RESUMO
Reducing the saturate content of vegetable oils is key to increasing their utility and adoption as a feedstock for the production of biofuels. Expression of either the FAT5 16 : 0-CoA desaturase from Caenorhabditis elegans, or an engineered cyanobacterial 16 : 0/18 : 0-glycerolipid desaturase, DES9*, in seeds of Arabidopsis (Arabidopsis thaliana) substantially lowered oil saturates. However, because pathway fluxes and regulation of oil synthesis are known to differ across species, translating this transgene technology from the model plant to crop species requires additional investigation. In the work reported here, we found that high expression of FAT5 in seeds of camelina (Camelina sativa) provided only a moderate decrease in saturates, from 12.9% of total oil fatty acids in untransformed controls to 8.6%. Expression of DES9* reduced saturates to 4.6%, but compromised seed physiology and oil content. However, the coexpression of the two desaturases together cooperatively reduced saturates to only 4.0%, less than one-third of the level in the parental line, without compromising oil yield or seedling germination and establishment. Our successful lowering of oil saturates in camelina identifies strategies that can now be integrated with genetic engineering approaches that reduce polyunsaturates to provide optimized oil composition for biofuels in camelina and other oil seed crops.
Assuntos
Arabidopsis , Brassicaceae , Biocombustíveis , Plantas Geneticamente Modificadas/genética , Brassicaceae/genética , Arabidopsis/genética , Ácidos Graxos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Sementes/genética , Óleos de Plantas/metabolismoRESUMO
Vegetable oils composed of triacylglycerols (TAG) are a major source of calories in human diets. However, the fatty acid compositions of these oils are not ideal for human nutrition and the needs of the food industry. Saturated fatty acids contribute to health problems, while polyunsaturated fatty acids (PUFA) can become rancid upon storage or processing. In this review, we first summarize the pathways of fatty acid metabolism and TAG synthesis and detail the problems with the oil compositions of major crops. Then we describe how transgenic expression of desaturases and downregulation of the plastid FatB thioesterase have provided the means to lower oil saturates. The traditional solution to PUFA rancidity uses industrial chemistry to reduce PUFA content by partial hydrogenation, but this results in the production of trans fats that are even more unhealthy than saturated fats. We detail the discoveries in the biochemistry and molecular genetics of oil synthesis that provided the knowledge and tools to lower oil PUFA content by blocking their synthesis during seed development. Finally, we describe the successes in breeding and biotechnology that are giving us new, high-oleic, low PUFA varieties of soybean, canola and other oilseed crops.
RESUMO
Hydroxy fatty acids (HFA) are industrially useful chemical feedstocks that accumulate in seed-storage triacylglycerols (TAG) of several plant species, including castor (Ricinus communis) and Physaria (Physaria fendleri). For researchers, HFA also offer a unique opportunity to trace fatty acid metabolism and modification. Past work producing HFA in Arabidopsis (Arabidopsis thaliana) has demonstrated the importance of isozymes of TAG synthesis from plants that evolved to store HFA and as a result have a high degree of specificity towards HFA substrates. Castor phospholipase A2α (RcPLA2) has specificity for HFA-containing phosphatidylcholine. However, expression of RcPLA2 in HFA-accumulating Arabidopsis line CL37-PLA2 reduced HFA content of TAG. This loss was interpreted as being due to poor ability of Arabidopsis longchain acyl-CoA synthetases (LACSs) to utilize HFAs substrates. LACS enzymes are essential to activate HFA to HFA-CoA for TAG synthesis. Physaria is a close relative of Arabidopsis in the Brassicaceae family. To test the hypothesis that this close relatedness would allow Physaria LACSs to interface successfully with Arabidopsis enzymes of seed lipid metabolism and thereby restore HFA accumulation, we transformed PfLACS4 and PfLACS8 constructs into the CL37-PLA2 line. However, HFA content was not recovered, and biochemical characterization of recombinant PfLACS4 and PfLACS8 indicated that these isozymes have substrate specificities and selectivities that are similar to their Arabidopsis orthologues. These and other results pose an important question about how HFA synthesized on phosphatidylcholine can be transferred into the acyl-CoA pool for TAG synthesis.
