Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants

We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate. We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems. Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli. The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination. We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions. Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.     

Glycerol catabolism in wild-type and mutant strains ofPseudomonas aeruginosa

Glycerol uptake, glycerol kinase (EC 2.7.1.30) and glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) activities are specifically induced during growth ofPseudomonas aeruginosa PAO on either glycerol or glycerol-3-phosphate. Mutants of strain PAO unable to grow on both glycerol and glycerol-3-phosphate were isolated. Mutant PFB 121 was deficient in an inducible, membrane-bound, pyridine nucleotide-independent, glycerol-3-phosphate dehydrogenase activity and PFB 82 was deficient in glycerol uptake and glycerol kinase and glycerol-3-phosphate dehydrogenase activities. Each mutant spontaneously reverted to wild phenotype, which indicates that each contained a single genetic lesion. These results demonstrate that membrane-bound, inducible glycerol-3-phosphate dehydrogenase is required for catabolism of both glycerol and glycerol-3-phosphate and provide suggestive evidence for a single regulatory locus that controls the synthesis of glycerol uptake, glycerol kinase, and glycerol-3-phosphate dehydrogenase inP. aeruginosa.     

Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities

We have transformed varieties of wheat with a Pisum sativum glycerol-3-phosphate acyltransferase gene, and also with an Arabidopsis thaliana acyl-ACP thioesterase gene. Morphological (growth, organelle development) and metabolic changes (fatty acid labelling of chloroplast and non-chloroplast lipids) have been observed in transgenics with altered gene expression for either enzyme.     

Relationship between glycerol-3-phosphate dehydrogenase,fatty acid synthase and fatty acid binding proteins in developing human placenta

The activities of the enzymes glycerol-3-phosphate dehydrogenase and fatty acid synthase are inhibited by palmitoyl-coenzyme A and oleate. The two isoforms of fatty acid binding proteins (PI 6.9 and PI 5.4) enhance the activities of glycerol-3-phosphate dehydrogenase and fatty acid synthase in the absence of palmitoyl-coenzyme A or oleate and also protect them against palmitoyl-coenzyme A or oleate inhibition. Levels of fatty acid binding proteins, the activities of the enzymes fatty acid synthase and glycerol-3-phosphate dehydrogenase increase with gestation showing a peak at term. However, the activity of fatty acid synthase showed the same trend up to the 30th week of gestation and then declined slightly at term. With the advancement of pregnancy when more lipids are required for the developing placenta, fatty acid binding proteins supply more fatty acids and glycerol-3-phosphate for the synthesis of lipids. Thus a correlation exists between glycerol-3-phosphate dehydrogenase, fatty acid synthase and fatty acid binding proteins in developing human placenta.     

Increasing seed oil content in oil-seed rape (Brassica napus L.) by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter

Previous attempts to manipulate oil synthesis in plants have mainly concentrated on the genes involved in the biosynthesis and use of fatty acids, neglecting the possible role of glycerol-3-phosphate supply on the rate of triacylglycerol synthesis. In this study, a yeast gene coding for cytosolic glycerol-3-phosphate dehydrogenase ( gpd 1) was expressed in transgenic oil-seed rape under the control of the seed-specific napin promoter. It was found that a twofold increase in glycerol-3-phosphate dehydrogenase activity led to a three- to fourfold increase in the level of glycerol-3-phosphate in developing seeds, resulting in a 40% increase in the final lipid content of the seed, with the protein content remaining substantially unchanged. This was accompanied by a decrease in the glycolytic intermediate dihydroxyacetone phosphate, the direct precursor of glycerol-3-phosphate dehydrogenase. The levels of sucrose and various metabolites in the pathway from sucrose to fatty acids remained unaltered. The results show that glycerol-3-phosphate supply co-limits oil accumulation in developing seeds. This has important implications for strategies that aim to increase the overall level of oil in commercial oil-seed crops for use as a renewable alternative to petrol.     

Substrate-induced dissociation of glycerol-3-phosphate dehydrogenase and its complex formation with fructose-bisphosphate aldolase

A threefold decrease in specific activity of glycerol-3-phosphate dehydrogenase was found on going from 800 nM to 10 nM enzyme concentration. According to ultracentrifugal analyses the dimeric glycerol-3-phosphate dehydrogenase (molecular weight 78,000) dissociates into monomers in the equilibrium mixture of its substrates and products. The concentration-dependent decrease in the specific activity is interpreted as a consequence of subunit dissociation and the estimated dissociation constants are 0.7 micro M and 3.5 micro M at 38 degrees C and 20 degrees C respectively. According to active-enzyme-band centrifugation experiments and kinetic analysis aldolase forms a complex with glycerol-3-phosphate dehydrogenase and this complex formation influences the specific activity of the dehydrogenase. The interaction between glycerol-3-phosphate dehydrogenase and aldolase can provide a regulatory mechanism at the branching point of glycolytic and lipid metabolic pathways.     

