bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular biology of cytosolic acetyl-CoA generation
Authors: Fatland B. , Anderson M. , Nikolau B. J. , Wurtele E. S.
Published: January 2005

ATP citrate lyase (ACL) catalyses the ATP-dependent reaction between citrate and CoA to form oxaloacetate and acetyl-CoA. Our molecular characterizations of the cDNAs and genes coding for the Arabidopsis ACL indicate that the plant enzyme is heteromeric, consisting of two dissimilar subunits. The A subunit is homologous to the N-terminal third of the animal ACL, and the B subunit is homologous to C-terminal two-thirds of the animal ACL. Using both ACL-A- and ACL-B-specific antibodies and activity assays we have shown that ACL is located in the cytosol, and is not detectable in the plastids, mitochondria or peroxisomes. During seed development, ACL-A and ACL-B mRNA accumulation is co-ordinated with the accumulation of the cytosolic homomeric acetyl-CoA carboxylase mRNA. Antisense Arabidopsis plants reduced in ATP citrate lyase activity show a complex phenotype, with miniaturized organs, small cell size, aberrant plastid morphology and reduced cuticular wax. Our results indicate that ACL generates the cytosolic pool of acetyl-CoA, which is the substrate required for the biosynthesis of a variety of phytochemicals, including cuticular waxes and flavonoids.
Molecular aspects of β-ketoacyl synthase (KAS) catalysis
Authors: von Wettstein-Knowles P. , Olsen J. Gotthardt , McGuire K. Arnvig , Larsen S.
Published: January 2005

Crystal structure data for Escherichia coli β-ketoacyl synthase (KAS) I with C10 and C12 fatty acid substrates bound in conjunction with results from mutagenizing residues in the active site leads to a model for catalysis. Differences from and similarities to the other Claisen enzymes carrying out decarboxylations reveal two catalytic mechanisms, one for KAS I and KAS II, the other for KAS III and chalcone synthase. A comparison of the structures of KAS I and KAS II does not reveal the basis of chain-length specificity. The structures of the Arabidopsis thaliana KAS family are compared.
Acyl carrier protein (ACP) inhibition and other differences between β-ketoacyl synthase (KAS) I and II
Authors: McGuire K. Arnvig , McGuire J. N. , von Wettstein-Knowles P.
Published: January 2005

Escherichia coli β-ketoacyl synthases (KAS) I and II carry out the elongation steps in fatty acid synthesis. Analyses using the cross-linker BSa [bis(sulphosuccinimidyl) suberate] and surfaceenhanced laser desorption/ionization-time-offlight MS disclosed only monomeric and dimeric forms of KAS II, whereas KAS I also forms higher multimers. The binding affinities for KAS I and KAS II to C14-acyl carrier protein (ACP) as well as for C14-ACP to KAS I and KAS II were determined. KAS I is sensitive to the ACP released during the transfer reaction, with 50% inhibition at 0.17 μM ACP close to the physiological concentration of ACP (0.13 μM). KAS I and II also differ in carrying out the decarboxylation step of the elongation reaction.
Protein interactions of fatty acid synthase II
Authors: Honeyman G. , Fawcett T.
Published: January 2005

We have used a yeast two-hybrid approach to detect direct protein interactions between fatty acid synthase components. Enoyl-acyl carrier protein (ACP) reductase was found to interact with stearoyl-ACP desaturase and acyl-ACP thioesterase, but none of these proteins interacted with ACP in the yeast nucleus.
Cloning of a palmitoyl-acyl carrier protein thioesterase from oil palm
Authors: Othman A. , Lazarus C. , Fraser T. , Stobart K.
Published: January 2005

A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northernblot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns.
Isolation of two putative acyl-acyl carrier protein desaturase enzymes from Kochia scoparia
Authors: Whitney H. , Sayanova O. , Lewis M. J. , Pickett J. , Napier J. A.
Published: January 2005

5-Hexadecenoic acid can be used to produce a semiochemical method to control the disease-carrying mosquito Culex quinquefasciatus. This ususual fatty acid is produced in the seed of Kochia scoparia. We have isolated two acyl-acyl carrier protein desaturases from this species and expressed them in Escherichia coli to facilitate functional characterisation.
Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal ω-3 fatty acid desaturases
Authors: Meesapyodsuk D. , Reed D. W. , Savile C. K. , Buist P. H. , Schäfer U. A. , Ambrose S. J. , Covello P. S.
Published: January 2005