Assuntos
Arabidopsis , Brassicaceae , Acil Coenzima A/metabolismo , Arabidopsis/metabolismo , Brassicaceae/genética , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Isoenzimas/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipases A2/análise , Fosfolipases A2/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes , Triglicerídeos/metabolismoRESUMO
A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from more than a dozen wild species, progress has been limited because expression of these enzymes in transgenic plants produces only low yields of the desired products. For example, fatty acid hydroxylase 12 (FAH12) from castor (Ricinus communis) produces only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis (Arabidopsis thaliana), compared with 90% HFAs in castor seeds. The transgenic plants also have reduced oil content and seed vigor. Here, we review experiments that have provided for steady increased HFA accumulation and oil content. This research has led to exciting new discoveries of enzymes and regulatory processes in the pathways of both seed oil synthesis and lipid metabolism in other parts of the plant. Recent investigations have revealed that HFA-accumulating seeds are unable to rapidly mobilize HFA-containing triacylglycerol (TAG) storage lipid after germination to provide carbon and energy for seedling development, resulting in reduced seedling establishment. These findings present a new opportunity to investigate a different, key area of lipid metabolism-the pathways of TAG lipolysis and ß-oxidation in germinating seedlings.
Assuntos
Arabidopsis , Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Plantas Geneticamente Modificadas/genética , Plântula , Sementes , Triglicerídeos/metabolismoRESUMO
Reactive oxygen species (ROS) contribute to cellular damage in several different contexts, but their role during chilling damage is poorly defined. Chilling sensitivity both limits the distribution of plant species and causes devastating crop losses worldwide. Our screen of chilling-tolerant Arabidopsis (Arabidopsis thaliana) for mutants that suffer chilling damage identified a gene (At4g03410) encoding a chloroplast Mpv17_PMP22 protein, MPD1, with no previous connection to chilling. The chilling-sensitive mpd1-1 mutant is an overexpression allele that we successfully phenocopied by creating transgenic lines with a similar level of MPD1 overexpression. In mammals and yeast, MPD1 homologs are associated with ROS management. In chilling conditions, Arabidopsis overexpressing MPD1 accumulated H2O2 to higher levels than wild-type controls and exhibited stronger induction of ROS response genes. Paraquat application exacerbated chilling damage, confirming that the phenotype occurs due to ROS dysregulation. We conclude that at low temperature increased MPD1 expression results in increased ROS production, causing chilling damage. Our discovery of the effect of MPD1 overexpression on ROS production under chilling stress implies that investigation of the nine other members of the Mpv17_PMP22 family in Arabidopsis may lead to new discoveries regarding ROS signaling and management in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Temperatura Baixa , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Analysis of plant lipids provides insights into a range of biological processes, from photosynthetic membrane function to oil seed engineering. Many lipid extraction protocols are tailored to fit a specific lipid class. Here we describe a procedure for extraction of glycerolipids from vegetative tissue. This procedure is designed for 1 gram of tissue per sample but maybe scaled for larger samples.
Assuntos
Lipídeos/isolamento & purificação , Extração Líquido-Líquido/métodos , Plantas/metabolismo , Clorofórmio/química , Glicerol/metabolismo , Lipídeos/análise , Metanol/química , Sementes/química , Solventes/química , Água/químicaRESUMO
The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)-a trait associated with chilling-sensitive plants-compared with <10% in wild-type Arabidopsis. Although they do not exhibit short-term chilling sensitivity when exposed to low temperatures (2°C to 6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. To test the relevance of HMP-PG to the fab1 phenotype, we used transgenic 16:0 desaturases targeted to the endoplasmic reticulum and the chloroplast to lower 16:0 in leaf lipids of fab1 plants. We produced two lines that had very similar lipid compositions except that one, ER-FAT5, contained high HMP-PG, similar to the fab1 parent, while the second, TP-DES9*, contained <10% HMP-PG, similar to the wild type. TP-DES9* plants, but not ER-FAT5 plants, showed strong recovery and growth following 75 d at 2°C, demonstrating the role of HMP-PG in low-temperature damage and death in fab1, and in chilling-sensitive plants more broadly.