Metabolome analysis of food-chain between plants and insects

Evolution has shown the co-dependency between host plants and predators (insects), especially inevitable dependency of predators on plant biomass for securing their energy sources. It was postulated that NAD⁺ source used for major energy producing pathway is the glycerol-3-phosphate shuttle in insects. Using high throughput metabolomics approach, we found that larva of leaf beetle (Gastrophysa atrocyanea), which feed oxalate-rich plants (Rumex obtusifolius), possessed a unique mechanism for accumulating unusually high amounts of lactate. Similarly, larva of butterfly (Papilio machaon) fed with fennel (Foeniculum vulgare) accumulated lactate. Same butterfly also showed the elevated level of glycerol-3-phosphate equivalent to lactate. These evidences provide new insights into the mechanism underlying metabolite alteration between host plants and insect herbivores.     

Description and applications of a rapid and sensitive non-radioactive microplate-based assay for maximum and initial activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase

D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the first step in photosynthetic carbon assimilation and represents the largest sink for nitrogen in plants. Improvement of its kinetic properties or the efficiency with which it is used in planta would benefit photosynthesis, nitrogen and water use efficiency, and yield. This paper presents a new non-radioactive microplate-based assay, which determines the product [3-phosphoglycerate (3-PGA)] in an enzymic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase. High sensitivity permits use of highly diluted extracts, and a short reaction time to avoid problems due to fall-off. Throughput was several hundreds of samples per person per day. Sensitivity and convenience compared favourably with radioisotopic assays, which were previously used to assay Rubisco. Its use is illustrated in three applications. (1) Maximal and initial activities and the K(m) for ribulose-1,5-bisphosphate were determined in raw extracts of leaves from several species. Similar values were obtained from those in the literature. (2) Diurnal changes were compared in rosettes of wild-type (WT) Arabidopsis and the starchless pgm mutant. Despite these dramatic differences in carbon metabolism, Rubisco activity and activation were similar in both genotypes. (3) A preliminary association mapping study was performed with 118 Arabidopsis accessions, using 183 markers that probably cover approximately 3-8% of the total genome. At a P-value < 0.005, two, two and no quantitative trait loci (QTL) were found for Rubisco maximal activity, initial activity and activation state, respectively. Inspection of the genomic regions that span these markers revealed these QTL involved genes not previously implicated in the regulation of Rubisco expression or activity.     

Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family,is essential for tapetum differentiation and male fertility

Membrane-bound glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) mediates the initial step of glycerolipid biosynthesis in the extraplastidic compartments of plant cells. Here, we report the molecular characterization of a novel GPAT gene family from Arabidopsis, designated AtGPAT. The corresponding polypeptides possess transmembrane domains and GPAT activity when expressed heterologously in a yeast lipid mutant. The functional significance of one isoform, AtGPAT1, is the focus of the present study. Disruption of the AtGPAT1 gene causes a massive pollen development arrest, and subsequent introduction of the gene into the mutant plant rescues the phenotype, illustrating a pivotal role for AtGPAT1 in pollen development. Microscopic examinations revealed that the gene lesion results in a perturbed degeneration of the tapetum, which is associated with altered endoplasmic reticulum profiles and reduced secretion. In addition to the sporophytic effect, AtGPAT1 also exerts a gametophytic effect on pollen performance, as the competitive ability of a pollen grain to pollinate is dependent on the presence of an AtGPAT1 gene. Deficiency in AtGPAT1 correlates with several fatty acid composition changes in flower tissues and seeds. Unexpectedly, however, a loss of AtGPAT1 causes no significant change in seed oil content.     

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction.

The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.     

Localization of myo-inositol-1-phosphate synthase to the endosperm in developing seeds of Arabidopsis

Expression and localization of myo-inositol-1-phosphate synthase (MIPS) in developing seeds of Arabidopsis thaliana was investigated. MIPS is an essential enzyme for production of inositol and inositol phosphates via its circularization of glucose-6-phosphate as the initial step. myo-inositol-6-phosphate (InsP(6) or phytic acid) is the predominant form of phosphorus found in seeds and accumulates as a consequence of MIPS action. Three MIPS genes have been identified in Arabidopsis, all of which were expressed not only in siliques but in both leaves and roots. Immunoelectron microscopy using a MIPS antibody showed that MIPS localizes to the cytosol primarily in the endosperm during seed development and not in the embryo. This is consistent with results obtained using fluorescent microscopy and western blot analysis that showed a similar pattern of localization. However, InsP(6), which is the final product of inositol phosphate metabolism, was present mainly in the embryo. This suggests that a complex interaction between the endosperm and embryo occurs during the synthesis and subsequent accumulation of InsP(6) in developing seeds of Arabidopsis.     

Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis

The activity of some enzymes of intermediary metabolism, including enzymes of glycolysis, the hexose monophosphate shunt, and polyol cryoprotectant synthesis, were measured in freeze-tolerant Eurosta solidaginis larvae over a winter season and upon entry into pupation. Flexible metabolic rearrangement was observed concurrently with acclimatization and development. Profiles of enzyme activities related to the metabolism of the cryoprotectant glycerol indicated that fall biosynthesis may occur from two possible pathways: 1. glyceraldehyde-phosphate glyceraldehyde glycerol, using glyceraldehyde phosphatase and NADPH-linked polyol dehydrogenase, or 2. dihydroxyacetonephosphate glycerol-3-phosphate glycerol, using glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. Clearance of glycerol in the spring appeared to occur by a novel route through the action of polyol dehydrogenase and glyceraldehyde kinase. Profiles of enzyme activities associated with sorbitol metabolism suggested that this polyol cryoprotectant was synthesized from glucose-6-phosphate through the action of glucose-6-phosphatase and NADPH-linked polyol dehydrogenase. Removal of sorbitol in the spring appeared to occur through the action of sorbitol dehydrogenase and hexokinase. Glycogen phosphorylase activation ensured the required flow of carbon into the synthesis of both glycerol and sorbitol. Little change was seen in the activity of glycolytic or hexose monophosphate shunt enzymes over the winter. Increased activity of the -glycerophosphate shuttle in the spring, indicated by greatly increased glycerol-3-phosphate dehydrogenase activity, may be key to removal and oxidation of reducing equivalents generated from polyol cryoprotectan catabolism.Abbreviations 6PGDH 6-Phosphogluconate dehydrogenase - DHAP dihydroxy acetone phosphate - F6P fructose-6-phosphate - F6Pase fructose-6-phospha-tase - FBPase fructose-bisphosphatase - G3P glycerol-3-phosphate - G3Pase glycerol-3-phosphate phophatase - G3PDH glycerol-3-phosphate dehydrogenase - G6P glucose-6-phosphate - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - GAK glyceraldehyde kinase - GAP glyceraldehyde-3-phosphate - GAPase glyceraldehyde-3-phosphatase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glycerol dehydrogenase - GPase glycogen phosphorylase - HMS hexose monophosphate shunt - LDH lactate dehydrogenase - NADP-IDH NADP⁺-dependent isocitrate dehydrogenase - PDHald polyol dehydrogenase, glyceraldehyde activity - PDHgluc polyol dehydrogenase, glucose activity - PFK phosphofructokinase - PGI phosphoglucoisomerase - PGK phosphoglycerate kinase - PGM phosphoglucomutase - PK pyruvate kinase - PMSF phenylmethylsulfonylfluoride - SoDH sorbitol dehydrogenase - V _max maximal enzyme activity - ww wet weight     

Model of early self-replication based on covalent complementarity for a copolymer of glycerate-3-phosphate and glycerol-3-phosphate

Glyceraldehyde-3-phosphate acts as the substrate in a model of early self-replication of a phosphodiester copolymer of glycerate-3-phosphate and glycerol-3-phosphate. This model of self-replication is based on covalent complementarity in which information transfer is mediated by a single covalent bond, in contrast to multiple weak interactions that establish complementarity in nucleic acid replication. This replication model is connected to contemporary biochemistry through its use of glyceraldehyde-3-phosphate, a central metabolite of glycolysis and photosynthesis.     

Differential Metabolite Profiles during Fruit Development in High-Yielding Oil Palm Mesocarp

To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.     

Fatty acid metabolism in sn-glycerol-3-phosphate acyltransferase (plsB) mutants.

Fatty acid metabolism was examined in Escherichia coli plsB mutants that were conditionally defective in sn-glycerol-3-phosphate acyltransferase activity. The fatty acids synthesized when acyl transfer to glycerol-3-phosphate was inhibited were preferentially transferred to phosphatidylglycerol. A comparison of the ratio of phospholipid species labeled with 32Pi and [3H]acetate in the presence and absence of glycerol-3-phosphate indicated that [3H]acetate incorporation into phosphatidylglycerol was due to fatty acid turnover. A significant contraction of the acetyl coenzyme A pool after glycerol-3-phosphate starvation of the plsB mutant precluded the quantitative assessment of the rate of phosphatidylglycerol fatty acid labeling. Fatty acid chain length in membrane phospholipids increased as the concentration of the glycerol-3-phosphate growth supplement decreased, and after the abrupt cessation of phospholipid biosynthesis abnormally long chain fatty acids were excreted into the growth medium. These data suggest that the acyl moieties of phosphatidylglycerol are metabolically active, and that competition between fatty acid elongation and acyl transfer is an important determinant of the acyl chain length in membrane phospholipids.     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.