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the ω-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16–20 carbons with a preference for ω-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of ω-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments. Whereas kH/kD at the ω-3 position was shown to be large, essentially no kinetic isotope effect at the ω-2 position was observed for the plant or the nematode enzymes. These results indicate that ω-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.
Mutagenesis of the borage Δ6 fatty acid desaturase
Authors: Sayanova O. , Beaudoin F. , Libisch B. , Shewry P. , Napier J.
Published: January 2005

The consensus sequence of the third histidine box of a range of Δ5, Δ6, Δ8 and sphingolipid desaturases differs from that of the membrane-bound non-fusion Δ12 and Δ15 desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b5 fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Δ6 desaturase.
An unusual desaturase in Aquilegia vulgaris
Authors: Longman A. J. , Michaelson L. V. , Sayanova O. , Napier J. A. , Stobart A. K.
Published: January 2005

Aquilegia vulgaris seed oil contains high levels of the rare fatty acid columbinic acid (18:3 Δ5,9,12), which is unusual in having the double bond at the Δ5 carbon in the trans configuration. Columbinic acid was found to be a seed-specific fatty acid not only present in the storage oil but also in membrane lipids. Several putative gene fragments have been isolated from plant RNA with sequences similar to previously characterized ‘front-end’ desaturases. Functional characterization of the Aquilegia cDNA is underway.
The inhibition of fatty acid elongation by pebulate can be effectively counteracted by the safener dichlormid
Authors: Baldwin A. , Francis D. , Rogers H. J. , Harwood J. L.
Published: January 2005

The thiocarbamate herbicide pebulate inhibits fatty acid elongation, which is necessary for surface lipid biosynthesis. As both barley and wild oats are susceptible to pebulate, the safener dichlormid was used to study the reversal of its herbicidal effect. Fatty acid elongation was restored by a dichlormid pretreatment in barley, but not in pebulate-expressed oats.
Cloning and functional expression of the first plant fatty acid elongase specific for Δ6-polyunsaturated fatty acids
Authors: Zank T. K. , Zähringer U. , Lerchl J. , Heinz E.
Published: January 2005

In order to elucidate the biosynthesis of longchain polyunsaturated fatty acids (PUFAs) in plants we searched for a cDNA encoding a Δ6-specific PUFA elongase from Physcomitrella patens which is known to contain high proportions of arachidonic acid (20:4 Δ5,8,11,14). An EST clone from P. patens was identified by its low homology to the yeast gene ELO1, which is required for the elongation of medium-chain fatty acids. We functionally characterized this cDNA by heterologous expression in Saccharomyces cerevisiae grown in the presence of several fatty acids. Analysis of the fatty acid profile of the transgenic yeast revealed that the cDNA encodes a protein that leads to the elongation of the C18 Δ6-polyunsaturated fatty acids γ-linolenic acid (18:3 Δ6,9,12) and stearidonic acid (18:4 Δ6,9,12,15), which were recovered to 45–51% as their elongation products. In contrast, linoleic and α-linolenic acids were hardly elongated and we could not measure any elongation of saturated and mono-unsaturated fatty acids (including 18:1 Δ6), indicating that the elongase is highly specific for the polyunsaturated nature of the fatty acid acting as substrate.
The role of plastidial transporters in developing embryos of oilseed rape (Brassica napus L.) for fatty acid synthesis
Authors: Kubis S. E. , Rawsthorne S.
Published: January 2005

The phosphoenolpyruvate transporter (PPT) is one of several important transporters for channelling carbon intermediates utilized for fatty acid synthesis and other plastidial pathways from the cytosol into the plastid. In this paper we show results on how the activity of the PPT changes between two important, physiologically different developmental stages of oilseed rape embryos.
Metabolic control analysis of de novo sunflower fatty acid biosynthesis
Authors: Martinez-Force E. , Garcés R.
Published: January 2005