Assuntos
Aclimatação/fisiologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Temperatura Baixa , Ácidos Graxos/biossíntese , Fosfatidilgliceróis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Ácidos Graxos/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Mutação , Fosfatidilgliceróis/genéticaRESUMO
Oilseeds produce abundant triacylglycerol (TAG) during seed maturation to fuel the establishment of photoautotrophism in the subsequent generation. Commonly, TAG contains 18-carbon polyunsaturated fatty acids (FA), but plants also produce oils with unique chemical properties highly desirable for industrial processes. Unfortunately, plants that produce such oils are poorly suited to agronomic exploitation, leading to a desire to reconstitute novel oil biosynthesis in crop plants. Here, we studied the production and incorporation of hydroxy-fatty acids (HFA) onto TAG in Arabidopsis (Arabidopsis thaliana) plants expressing the castor (Ricinus communis) FAH12 hydroxylase. One factor limiting HFA accumulation in these plants is the inefficient removal of HFA from the site of synthesis on phosphatidylcholine (PC). In Arabidopsis, lysophosphatidic acid acyltransferase (LPCAT) cycles FA to and from PC for modification. We reasoned that the castor LPCAT (RcLPCAT) would preferentially remove HFA from PC, resulting in greater incorporation onto TAG. However, expressing RcLPCAT in Arabidopsis expressing FAH12 alone (line CL37) or together with castor acyl:coenzyme A:diacylglycerol acyltransferase2 reduced HFA and total oil yield. Detailed analysis indicated that RcLPCAT reduced the removal of HFA from PC, possibly by competing with the endogenous LPCAT isozymes. Significantly, coexpressing RcLPCAT with castor phospholipid:diacylglycerol acyltransferase increased novel FA and total oil contents by transferring HFA from PC to diacylglycerol. Our results demonstrate that a detailed understanding is required to engineer modified FA production in oilseeds and suggest that phospholipase A2 enzymes rather than LPCAT mediate the highly efficient removal of HFA from PC in castor seeds.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Aciltransferases/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Ricinus/genética , Triglicerídeos/biossíntese , Aciltransferases/metabolismo , Arabidopsis , Fosfatidilcolinas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismo , EstereoisomerismoRESUMO
Many pathways of primary metabolism are substantially conserved within and across plant families. However, significant differences in organization and fluxes through a reaction network may occur, even between plants in closely related genera. Assessing and understanding these differences is key to appreciating metabolic diversity, and to attempts to engineer plant metabolism for higher crop yields and desired product profiles. To better understand lipid metabolism and seed oil synthesis in canola (Brassica napus), we have characterized four canola homologues of the Arabidopsis (Arabidopsis thaliana) ROD1 gene. AtROD1 encodes phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), the enzyme that catalyzes a major flux of polyunsaturated fatty acids (PUFAs) in oil synthesis. Assays in yeast indicated that only two of the canola genes, BnROD1.A3 and BnROD1.C3, encode active isozymes of PDCT, and these genes are strongly expressed during the period of seed oil synthesis. Loss of expression of BnROD1.A3 and BnROD1.C3 in a double mutant, or by RNA interference, reduced the PUFA content of the oil to 26.6% compared with 32.5% in the wild type. These results indicate that ROD1 isozymes in canola are responsible for less than 20% of the PUFAs that accumulate in the seed oil compared with 40% in Arabidopsis. Our results demonstrate the care needed when translating results from a model species to crop plants.
Assuntos
Brassica napus/metabolismo , Triglicerídeos/biossíntese , Arabidopsis/metabolismo , Brassica napus/enzimologia , Brassica napus/genética , Ácidos Graxos Insaturados/metabolismo , Genes de Plantas , Redes e Vias Metabólicas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Transcriptoma , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
The Arabidopsis male-sterile phenotype has been a wonderful model for jasmonate action in plants. It has allowed us to identify transcription factors that control gene expression during stamen and pollen maturation and provided for the discovery of the JAZ repressor proteins and the mechanism of jasmonate signaling. More recently, it has revealed intriguing details of the spatial localization of jasmonate synthesis and perception in stamen tissues. The extensive and thoughtful application of protein-protein interaction assays to identify JAZ-interacting partners has led to a much richer appreciation of the mechanisms by which jasmonate integrates with the actions of other hormones to regulate plant growth and physiological responses. This integration is strikingly evident in stamen and pollen development in Arabidopsis, which requires the actions of many hormones. Just as importantly, it is now evident that jasmonate has very different actions during flower development and reproduction in other plant species. This integration and diversity of action indicates that many exciting discoveries remain to be made in this area of jasmonate hormone signaling and response.
