularly, 3 for selective and controlled cancer photochemotherapy. The reactivity of the Ru complexes against DNA under UV-Vis irradiation was studied by analysing plasmid mobility. Experimental data shows that 4 unfolds supercoiled DNA (SC DNA) both in the dark and under visible irradiation, while 1 and 3 are only active under light, being 2 inactive in either case. The unfolding activities of complexes 3 and 4 were dependent on the air present in the reaction. The measured intracellular levels of reactive oxygen species (ROS) upon irradiation with complexes 1, 2 and 3 suggest that their mechanism of action is related to oxidative stress.

Analysis of Fifty Years of Severe Malaria Worldwide Research.

This study analyzed fifty years of severe malaria research worldwide. Malaria is a parasitic disease that continues to have a significant impact on global health, particularly in sub-Saharan Africa. Severe malaria, a severe and often fatal form of the disease, is a major public health concern. The study used different bibliometric indicators such as the number of publications, citations, authorship, and keywords to analyze the research trends, patterns, and progress made in the field of severe malaria. The study covers the period from 1974 to 2021 and includes articles from Scopus. The results of the study indicated that there has been a steady increase in the number of publications on severe malaria over the past fifty years, with a particular increase in the last decade. The study also showed that most of the publications are from USA and Europe, while the disease occurs in Africa, South-East Asia, and the Americas. The study also identified the most frequent keywords used in the publications, and the most influential journals and authors in the field. In conclusion, this bibliometric study provides a comprehensive overview of the research trends and patterns in the field of severe malaria over the past fifty years and highlights the areas that need more attention and research efforts.

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1.

Cloning and molecular characterization of a glycerol-3-phosphate O-acyltransferase (GPAT) gene from Echium (Boraginaceae) involved in the biosynthesis of cutin polyesters.

The glycerol-based lipid polyester called cutin is a main component of cuticle, the protective interface of aerial plant organs also controlling compound exchange with the environment. Though recent progress towards understanding of cutin biosynthesis has been made in Arabidopsis thaliana, little is known in other plants. One key step in this process is the acyl transfer reaction to the glycerol backbone. Here we report the cloning and molecular characterization of EpGPAT1, a gene encoding a glycerol-3-phosphate O-acyltransferase (GPAT) from Echium pitardii (Boraginaceae) with high similarity to the AtGPAT4/AtGPAT8 of Arabidopsis. Quantitative analysis by qRT-PCR showed highest expression of EpGPAT1 in seeds, roots, young leaves and flowers. Acyltransferase activity of EpGPAT1 was evidenced by heterologous expression in yeast. Ectopic expression in leaves of tobacco plants lead to an increase of C16 and C18 hydroxyacids and alpha,omega-diacids in the cell wall fraction, indicating a role in the biosynthesis of polyesters. Analysis of the genomic organization in Echium revealed the presence of EpGPAT2, a closely related gene which was found to be mostly expressed in developing leaves and flowers. The presence of a conserved HAD-like domain at the N-terminal moiety of GPATs from Echium, Arabidopsis and other plant species suggests a possible phosphohydrolase activity in addition to the reported acyltransferase activity. Evolutive implications of this finding are discussed.

Plasmodium vivax Cysteine-Rich Protective Antigen Polymorphism at Exon-1 Shows Recombination and Signatures of Balancing Selection.

Plasmodium vivax Cysteine-Rich Protective Antigen (CyRPA) is a merozoite protein participating in the parasite invasion of human reticulocytes. During natural P. vivax infection, antibody responses against PvCyRPA have been detected. In children, low anti-CyRPA antibody titers correlated with clinical protection, which suggests this protein as a potential vaccine candidate. This work analyzed the genetic and amino acid diversity of pvcyrpa in Mexican and global parasites. Consensus coding sequences of pvcyrpa were obtained from seven isolates. Other sequences were extracted from a repository. Maximum likelihood phylogenetic trees, genetic diversity parameters, linkage disequilibrium (LD), and neutrality tests were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. In 22 sequences from Southern Mexico, two synonymous and 21 nonsynonymous mutations defined nine private haplotypes. These parasites had the highest LD-R2 index and the lowest nucleotide diversity compared to isolates from South America or Asia. The nucleotide diversity and Tajima's D values varied across the coding gene. The exon-1 sequence had greater diversity and Rm values than those of exon-2. Exon-1 had significant positive values for Tajima's D, ß-α values, and for the Z (HA: dN > dS) and MK tests. These patterns were similar for parasites of different origin. The polymorphic amino acid residues at PvCyRPA resembled the conformational B-cell peptides reported in PfCyRPA. Diversity at pvcyrpa exon-1 is caused by mutation and recombination. This seems to be maintained by balancing selection, likely due to selective immune pressure, all of which merit further study.