We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower control line RHA-274. For this simulation, we have used data from the evolution of fatty acids during seed formation and from the biochemical characterization of β-keto-acyl-ACP synthetase II (FASII), stearoyl-ACP desaturase (SAD) and acyl-ACP thioesterase activities and the program GEPASI (based on the metabolic control-analysis theory). When physiological data from high- and mediumstearic acid mutants seed development were used with this model the predicted changes in SAD and TE were very similar to those actually found in the biochemical characterization of these mutants. However, the model had to be modified when results from high-palmitic mutants, accumulating unusual fatty acids like palmitoleic, asclepic and palmitolinoleic acids, were used. The emerging model, that fits all of our results, predicts the existence of a dynamic channelling between the FASII complex and SAD, that channelling being responsible for the alternative pathway starting with the desaturation of palmitic acid by the stearoyl-ACP desaturase. This channelling is consistent with our previous results. For instance, the determination of SAD activity on sunflower seed crude extracts only rendered oleic acid when the stearic acid used as a substrate was obtained from a KASII assay, but not when the stearic acid came from in vitro synthesis using acyl-ACP synthetase from Escherichia coli. This theoretical approximation will be very useful in predicting the evolution of the system when introducing new or modified activities; similar approximations in other oil-seed crops could be of great interest.
Role of acyl-CoAs and acyl-CoA-binding protein in regulation of carbon supply for fatty acid biosynthesis
Authors: Fox S. R. , Rawsthorne S. , Hills M. J.
Published: January 2005

Acyl-CoA esters inhibit the plastidial glucose 6-phosphate (Glc-6-P) transporter and the adenylate transporter; the IC50 values for the inhibition by oleoyl-CoA (18:1-CoA) are 200–400 nM and 1–2 μM respectively. The inhibition of either of these processes significantly reduces the flux of carbon from Glc-6-P or from acetate into longchain fatty acids. The effect is dependent on the acyl chain length, e.g. lauryl-CoA is less inhibitory than oleoyl-CoA, causing 34 and 68% inhibition respectively of Glc-6-P uptake after 30 s. The inhibition of Glc-6-P and ATP transport is alleviated by addition of an equivalent concentration of acyl-CoA-binding protein (ACBP) or BSA. Acyl-CoAs do not inhibit pyruvate or glucose transporters. The endogenous concentrations of acyl-CoAs and ACBP are similar during embryo maturation.
Expression and properties of diacylglycerol acyltransferase from cell-suspension cultures of oilseed rape
Authors: Weselake R. J. , Nykiforuk C. L. , Laroche A. , Patterson N. A. , Wiehler W. B. , Szarka S. J. , Moloney M. M. , Tari L. W. , Derekh U.
Published: January 2005

The expression of diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) with predicted molecular mass of 56.9. kDa (BnDGAT1) was examined using microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf). As well, a recombinant histidine-tagged N-terminal fragment of BnDGAT1 [BnDGAT1(1–116)His6], which was relatively hydrophilic, was partially characterized. A temporal increase in DGAT activity occurred within a 24 h period following transfer of cells from 6% (w/v) sucrose to 14% (w/v) sucrose. Western blotting indicated that the abundance of BnDGAT1 protein was closely correlated with DGAT activity. BnDGAT1 mRNA also exhibited a temporal increase within the 24 h period following transfer of cells into higher sucrose concentrations, but the transcript level was not closely associated with DGAT activity as BnDGAT1 protein. The fragment BnDGAT1(1–116)His6, interacted with [1-14C] oleoyl-CoA, suggesting that the N-terminal region of BnDGAT1 may have a role in binding cellular acyl-CoA.
Diacylglycerol acyltransferase in maturing sunflower seeds
Authors: Triki S. , Hamida J. Ben , Mazliak P.
Published: January 2005

Developing sunflower seeds exhibit a high diacylglycerol acyltransferase (DAGAT, EC 2.3.1.20) activity. The distribution of the enzyme has been studied in subcellular fractions prepared by differential centrifugation of seed homogenate. Its activity was characterized using [1-14C]oleoyl-CoA and diolein dispersed in Tween 20. Some properties of the microsomal fraction of DAGAT were investigated. Hyperbolic kinetics were observed, the apparent Km was 60 μM and the specific activity of the reaction 15 pmol/min/mg of protein. Addition of BSA (0.1%) stimulated oleate incorporation, which was not dependent on the presence of exogenous diacylglycerol. Detergents which might solubilize DAGAT, Triton X-100 and CHAPS, were tested for enzyme inhibition, and CHAPS was found to be the least denaturing.
An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Sacchoromyces cerevisiae
Authors: Sandager L. , Dahlqvist A. , Banaś A. , Ståhl U. , Lenman M. , Gustavsson M. , Stymne S.
Published: January 2005

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA: diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA: cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.
Accumulation of storage products in oat during kernel development
Authors: Banaś A. , Dahlqvist A. , Dêbski H. , Gummeson P.-O. , Stymne S.
Published: January 2005