RESUMO
Understanding the biochemistry of triacylglycerol (TAG) assembly is critical in tailoring seed oils to produce high-value products. Hydroxy-fatty acid (HFA) is one such valuable modified fatty acid, which can be produced at low levels in Arabidopsis (Arabidopsis thaliana) seed through transgenic expression of the castor (Ricinus communis) hydroxylase. The resulting plants have low seed oil content and poor seedling establishment, indicating that Arabidopsis lacks efficient metabolic networks for biosynthesis and catabolism of hydroxy-containing TAG. To improve utilization of such substrates, we expressed three castor acyltransferase enzymes that incorporate HFA at each stereochemical position during TAG synthesis. This produced abundant tri-HFA TAG and concentrated 44% of seed HFA moieties into this one TAG species. Ricinoleic acid was more abundant than any other fatty acid in these seeds, which had 3-fold more HFA by weight than that in seeds following simple hydroxylase expression, the highest yet measured in a nonnative plant. Efficient utilization of hydroxy-containing lipid substrates increased the rate of TAG synthesis 2-fold, leading to complete relief of the low-oil phenotype. Partition of HFA into specific TAG molecules increased the storage lipid available for mobilization during seedling development, resulting in a 1.9-fold increase in seedling establishment. Expression of a complete acyltransferase pathway to efficiently process HFA establishes a benchmark in the quest to successfully produce modified oils in plants.
Assuntos
Aciltransferases/metabolismo , Arabidopsis/metabolismo , Proteínas de Plantas/fisiologia , Triglicerídeos/biossíntese , Aciltransferases/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Engenharia Genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Ricinus/genética , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
Producing healthy, high-oleic oils and eliminating trans-fatty acids from foods are two goals that can be addressed by reducing activity of the oleate desaturase, FAD2, in oilseeds. However, it is essential to understand the consequences of reducing FAD2 activity on the metabolism, cell biology and physiology of oilseed crop plants. Here, we translate knowledge from studies of fad2 mutants in Arabidopsis (Arabidopsis thaliana) to investigate the limits of non-GMO approaches to maximize oleic acid in the seed oil of canola (Brassica napus), a species that expresses three active FAD2 isozymes. A series of hypomorphic and null mutations in the FAD2.A5 isoform were characterized in yeast (Saccharomyes cerevisiae). Then, four of these were combined with null mutations in the other two isozymes, FAD2.C5 and FAD2.C1. The resulting mutant lines contained 71-87% oleic acid in their seed oil, compared with 62% in wild-type controls. All the mutant lines grew well in a greenhouse, but in field experiments we observed a clear demarcation in plant performance. Mutant lines containing less than 80% oleate in the seed oil were indistinguishable from wild-type controls in growth parameters and seed oil content. By contrast, lines with more than 80% oleate in the seed oil had significantly lower seedling establishment and vigor, delayed flowering and reduced plant height at maturity. These lines also had 7-11% reductions in seed oil content. Our results extend understanding of the B. napusFAD2 isozymes and define the practical limit to increasing oil oleate content in this crop species.
Assuntos
Brassica napus/genética , Ácidos Graxos Dessaturases/metabolismo , Ácido Oleico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Óleos de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Brassica napus/metabolismo , Produtos Agrícolas , Ácidos Graxos Dessaturases/genética , Isoenzimas , Mutação com Perda de Função , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sementes/genética , Sementes/metabolismoRESUMO
Researchers have long endeavored to produce modified fatty acids in easily managed crop plants where they are not natively found. An important step toward this goal has been the biosynthesis of these valuable products in model oilseeds. The successful production of such fatty acids has revealed barriers to the broad application of this technology, including low seed oil and low proportion of the introduced fatty acid and reduced seed vigor. Here, we analyze the impact of producing hydroxy-fatty acids on seedling development. We show that germinating seeds of a hydroxy-fatty acid-accumulating Arabidopsis (Arabidopsis thaliana) line produce chlorotic cotyledons and suffer reduced photosynthetic capacity. These seedlings retain hydroxy-fatty acids in polar lipids, including chloroplast lipids, and exhibit decreased fatty acid synthesis. Triacylglycerol mobilization in seedling development also is reduced, especially for lipids that include hydroxy-fatty acid moieties. These developmental defects are ameliorated by increased flux of hydroxy-fatty acids into seed triacylglycerol created through the expression of either castor (Ricinus communis) acyltransferase enzyme ACYL-COA:DIACYLGLYCEROL ACYLTRANSFERASE2 or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1A. Such expression increases both the level of total stored triacylglycerol and the rate at which it is mobilized, fueling fatty acid synthesis and restoring photosynthetic capacity. Our results suggest that further improvements in seedling development may require the specific mobilization of triacylglycerol-containing hydroxy-fatty acids. Understanding the defects in early development caused by the accumulation of modified fatty acids and providing mechanisms to circumvent these defects are vital steps in the development of tailored oil crops.
