RESUMEN

BACKGROUND: Cognitive behavioural therapy (CBT) is a key intervention, enabling workers on sick leave with common mental health problems to return to work. It can be delivered by a variety of methods including face-to-face therapy and the Internet. It is not known which is the optimal method of delivery. AIMS: To establish the optimum method of delivering CBT to workers with common mental health problems. METHODS: We undertook a systematic search of the OvidMEDLINE and EMBASE biomedical databases from the start of electronic records to 31 July 2013 for randomized trials comparing one method of delivering CBT with another for treatment of mild-to-moderate depression, anxiety and adjustment disorders. We included publications that assessed at least one of four outcomes: clinical or cost-effectiveness, accessibility and acceptability. A scoping search found no studies in the workplace. We therefore focussed on interventions in the 18-65 year age group. RESULTS: We found six studies comparing methods of delivery of CBT for anxiety disorders but found no trials which compared methods of delivery for mild-to-moderate depression. All delivery methods led to an improvement in anxiety symptoms. Internet-delivered CBT with some input from a therapist was found to be as clinically effective as face-to-face CBT and more cost-effective. CONCLUSIONS: Internet CBT should be made available in workplaces for workers with anxiety disorders as part of a stepped care plan.

Asunto(s)

Trastornos de Ansiedad/terapia , Terapia Cognitivo-Conductual , Trastorno Depresivo/terapia , Enfermedades Profesionales/terapia , Salud Laboral , Reinserción al Trabajo/psicología , Trastornos de Ansiedad/fisiopatología , Trastornos de Ansiedad/psicología , Terapia Cognitivo-Conductual/métodos , Trastorno Depresivo/fisiopatología , Trastorno Depresivo/psicología , Humanos , Enfermedades Profesionales/fisiopatología , Enfermedades Profesionales/psicología , Ausencia por Enfermedad , Resultado del Tratamiento , Lugar de Trabajo/psicología

RESUMEN

Unlike flapping birds and insects, bats possess membrane wings that are more similar to many gliding mammals. The vast majority of the wing is composed of a thin compliant skin membrane stretched between the limbs, hand, and body. Membrane wings are of particular interest because they may offer many advantages to micro air vehicles. One critical feature of membrane wings is that they camber passively in response to aerodynamic load, potentially allowing for simplified wing control. However, for maximum membrane wing performance, tuning of the membrane structure to aerodynamic conditions is necessary. Bats possess an array of muscles, the plagiopatagiales proprii, embedded within the wing membrane that could serve to tune membrane stiffness, or may have alternative functions. We recorded the electromyogram from the plagiopatagiales proprii muscles of Artibeus jamaicensis, the Jamaican fruit bat, in flight at two different speeds and found that these muscles were active during downstroke. For both low- and high-speed flight, muscle activity increased between late upstroke and early downstroke and decreased at late downstroke. Thus, the array of plagiopatagiales may provide a mechanism for bats to increase wing stiffness and thereby reduce passive membrane deformation. These muscles also activate in synchrony, presumably as a means to maximize force generation, because each muscle is small and, by estimation, weak. Small differences in activation timing were observed when comparing low- and high-speed flight, which may indicate that bats modulate membrane stiffness differently depending on flight speed.

Asunto(s)

Biomimética/métodos , Quirópteros/fisiología , Vuelo Animal/fisiología , Modelos Biológicos , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Alas de Animales/fisiología , Animales , Simulación por Computador , Módulo de Elasticidad/fisiología , Membranas/fisiología , Esfuerzo Físico/fisiología , Estrés Mecánico , Resistencia a la Tracción/fisiología

RESUMEN

Nervonic acid 24:1 Delta15 (cis-tetracos-15-enoic acid) is a very long-chain monounsaturated fatty acid and exists in nature as an elongation product of oleic acid. There is an increasing interest in production of high nervonic acid oils for pharmaceutical, nutraceutical and industrial applications. Using a polymerase chain reaction approach, we have isolated a gene from Cardamine graeca L., which encodes a 3-ketoacyl-CoA synthase (KCS), the first component of the elongation complex involved in synthesis of nervonic acid. Expression of the Cardamine KCS in yeast resulted in biosynthesis of nervonic acid, which is not normally present in yeast cells. We transformed Arabidopsis and Brassica carinata with the Cardamine KCS under the control of the seed-specific promoter, napin. The T(3) generations of transgenic Arabidopsis and B. carinata plants expressing the Cardamine KCS showed that seed-specific expression resulted in relatively large comparative increases in nervonic acid proportions in Arabidopsis seed oil, and 15-fold increase in nervonic acid proportions in B. carinata seed oil. The highest nervonic acid level in transgenic B. carinata lines reached 44%, with only 6% of residual erucic acid. In contrast, similar transgenic expression of the Cardamine KCS in high erucic B. napus resulted in 30% nervonic acid but with 20% residual erucic acid. Experiments using the Lunaria KCS gene gave results similar to the latter. In both cases, the erucic acid content is too high for human or animal consumption. Thus, the Cardamine KCS: B. carinata high nervonic/highly reduced erucic transgenic seed oils will be the most suitable for testing in pharmaceutical/nutraceutical applications to improve human and animal health.