Lipids, proteins and starch are the main storage products in oat seeds. As a first step in elucidating the regulatory mechanisms behind the deposition of these compounds, two different oat varieties, ‘Freja’ and ‘Matilda’, were analysed during kernel development. In both cultivars, the majority of the lipids accumulated at very early stage of development but Matilda accumulated about twice the amount of lipids compared to Freja. Accumulation of proteins and starch started also in the early stage of kernel development but, in contrast to lipids, continued over a considerably longer period. The high-oil variety Matilda also accumulated higher amounts of proteins than Freja. The starch content in Freja kernels was higher than in Matilda kernels and the difference was most pronounced during the early stage of development when oil synthesis was most active. Oleosin accumulation continued during the whole period of kernel development.
Oil biosynthesis in microsomal membrane preparations from Mortierella alpina
Authors: Chatrattanakunchai S. , Fraser T. , Stobart K.
Published: January 2005

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [14C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [14C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.
New insights into the mechanisms of lipid-body biogenesis in plants and other organisms
Authors: Murphy D.J. , Hernendez-Pinzon I. , Patel K. , Hope R. G. , McLauchlan J.
Published: January 2005

A comparative approach has been used to study the role of several lipid-body-binding proteins in plants and animals. Caleosins are a newly discovered class of calcium-binding lipid-body proteins found in plants and fungi, which we now report to have separate endoplasmic reticulum and lipid-body-associated isoforms. We also compare the lipid-body targeting of oleosin from plants and the core protein of the hepatitis C virus when they were expressed separately in lipid-accumulating animal cell lines. This is a novel and powerful approach to investigating the factors that determine the lipid-body targeting of a wide range of proteins.
The multigenic family of monogalactosyl diacylglycerol synthases
Authors: Maréchal E. , Awai K. , Block M. A. , Brun D. , Masuda T. , Shimada H. , Takamiya K.-i. , Ohta H. , Joyard J.
Published: January 2005

Because the synthesis of monogalactosyldiacylglycerol (MGDG) is unique to plants, identified as an important marker of the plastid envelope, involved in a key step of plastid biogenesis and is the most abundant lipid on earth, MGDG synthase activity was extensively analysed at the biochemical and physiological levels. In the present paper, we present our current knowledge on the MGDG synthase's function, structure and topology in envelope membranes, and discuss possible roles in plant cell glycerolipid metabolism. The recent discovery of a multigenic family of MGDG synthases raised the possibility that multiple isoenzymes might carry out MGDG synthesis in various tissues and developmental stages.
Transcriptional regulation by light and phytohormones of the MGD gene in cucumber
Authors: Yamaryo Y. , Kanai D. , Awai K. , Masuda T. , Shimada H. , Takamiya K.-i. , Ohta H.
Published: January 2005

Monogalactosyldiacylglycerol (MGDG) synthase catalyses formation of MGDG, a major structural lipid of chloroplasts. We have already cloned a cDNA for the synthase from a cucumber cDNA library and shown that expression of this gene is regulated by light and a phytohormone, cytokinin. In the present study, we report the molecular basis for transcriptional regulation by light and cytokinin in detail. First, in terms of the enzyme activity, gradual increases in activity mediated by light and cytokinin treatments were observed. At the same time, however, the changes in the mRNA level showed different profiles, with a transient peak during the early stages of light and cytokinin treatment. The interval between the peak level of mRNA and enzyme activation implies the existence of a post-transcriptional regulatory system. In addition, a genomic clone of MGDG synthase isolated from a cucumber genome library was used for a motif search in databases, and this revealed that putative cis-acting elements for light and phytohormones exist in the 5Î-upstream region of the MGD cucumber gene. Detailed analysis of this region for light- and cytokinin-responsive activity was performed using a – 90 truncated 35 S minimal promoter/luciferase (LUC) reporter gene. It resulted in high levels of LUC expression in etiolated cucumber cotyledons in response to illumination and cytokinin treatment. These results indicate that this 5Î-upstream region is involved in light- and cytokinin-enhanced MGD gene expression, and that light- and cytokinin-responsive enhancements of the MGDG synthase activity are regulated, at least in part, at the level of transcription.
Cloning and characterization of a cDNA encoding serine palmitoyltransferase in Arabidopsis thaliana
Authors: Tamura K. , Nishiura H. , Mori J. , Imai H.
Published: January 2005