Assuntos
Aciltransferases/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Sementes/metabolismo , Aciltransferases/genética , Arabidopsis/genética , Clorofila/metabolismo , Ácidos Graxos/genética , Regulação da Expressão Gênica de Plantas , Germinação , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Metabolismo dos Lipídeos/genética , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Ricinus/genética , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Triglicerídeos/metabolismoRESUMO
While plant oils are an important source of food, plants also produce oils containing specialized fatty acids with chemical and physical properties valued in industry. Ricinoleic acid, a hydroxy fatty acid (HFA) produced in the seed of castor (Ricinus communis), is of particular value, with a wide range of applications. Since castor cultivation is currently successful only in tropical climates, and because castor seed contain the toxin ricin, there are ongoing efforts to develop a temperate crop capable of HFA biosynthesis. In castor, ricinoleic acid is incorporated into triacylglycerol (TAG) which accumulates in the seed lipid droplets. Research in the model plant Arabidopsis (Arabidopsis thaliana) has successfully produced HFA constituting 30% of the total seed oil, but this is far short of the level required to engineer commercially viable crops. Strategies to increase HFA have centered on co-expression of castor TAG biosynthesis enzymes. However, since lipid droplets are the location of neutral lipid storage, manipulating droplets offers an alternative method to increase oil that contains specialized fatty acids. The Arabidopsis Seipin1 protein modulates TAG accumulation by affecting lipid droplet size. Here, we overexpress Seipin1 in a hydroxylase-expressing Arabidopsis line, increasing seed HFA by 62% and proportionally increasing total oil. Increased seed oil was concomitant with a 22% increase in single seed weight and a 69% increase in harvest weight, while seed germination rose by 45%. Because Seipin1 function is unaffected by the structure of the HFA, these results demonstrate a novel strategy that may increase accumulation of many specialized seed oils.
Assuntos
Regulação da Expressão Gênica de Plantas , Óleos de Plantas/metabolismo , Proteínas de Plantas/genética , Ácidos Ricinoleicos/metabolismo , Sementes/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Ricinus communis/genética , Ricinus communis/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/metabolismo , Triglicerídeos/metabolismoRESUMO
Chilling stress is a major factor limiting plant development and crop productivity. Because the plant response to chilling is so complex, we are far from understanding the genes important in the response to chilling. To identify new genes important in chilling tolerance, we conducted a novel mutant screen, combining a confirmed SALK T-DNA insertion collection with traditional forward genetics. We screened a pool of more than 3700 confirmed homozygous SALK T-DNA insertion lines for visible defects under prolonged growth at 5°C. Of the chilling-sensitive mutants we observed, mutations at one locus were characterized in detail. This gene, At1g45231, encodes an Arabidopsis (Arabidopsis thaliana) trimethylguanosine synthase (TGS1), previously uncharacterized in the plant kingdom. We confirmed that Arabidopsis TGS1 is a functional ortholog of other trimethylguanosine synthases based both on its in vitro methyltransferase activity and on its ability to rescue the cold-growth inhibition of a Saccharomyces cerevisiae tgs1Δ mutant in vivo. While tgs1 mutant plants grew normally at 22°C, their vegetative and reproductive growth was severely compromised under chilling conditions. When we transgenically expressed TGS1 in the mutant plants, the chilling-sensitive phenotype was relieved, demonstrating that TGS1 is required for chilling tolerance.
Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Temperatura Baixa , Metiltransferases/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Metiltransferases/química , Metiltransferases/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/metabolismo , Reprodução , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Directed evolution of a cyanobacterial Δ9 fatty acid desaturase (DSG) from Synechococcus elongatus, PCC6301 created new, more productive desaturases and revealed the importance of certain amino acid residues to increased desaturation. A codon-optimized DSG open reading frame with an endoplasmic-reticulum retention/retrieval signal appended was used as template for random mutagenesis. Increased desaturation was detected using a novel screen based on complementation of the unsaturated fatty acid auxotrophy of Saccharomyces cerevisiae mutant ole1Δ. Amino acid residues whose importance was discovered by the random processes were further examined by saturation mutation to determine the best amino acid at each identified location in the peptide chain and by combinatorial analysis. One frequently-detected single amino acid change, Q240R, yielded a nearly 25-fold increase in total desaturation in S. cerevisiae. Several other variants of the protein sequence with multiple amino acid changes increased total desaturation more than 60-fold. Many changes leading to increased desaturation were in the vicinity of the canonical histidine-rich regions known to be critical for electron transfer mediated by these di-iron proteins. Expression of these evolved proteins in the seed of Arabidopsis thaliana altered the fatty acid composition, increasing monounsaturated fatty acids and decreasing the level of saturated fatty acid, suggesting a potential application of these desaturases in oilseed crops. Biotechnol. Bioeng. 2016;113: 1522-1530. © 2016 Wiley Periodicals, Inc.
Assuntos
Cianobactérias/enzimologia , Evolução Molecular Direcionada/métodos , Ácidos Graxos Dessaturases/genética , Ácidos Graxos/metabolismo , Engenharia Metabólica/métodos , Saccharomyces cerevisiae/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianobactérias/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/química , Ácidos Graxos/genética , Óleos de Plantas/análise , Óleos de Plantas/química , Óleos de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C-6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Temperatura Baixa , Genes Supressores , Mutação/genética , Células Procarióticas/metabolismo , Alelos , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Fenótipo , Fosfatidilgliceróis/química , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop.
Assuntos
Brassica napus/genética , Diacilglicerol O-Aciltransferase/genética , Sementes/metabolismo , Acil Coenzima A/metabolismo , Brassica napus/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Óleos de Plantas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
In all eukaryotes, NADH:cytochrome b5 reductase provides electrons, via cytochrome b5, for a range of biochemical reactions in cellular metabolism, including for fatty acid desaturation in the endoplasmic reticulum. Studies in mammals, yeast, and in vitro plant systems have shown that cytochrome b5 can, at least in some circumstances, also accept electrons from NADPH:cytochrome P450 reductase, potentially allowing for redundancy in reductase function. Here, we report characterization of three T-DNA insertional mutants of the gene encoding cytochrome b5 reductase in Arabidopsis thaliana, CBR1. The progeny of plants heterozygous for the cbr1-2 allele segregated 6% homozygous mutants, while cbr1-3 and cbr1-4 heterozygotes segregated 1:1 heterozygous:wild type, indicating a gametophyte defect. Homozygous cbr1-2 seeds were deformed and required Suc for successful germination and seedling establishment. Vegetative growth of cbr1-2 plants was relatively normal, and they produced abundant flowers, but very few seeds. The pollen produced in cbr1-2 anthers was viable, but when germinated on cbr1-2 or wild-type stigmas, most of the resulting pollen tubes did not extend into the transmitting tract, resulting in a very low frequency of fertilization. These results indicate that cytochrome b5 reductase is not essential during vegetative growth but is required for correct pollen function and seed maturation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Citocromo-B(5) Redutase/metabolismo , Pólen/enzimologia , Alelos , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Segregação de Cromossomos/genética , Cruzamentos Genéticos , DNA Bacteriano , Fertilização , Teste de Complementação Genética , Germinação , Homozigoto , Mutagênese Insercional/genética , Mutação/genética , Fenótipo , Pólen/citologia , Pólen/crescimento & desenvolvimento , Tubo Polínico/citologia , Tubo Polínico/enzimologia , Tubo Polínico/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2(Δ9,12) substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b5 protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b5 domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b5s were coexpressed with the desaturase. Coexpression of a cytochrome b5 from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b5 proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.