Asunto(s)

3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Brassica/metabolismo , Cardamine/genética , Ácidos Grasos Monoinsaturados/metabolismo , Aceites de Plantas/análisis , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Brassica/genética , Cardamine/enzimología , Clonación Molecular , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Transformación Genética

RESUMEN

In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be essential for growth and metabolism of yeast cells, they may play an important role in cell survival by metabolizing excess intracellular lysophospholipids. To test our hypothesis, we overexpressed a plant phospholipase A2 (PLA2) in both WT and plb123 cells, producing a pool of lysophosphatidylcholine (lysoPtdCho) in both transformants. In ¹4C acetate labeling experiments, WT cells were able to catabolize the resultant labeled lysoPtdCho, preventing accumulation, and the cells grew normally. In contrast, in the triple plb123 mutant PLA2 transformant, lysoPtDCho accumulated more than 4-fold to a toxic level, inhibiting cell growth. However, this growth inhibition was complemented by co-expression of either PLB1, PLB2 or PLB3 in the plb123 triple mutant already expressing the plant PLA2. Furthermore, in labeling experiments, the rescued cells exhibited a 60-75% reduction in 14C-lysoPtdCho build-up compared to plb123PLA2 cells. Our data provides conclusive evidence that yeast PLBs can metabolize intracellular lysoPtdCho produced by plant PLA2 overexpression in yeast. Our experiments indicate the utility of ectopic plant phospholipase A gene expression to characterize poorly-understood phospholipid metabolism mutants in yeast or other organisms.

RESUMEN

BACKGROUND: Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed. RESULTS: Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development. CONCLUSION: Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

Asunto(s)

Brassica/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Aceites de Plantas/metabolismo , Semillas/enzimología , Triglicéridos/biosíntesis , Ácido Abscísico/metabolismo , Brassica/enzimología , Citocininas/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Metaboloma , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , ARN de Planta/genética , Semillas/genética

RESUMEN

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.

Asunto(s)

Acilcoenzima A/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Aceites de Plantas/metabolismo , Tropaeolum/enzimología , Tropaeolum/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , ADN de Plantas/genética , Ácidos Erucicos , Biblioteca de Genes , Genes de Plantas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Transformación Genética , Triglicéridos/biosíntesis

RESUMEN

A genomic fatty acid elongation 1 (FAE1) clone was isolated from Crambe abyssinica. The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20:1(delta11)) and erucic (22:1(delta13)) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content.

Asunto(s)

Acetiltransferasas/genética , Crambe (Planta)/genética , Proteínas de Plantas/genética , Acetiltransferasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Brassica/genética , Brassica/metabolismo , Clonación Molecular , Crambe (Planta)/enzimología , Crambe (Planta)/metabolismo , ADN de Plantas/química , ADN de Plantas/genética , Ácidos Erucicos/metabolismo , Elongasas de Ácidos Grasos , Ácidos Grasos Insaturados/metabolismo , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN

RESUMEN

Extramedullary hematopoiesis is a rare cause of an intrathoracic mass in individuals with hemolytic disorders. It can be clinically confused with other tumors of the mediastinum. While radiologic studies often demonstrate findings suggesting intrathoracic extramedullary hematopoiesis, histology is usually required for diagnostic purposes. Thoracotomy was the mainstay procedure for obtaining tissue diagnosis and resection. However, video-assisted thoracoscopy (VATS) is an amendable and less-invasive means of tumor removal. We report a case of a posterior mediastinal extramedullary hematopoietic mass in a forty-two year old male in which VATS was utilized for diagnosis and resection.