The first and committed step in de novo sphingo-lipid synthesis is catalysed by serine palmitoyl-transferase (EC 2.3.1.50), which condenses serine and palmitoyl-CoA to form 3-ketosphinganine in a pyridoxal-5Î-phosphate-dependent reaction. We have isolated and characterized a cDNA clone from Arabidopsis thaliana that is homologous to yeast and mammalian LCB2. For a functional identification, the A. thaliana homologous cDNA was expressed in Escherichia coli which resulted in significant production of new sphinganine in E. coli cells.
Fatty acid breakdown in developing embryos of Brassica napus L.
Authors: Chia T. , Rawsthorne S.
Published: January 2005

Developing Brassica napus embryos are primarily concerned with the accumulation of storage products, namely oil, starch and protein. The presence of fatty acid catabolic pathways in the background of this biosynthetic activity was investigated. Enzymes involved in the process of lipid mobilization, such as malate synthase and isocitrate lyase, are detectable towards the late stages of embryo development. [14C]Acetate feeding experiments also reveal that fatty acid catabolism becomes increasingly functional as the embryo matures.
The Arabidopsis acyl-CoA oxidase gene family
Authors: Eastmond P. J. , Hooks M. , Graham I. A.
Published: January 2005

A family of acyl-CoA oxidase isozymes catalyse the first step in the peroxisomal fatty acid β-oxidation spiral. Our group and others have recently characterized four genes from this family in the model oilseed Arabidopsis. These genes encode isozymes with different acyl-CoA substrate specificities, which together encompass the full range of fatty acid chain lengths that exist in vivo. Here we review the biochemical properties and physiological roles of the acyl-CoA oxidase isozymes.
Contribution of mitochondria and peroxisomes to palmitate oxidation in pea tissues
Authors: Masterson C. , Wood C.
Published: January 2005

Total, mitochondrial and peroxisomal palmitate oxidation capacities were compared in pea, from the dry seed to 14 days after imbibition. Total β-oxidation varied over the measured time period and showed four peaks of activity at day 2, days 5–6, day 10 and days 12–13. The contribution of peroxisomal and mitochondrial β-oxidation to this overall β-oxidation varied. Over the first 48 h of seed germination, peroxisomal β-oxidation accounted for 80–100% of the total observed β-oxidation. The larger peaks of β-oxidation at days 5–6, day 10 and days 12–13 were due primarily to mitochondrial β-oxidation activity, which accounted for 70–90% of the observed total β-oxidation at these times. The peaks of activity are related to observed stages in seedling development.
Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth
Authors: Masterson C. , Blackburn A. , Wood C.
Published: January 2005

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C4, C12 and C16 acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C16- handling enzyme first peaked at 3–4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.
Arabidopsis thaliana mutants disrupted in lipid mobilization
Authors: Lange P. R. , Graham I.
Published: January 2005

To isolate mutants in the process of lipid mobilization during post-germinative growth we employed a screen using the pro-herbicide 2,4-dichlorophenoxybutyric acid (2,4-DB). The phenotypes of a number of 2,4-DB-resistant mutants are compared with previously characterized mutants disrupted in β-oxidation or the glyoxylate cycle. We conclude that the strength of 2,4-DB resistance and the ability of the seedlings to grow in the absence of exogenous sugar are inversely correlated. Sugar dependence of 2,4-DB-resistant seedlings is a consequence of impaired storage-lipid mobilization.
Screening for lipase activity in the oil palm
Authors: Sambanthamurthi R. , Rajanaidu N. , Parman S. Hasnah
Published: January 2005

The oil palm mesocarp contains an endogenous lipase which is strongly activated at low temperature. Lipase activity is thus very conveniently assayed by prior exposure of the fruits to low temperature. More than 100 oil palm samples from the germplasm collection of the Palm Oil Research Institute of Malaysia (now known as the Malaysian Palm Oil Board) were screened for nonesterified fatty acid activity using both the lowtemperature activation assay and a radioactivity assay. The results showed good correlation between assay procedures. The different samples had a very wide range of lipase activity. Elaeis oleifera samples had significantly lower lipase activity compared with E. guineensis (var. tenera) samples. Even within E. guineensis (var. tenera), there was a wide range of activity. The results confirmed that lipase activity is genotype-dependent. Selection for lipase genotypes is thus possible and this will have obvious commercial value.
Lipase activity in germinating sunflower seedlings
Authors: Bahri S.
Published: January 2005