Delta6-desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae).

The oceanic islands of Macaronesia provide an ideal temporal and spatial context to test hypotheses of plant evolution using a novel set of phylogenetic markers, Delta(6)-desaturase sequences. In contrast to the limited resolution of standard molecular markers (nrDNA and plastid sequences), the Delta(6)-desaturase sequence phylogeny of Echium unequivocally reconstructs its active colonization across islands and archipelagos (Madeira, the Canary Islands, and Cape Verde), as well as its subsequent geographical and ecological speciation. Molecular-clock estimates using penalized likelihood and Bayesian inference reveal two radiation processes coincident with two dramatic climatic changes recorded in the region: the advent of the cold Canarian sea current (ca. 4 Ma) and the establishment of a strong seasonality in the Pleistocene (1.8 Ma). Though Echium had available all the diversity of present-day Macaronesian environments (xeric and mesic scrub, laurisilva, pine forest, and subalpine habitats) in the Miocene, evolutionary divergence appears to have been triggered by an extension of fluctuating xeric and mesic habitats with the advent of Pliocene conditions. These Echium radiations not only fulfill traditional predictions of adaptive radiation (i.e., common ancestry, rapid speciation, and phenotype-environment correlation), but also, uniquely among Macaronesian species, trait utility of woodiness. A Pliocene transition from annuality to a bush or tree-like condition occurred in early Echium lineages. Maintenance of woodiness in major lineages, and reversal to an herbaceous condition by three independent events, is reported for the first time in plants of oceanic islands.

Kinetic study on the irreversible thermal denaturation of Schistosoma japonicum glutathione S-transferase.

The thermal unfolding pathway of the Schistosoma japonicum glutathione S-transferase (Sj26GST) was previously interpreted by applying equilibrium thermodynamics and a reversible two-state model (Kaplan et al., (1997) Protein Science, 6, 399-406), though weak support for this interpretation was provided. In our study, thermal denaturation of Sj26GST has been re-examined by differential scanning calorimetry in the pH range of 6.5-8.5 and in the presence of the substrate and S-hexylglutathione. Calorimetric traces were found to be irreversible and highly scan-rate dependent. Thermogram shapes, as well as their scan-rate dependence, can be globally explained by assuming that thermal denaturation takes place according to one irreversible step described by a first-order kinetic constant that changes with temperature, as given by an Arrhenius equation. On the basis of this model, values for the rate constant as a function of temperature and the activation energy have been determined. Data also indicate that binding of GSH or S-hexylglutathione just exert a very little stabilising effect on the dimeric structure of the molecule.

Substrate specificity of acyl-Delta(6)-desaturases from Continental versus Macaronesian Echium species.

Echium (Boraginaceae) species from the Macaronesian islands exhibit an unusually high level of gamma-linolenic acid (18:3n-6; GLA) and relatively low content of octadecatetraenoic acid (18:4n-3; OTA) in the seed, while the amounts of both fatty acids in their Continental (European) relatives are rather similar. We have tested the hypothesis of whether a different specificity of the acyl-Delta(6)-desaturases (D6DES) towards their respective usual substrates, linoleic acid (18:2n-6; LA) for GLA and alpha-linolenic acid (18:3n-3; ALA) for OTA, was partly responsible for this composition pattern. To this aim we have expressed in yeast the coding sequences of the D6DES genes for the Continental species Echium sabulicola, and the Macaronesian Echium gentianoides. When the yeast cultures are supplemented with the two fatty acid substrates (LA and ALA), a similar utilization of both compounds was found for the D6DES of E. sabulicola, while a preference for LA over ALA was observed for the enzyme of E. gentianoides. This substrate preference must contribute to the increased accumulation of GLA in the seeds of the Macaronesian Echium species. Comparison among the amino acid sequences of these desaturases and other related enzymes, allowed us the discussion about the possible involvement of some specific positions in the determination of substrate specificity.