RESUMEN

Erucic acid and its derivatives represent important industrial feedstock compounds, and there is an increasing demand for the production of high erucate oils in this regard. Our goal therefore, is to develop high erucic acid (HEA) Brassicaceae lines with increased proportions of erucic acid and very long-chain fatty acids (VLCFAs). We proposed that oleate availability may be a rate-limiting factor in the biosynthesis of erucic acid. We have tried to address this question by manipulating the expression of the endogenous FAD2 gene in B. carinata using co-supression and antisense approaches. Both methods resulted in transgenic lines exhibiting decreased proportions of polyunsaturated C18 fatty acids (18:2+18:3) and concomitant and significantly increased proportions of 18:1, 22:1 and total VLCFAs. Co-suppressed FAD2 B. carinata lines exhibited 3-18% decreases in 18:2, 22-49% decreases in 18:3 and significantly increased proportions of 18:1 (36-99%), 22:1 (12-27%) and VLCFAs (6-15%). Transgenic B. carinata lines developed using an antisense FAD2 approach exhibited decreased proportions of 18:2 and 18:3 (9-39% and 33-48%, respectively) and significantly increased proportions of 18:1 (54-130%), 22:1 (5-19%) and VLCFAs (6-21%). The possibility of using these approaches to produce prototype transgenic germplasm of the Brassicaceae accumulating seed oils with improved proportions of erucic and other VLCFAs is discussed.

Asunto(s)

Brassica/genética , Brassica/metabolismo , Ácidos Erucicos/metabolismo , Ácido Graso Desaturasas/deficiencia , Ácido Graso Desaturasas/genética , Ácidos Grasos/metabolismo , Mejoramiento Genético/métodos , Ingeniería de Proteínas/métodos , Silenciador del Gen/fisiología , ARN sin Sentido/genética , Supresión Genética/genética

RESUMEN

The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.

Asunto(s)

Acetiltransferasas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Erucicos/metabolismo , Genes de Plantas , Nasturtium/enzimología , Nasturtium/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/enzimología , Secuencia de Bases , ADN Complementario/genética , ADN de Plantas/genética , Elongasas de Ácidos Grasos , Dosificación de Gen , Expresión Génica , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Semillas/enzimología , Semillas/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Nicotiana/enzimología , Nicotiana/genética

RESUMEN

To gain some insight whether there is an absolute requirement for the serine 282 to yield a functional fatty acid elongase 1 condensing enzyme we have introduced point mutations in the FAE1 coding sequence which led to the substitution of serine 282 with several aliphatic or aromatic amino acids. The mutated FAE1 polypeptides were expressed in yeast. Gas chromatography analyses of the fatty acid methyl esters from yeast lysates and fatty acid elongase activity assays demonstrated that there is not an absolute requirement for serine at position 282 to yield a functional FAE1 condensing enzyme.

Asunto(s)

Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Brassica napus/enzimología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Serina/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Brassica napus/genética , Elongasas de Ácidos Grasos , Estructura Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.

Asunto(s)

Acetiltransferasas/química , Brassica napus/enzimología , Brassica napus/metabolismo , Ácidos Erucicos/metabolismo , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Secuencia de Aminoácidos , Cromatografía de Gases , Clonación Molecular , Elongasas de Ácidos Grasos , Immunoblotting , Microsomas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/química , Fenilalanina/química , Regiones Promotoras Genéticas , Serina/química , Factores de Tiempo

RESUMEN

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.

Asunto(s)

Cycadopsida/enzimología , Estearoil-CoA Desaturasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Cinética , Fragmentos de Péptidos/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estearoil-CoA Desaturasa/química , Estearoil-CoA Desaturasa/genética , Árboles/enzimología

RESUMEN

We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

Asunto(s)

Aciltransferasas/genética , Arabidopsis/genética , Aceites de Plantas/análisis , Semillas/enzimología , Acilcoenzima A/metabolismo , Aciltransferasas/química , Secuencia de Aminoácidos , Arabidopsis/enzimología , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Diacilglicerol O-Acetiltransferasa , Prueba de Complementación Genética , Datos de Secuencia Molecular , Semillas/química , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.

Asunto(s)

Acetiltransferasas/genética , Arabidopsis/genética , Brassica/química , Brassica/crecimiento & desarrollo , Colágeno/genética , Ácidos Erucicos/análisis , Ácidos Grasos/análisis , Aceites de Plantas/química , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Arabidopsis/enzimología , Elongasas de Ácidos Grasos , Ingeniería Genética/métodos , Plantas Modificadas Genéticamente/química , Complejo de la Endopetidasa Proteasomal , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae

RESUMEN

Land plants secrete a layer of wax onto their aerial surfaces that is essential for survival in a terrestrial environment. This wax is composed of long-chain, aliphatic hydrocarbons derived from very-long-chain fatty acids (VLCFAs). Using the Arabidopsis expressed sequence tag database, we have identified a gene, designated CUT1, that encodes a VLCFA condensing enzyme required for cuticular wax production. Sense suppression of CUT1 in transgenic Arabidopsis plants results in waxless (eceriferum) stems and siliques as well as conditional male sterility. Scanning electron microscopy revealed that this was a severe waxless phenotype, because stems of CUT1-suppressed plants were completely devoid of wax crystals. Furthermore, chemical analyses of waxless plants demonstrated that the stem wax load was reduced to 6 to 7% of wild-type levels. This value is lower than that reported for any of the known eceriferum mutants. The severe waxless phenotype resulted from the downregulation of both the decarbonylation and acyl reduction wax biosynthetic pathways. This result indicates that CUT1 is involved in the production of VLCFA precursors used for the synthesis of all stem wax components in Arabidopsis. In CUT1-suppressed plants, the C24 chain-length wax components predominate, suggesting that CUT1 is required for elongation of C24 VLCFAs. The unique wax composition of CUT1-suppressed plants together with the fact that the location of CUT1 on the genetic map did not coincide with any of the known ECERIFERUM loci suggest that we have identified a novel gene involved in wax biosynthesis. CUT1 is currently the only known gene with a clearly established function in wax production.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/genética , Genes de Plantas , Ceras/metabolismo , Acetiltransferasas/química , Acetiltransferasas/genética , Aciltransferasas , Secuencia de Aminoácidos , Arabidopsis/enzimología , Mapeo Cromosómico , Regulación hacia Abajo , Elongasas de Ácidos Grasos , Fertilidad , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Datos de Secuencia Molecular , Fenotipo , Tallos de la Planta/metabolismo , Polen , Conformación Proteica , ARN Mensajero/aislamiento & purificación , Homología de Secuencia de Aminoácido , Supresión Genética , Transgenes

RESUMEN

A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs.

Asunto(s)

Aciltransferasas/genética , Brassica/enzimología , Proteínas Fúngicas/genética , Aceites de Plantas/análisis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Semillas/química , 1-Acilglicerol-3-Fosfato O-Aciltransferasa , Acilación , Aciltransferasas/biosíntesis , Aciltransferasas/metabolismo , Alelos , Arabidopsis , Brassica/genética , Brassica/metabolismo , Clonación Molecular , Dineínas , Ácidos Grasos/análisis , Proteínas Fúngicas/biosíntesis , Genes de Plantas , Genes Supresores , Plantas Modificadas Genéticamente/genética , Saccharomyces cerevisiae/genética , Triglicéridos/análisis

RESUMEN

Developing seeds from Brassica oleracea (L.) var botrytis cv Sesam were examined for the ability to biosynthesize and incorporate erucic acid into triacylglycerols (TAGs). Seed embryos at mid-development contained a high concentration of erucic acid in diacylglycerols and TAGs, and substantial levels were also detected in free fatty acids, acyl-coenzyme A (CoA), phosphatidic acid, and phosphatidylcholine. Homogenates and microsomal fractions from seeds at mid-development produced [14C]eicosenoyl- and [14C]erucoyl-CoAs from [14C]oleoyl-CoA in the presence of malonyl-CoA and reducing equivalents in vitro. These fatty acids were incorporated into TAGs via the Kennedy pathway. However, unlike most Brassicaceae, the B. oleracea was able to insert significant erucic acid into the sn-2 position of TAGs. It was shown that the lyso-phosphatidic acid acyltransferase (LPAT) incorporated erucic acid into the sn-2 position of lyso-phosphatidic acid. The erucoyl-CoA:LPAT activity during seed development and the sn-2 erucic acid content of the TAG fraction in mature seed were compared to those in B. napus, Tropaeolum majus, and Limnanthes douglasii. There was a correlation between the in vitro erucoyl-CoA:LPAT activity and the sn-2 erucic acid content in seed TAGs. To our knowledge, this is the first member of the Brassicaceae reported to have an LPAT able to use erucoyl-CoA. This observation has important implications for efforts being made to increase the erucic acid content in B. napus, to supply strategic industrial feedstocks.

RESUMEN

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids.

Asunto(s)

Aciltransferasas/metabolismo , Arabidopsis/metabolismo , Mapeo Cromosómico , Metanosulfonato de Etilo/farmacología , Ácidos Grasos/metabolismo , Aciltransferasas/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Secuencia de Bases , Núcleo Celular/metabolismo , Cartilla de ADN , Diacilglicerol O-Acetiltransferasa , Ácidos Grasos/análisis , Genes de Plantas , Cinética , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Hojas de la Planta , Raíces de Plantas , Reacción en Cadena de la Polimerasa , Semillas/metabolismo , Especificidad de la Especie

RESUMEN

The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, (14)C 18∶1-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 18∶1 to 18∶2 and to a lesser extent, 18∶3. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (20∶1 and 22∶1) from 18∶1-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with (14)C 20∶1-CoA or (14)C 22∶1-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.