Studying lipase in germinating sunflower seedlings, we looked for an activator of the lipolytic activity. In the presence of 1.25 mM ATP, the enzyme activity increased 2-fold. Lipid-body lipase solubilization was realized using two detergents: Tween 80 and CHAPS. Lipolytic activity was increased 10-fold in the presence of 2% (w/v) CHAPS, showing the probable ‘complexity’ of the enzyme. Looking for the possible lipolytic activity of the 10000g pellet we detected the presence of the enzyme. The pellet extract was mixed, in a range of concentrations, with the oilbody fraction. The resulting lipolytic activity was 4-fold higher. These results give clues as to the subcellular distribution of lipase and its intracellular transport.
Selective increase in acyl hydrolase activity by graminicides in wheat
Authors: Banaś A. , Banaś W. , Stenlid G. , Stymne S.
Published: January 2005

Seedlings of wheat were grown for 24 h in control nutrient solution and in solutions containing haloxyfop, alloxydim, diquat or paraquat, and thereafter the roots were used for microsomal preparations. Phosphatidylcholine or diacyl-glycerol with various 1-14C-labelled fatty acids (oleic, linoleic, linolenic or ricinoleic acids) in position sn-2 were added to the prepared microsomes. After incubation for 2 h at 30 °C, the lipids were extracted and the distribution of radioactivity among lipid classes was determined. In the microsomal preparations of plants treated with diquat and paraquat, the amounts of fatty acids released were similar to the control, whereas they were 1.4–2 times higher in the microsomal preparation of plants treated with haloxyfop and alloxydim. Thus, the data indicate that graminicides could increase lipid catabolism in sensitive plants and that this is not a general phenomenon connected with inhibition of growth.
Polyprenols in hairy roots of Coluria geoides
Authors: Skorupińska-Tudek K. , Hung V. S. , Olszowska O. , Furmanowa M. , Chojnacki T. , Świezwska E.
Published: January 2005

Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms. They were found mostly in plants, bacteria, yeasts and mammalian cells. In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes. Growth was optimal at 0.75% (w/v) glucose and at 22 °C. Dry samples of roots were extracted and lipid content was analysed by HPLC. According to our estimation, polyprenols are accumulated in roots of C. geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units. The content of polyprenols in tissue was approx. 300 μg/g of dry weight.
Properties and inhibition of the first two enzymes of the non-mevalonate pathway of isoprenoid biosynthesis
Authors: Mueller C. , Schwender J. , Zeidler J. , Lichtenthaler H. K.
Published: January 2005

Enzymes of the 1-deoxy-D-xylulose 5-phosphate/2-C-methylerythritol 4-phosphate (DOXP/MEP) pathway are targets for new herbicides and antibacterial drugs. Until now, no inhibitors for the DOXP synthase have been known of. We show that one of the breakdown products of the herbicide clomazone affects the DOXP synthase. One inhibitor of the non-mevalonate pathway, fosmidomycin, blocks the DOXP reductoisomerase (DXR) of plants and bacteria. The I50 values of plants are, however, higher than those found for the DXR of Escherichia coli. The DXR of plants, isolated from barley seedlings, shows a pH optimum of 8.1, which is typical for enzymes active in the chloroplast stroma.
Effect of monogalactosyldiacylglycerol and other thylakoid lipids on violaxanthin de-epoxidation in liposomes
Authors: Latowski D. , Kostecka A. , Strzalka K.
Published: January 2005

In this study we present evidence that one of two reactions of the xanthophyll cycle, violaxanthin de-epoxidation, may occur in unilamellar egg phosphatidylcholine vesicles supplemented with monogalactosyldiacylglycerol (MGDG). Activity of violaxanthin de-epoxidase (VDE) in this system was found to be strongly dependent on the content of MGDG in the membrane; however, only to a level of 30 mol%. Above this concentration the rate of violaxanthin de-epoxidation decreased. The effect of individual thylakoid lipids on VDE-independent violaxanthin transformation was also investigated and unspecific effects of phosphatidylglycerol and sulphoquinovosyldiacyglycerol, probably related to the acidic character of these lipids, were found. The presented results suggest that violaxanthin de-epoxidation most probably takes place inside MGDG-rich domains of the thylakoid membrane. The described activity of the violaxanthin de-epoxidation reaction in liposomes opens new possibilities in the investigation of the xanthophyll cycle and may contribute to a better understanding of this process.
Determining functions of multiple phospholipase Ds in stress response of Arabidopsis
Authors: Wang X. , Wang C. , Sang Y. , Zheng L. , Qin C.
Published: January 2005