Type I Diacylglycerol Acyltransferase (MtDGAT1) from Macadamia tetraphylla: Cloning, Characterization, and Impact of Its Heterologous Expression on Triacylglycerol Composition in Yeast.

Acyltransferase enzymes have been reported as useful biotechnological tools in order to increase oil yield and modify fatty acid composition. Macadamia species are able to accumulate unusually high levels of palmitoleic acid that besides oleic acid amounts to over 80% of monounsaturated fatty acids in the seed oil. In this work, a gene encoding a type 1 acyl-CoA:diacylglycerol acyltransferase (DGAT1) was cloned from M. tetraphylla. DGAT activity of the protein encoded by MtDGAT1 was confirmed by heterologous expression in a yeast mutant. Fatty acid composition of triacylglycerols synthesized by MtDGAT1 was compared to that of DGAT1 enzymes from Arabidopsis and Echium, with the results suggesting a substrate preference for monounsaturated over polyunsaturated fatty acids. Characteristics of MtDGAT1 may contribute to biochemical mechanisms determining the particular fatty acid composition of Macadamia oil and also indicate the possibility of using this enzyme in biotechnological approaches where a reduction of polyunsaturated fatty acids in the oil is desired.

Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase.

The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K(d)=3.2 microM) than that for ANS (K(d)=195 microM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaG degrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degrees C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p) degrees =(-580.3+/-54.2) cal x K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p) degrees =(-294.8+/-15.8) cal x K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic.

Cloning and molecular characterization of the delta6-desaturase from two echium plant species: production of GLA by heterologous expression in yeast and tobacco.

The synthesis of GLA (delta6,9,12-1-8:3) is carried out in a number of plant taxa by introducing a double bond at the delta6 position of its precursor, linoleic acid (delta9,12-18:2), through a reaction catalyzed by a delta6-desaturase enzyme. We have cloned genes encoding the delta6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b(5) domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other delta6-desaturases in a different cluster from that of the highly related delta8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Molecular characterization of a lysophosphatidylcholine acyltransferase gene belonging to the MBOAT family in Ricinus communis L.

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) catalyzes acylation of lysophosphatidylcholine (lysoPtdCho) to produce phosphatidylcholine (PtdCho), the main phospholipid in cellular membranes. This reaction is a key component of the acyl-editing process, involving recycling of the fatty acids (FA) mainly at the sn-2 position of PtdCho. Growing evidences indicate that the LPCAT reaction controls the direct entry of newly synthesized FA into PtdCho and, at least in some plant species, it has an important impact on the synthesis and composition of triacylglycerols. Here we describe the molecular characterization of the single LPCAT gene found in the genome of Ricinus communis (RcLPCAT) that is homologous to LPCAT genes of the MBOAT family previously described in Arabidopsis and Brassica. RcLPCAT is ubiquitously expressed in all organs of the castor plant. Biochemical properties have been studied by heterologous expression of RcLPCAT in the ale1 yeast mutant, defective in lysophospholipid acyltransferase activity. RcLPCAT preferentially acylates lysoPtdCho against other lysophospholipids (lysoPL) and does not discriminates the acyl chain in the acceptor, displaying a strong activity with alkyl lysoPL. Regarding the acyl-CoA donor, RcLPCAT uses monounsaturated fatty acid thioesters, such as oleoyl-CoA (18:1-CoA), as preferred donors, while it has a low activity with saturated fatty acids and shows a poor utilization of ricinoleoyl-CoA (18:1-OH-CoA). These characteristics are discussed in terms of a possible role of RcLPCAT in regulating the entry of FA into PtdCho and the exclusion from the membranes of the hydroxylated FA.