Phospholipase D (PLD) is encoded by a multiple gene family, and several PLDs from Arabidopsis have been characterized at the molecular biological and biochemical levels. PLDα is the most abundant plant PLD and exhibits a number of different biochemical properties to the other isoforms. The other PLDs have many overlapping catalytic properties but display some unique patterns of expression during development and in response to stress cues. Accumulating data indicate that different PLDs have multiple and different roles in plant responses to stress.
Creating lipoxygenases with new positional specificities by site-directed mutagenesis
Authors: Hornung E. , Rosahl S. , Kühn H. , Feussner I.
Published: January 2005

In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a Δ-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid hetero-geneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngräber determinants) [3].
Lipoxygenase isoforms in elicitor-treated parsley cell suspension cultures
Authors: Noehringer C. , Scheel D. , Blée E.
Published: January 2005

Treatment of parsley cell cultures with a fungal elicitor triggered the induction of a lipoxygenase isoform which may be involved in the de novo synthesis of defence-response inducers, such as jasmonic acid or 12-oxo-phytodienoic acid.
Characterization of lipoxygenase isoforms in olive callus cultures
Authors: Williams M. , Harwood J. L.
Published: January 2005

Lipoxygenase activity is critical for the development of flavours and aromas in olive oils. We have partly purified isoforms of molecular mass 95 kDa that have activity against linoleic or α-linolenic acids by a simple procedure from olive callus cultures.
Lipoxygenases in early Brassica napus germination
Authors: Terp N. , Brandt A.
Published: January 2005

We have identified transcripts of two lipoxygenase (LOXs) from 3-day-germinated etiolated coty-ledons. One encodes a soluble and the other encodes a plastid LOX. They are 2520 bp and 2676 bp in size, respectively. Antibodies raised against the C-terminal 500 amino acids of the plastid LOX recognize a 90 kDa LOX in 5-day-old etiolated and green cotyledons. In mature leaves a LOX of 95 kDa is detected. LOX activity and protein was mainly located in the cytosol.
Lipid hydroperoxides in plants
Authors: Griffiths G. , Leverentz M. , Silkowski H. , Gill N. , Sánchez-Serrano J. J.
Published: January 2005

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and were determined spectrophotometrically based on their reaction with an excess of Fe2+ at low pH in the presence of the dye Xylenol Orange. Triphenyl-phosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in a range of plant tissues including Phaseolus hypocotyls (26 ± 5 nmol.g of fresh weight-1; mean ± S.D.), Alstroemeria floral tissues (sepals, 66±13 nmol.g of fresh weight-1; petals, 49±6 nmol.g of fresh weight-1), potato leaves (334±75 nmol.g of fresh weight-1), broccoli florets (568±68 nmol.g of fresh weight-1) and Chlamydomonas cells (602±40 nmol.g of wet weight-1). Relative to the total fatty acid content of the tissues, the percentage hydroperoxide content was within the range of 0.6–1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. Leaves of transgenic potato with the fatty acid hydroperoxide lyase enzyme expressed in the antisense orientation were elevated by 38%, indicating a role for this enzyme in the maintenance of cellular levels of lipid hydroperoxides.
Lipoxygenase pathway in tulip: biosynthesis of ketols
Authors: Grechkin A. N. , Mukhtarova L. S. , Hamberg M.
Published: January 2005

The metabolism in vitro of [1-14C]linoleate, [1-14C]linolenate and their 9(S)-hydroperoxides in tulip (Tulipa gesneriana) was found to be under the control of 9-lipoxygenase and allene oxide synthase, and directed towards α-ketol, γ-ketol and the novel compound (12Z)-10-oxo-11-hydroxy- 12-octadecadienoic acid (10,11-ketol). Potent activity of allene oxide cyclase (in bulbs) and a new enzyme, γ-ketol reductase (in bulbs and leaves), was detected. Metabolism in flowers is directed predominantly towards α-ketol hydroperoxide.
Potato tubers exhibit both homolytic and heterolytic hydroperoxide fatty acid-cleaving activities
Authors: Fauconnier M.-L , Delcarte J. , Hoyaux P. , du Jardin P. , Marlier M.
Published: January 2005

The action of a crude potato-tuber extract on 9- and 13-hydroperoxides of linoleic and linolenic acids was investigated. HPLC analysis revealed that 50% of the 9-hydroperoxide isomers and almost all the 13-hydroperoxide isomers were rapidly enzymically metabolized. No degradation of fatty acid hydroperoxides was observed with a thermally denatured enzymic extract. GC-MS identification of the volatiles formed by the reaction revealed that no volatiles were detected from the 9-hydroperoxide isomers, whereas 13-hydroperoxide of linolenic acid was cleaved into (Z)-3-hexenal, pentenols or dimers of pentene.
Metabolic profiling of oxylipins upon sorbitol treatment in barley leaves
Authors: Weichert H. , Kohlmann M. , Wasternack C. , Feussner I.
Published: January 2005

In barley leaves 13-lipoxygenases (LOXs) are induced by salicylate and jasmonate. Here, we analyse by metabolic profiling the accumulation of oxylipins upon sorbitol treatment. Although 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway, accumulation is much later than in the cases of salicylate and jasmonate treatment. In addition, under these conditions only the accumulation of jasmonates as additional products of the LOX pathway has been found.
Genome-wide expression-monitoring of jasmonate-responsive genes of Arabidopsis using cDNA arrays
Authors: Sasaki Y. , Asamizu E. , Shibata D. , Nakamura Y. , Kaneko T. , Awai K. , Masuda T. , Shimada H. , Takamiya K-i. , Tabata S. , Ohta H.
Published: January 2005

Jasmonates are generally considered to mediate signalling, such as defence responses, flowering and senescence. However, factors involved in the jasmonate signal-transduction pathway remain unclear. To clarify the functions and signalling mechanisms of jasmonates on a genome-wide level, we adopted a cDNA macroarray technique. We prepared nylon filters of a cDNA macroarray on which 2880 independent expressed sequence tag clones of Arabidopsis were blotted, and hybridized 33P-labelled single-strand DNAs synthesized from mRNAs of methyl jasmonate (MeJA)- treated and untreated Arabidopsis plants to the nylon filters. By analysing the data from the cDNA macroarray, we identified many function-known and unknown genes as MeJA-responsive genes, and confirmed that the profiles of the expression showed good agreement with Northernblot analysis. These results demonstrate the efficiency of the cDNA macroarray for systematically analysing jasmonate-responsive genes on a genome-wide scale.
Extremely rapid effects of polyunsaturated fatty acids and N-acetylglucosamine on free-radical metabolism in cultured potato plant cells
Authors: Kashulin P. A. , Merzlyak M. N. , Zhiboedov P. M. , Zhirov V. K.
Published: January 2005

The effects of arachidonic and linoleic acids, separately and in co-operation with N-acetylglucosamine oligomers, on Solanum tuberosum plant suspension cell cultures were investigated in terms of the fluorescent oxygen-activated-species-sensitive dye 2Î,7Î-dichlorofluorescin diacetate. The inductors used triggered extremely rapid (within 2–10 min) generation of H2O2 in the cells; the majority was expressed in cultures treated with combined polyunsaturated fatty acid and N-acetylglucosamine oligomers. The stimulation of free-radical generation may be related to defensive mechanisms modulating a plant-pathogenicmicro-organism interaction.
ω-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94AI: possible involvement in plant defence
Authors: Pinot F. , Skrabs M. , Compagnon V. , Salaun J.-P. , Benveniste I. , Schreiber L. , Durst F.
Published: January 2005

The C18 fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S, 10R and 9R, 10S enantiomers were incubated separately. We determined respective Km and Vmax values of 1.2±0.1 μM and 19.2±0.3 nmol/min per nmol of cytochrome P450 for the 9R, 10S enantiomer and of 5.9±0.1 μM and 20.2±1.0 nmol/min per nmol of cytochrome P450 for the 9S, 10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R, 10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C16 fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.
Differential signalling and plant-volatile biosynthesis
Authors: Engelberth J.
Published: January 2005

At least two different signalling pathways have been identified that result in clearly distinguishable volatile profiles in response to pathogens and herbivores in the lima bean Phaseolus lunatus. Alamethicin, a voltage-gated ion-channel-forming peptide from Trichoderma viride is a potent inducer of volatile biosynthesis in the lima bean. Unlike elicitation with cellulysin or herbivore damage, which act through the jasmonic acid pathway and result in a complex pattern of volatile compounds, the emitted blend comprises only the two homoterpens, 4,11-dimethylnona-1,3,7-triene and 4,8,12-trimethyltrideca-1,3,7,11- tetraene, and methyl salicylate. Both pathways, represented by jasmonic acid and alamethicin, depend on lipid-derived signalling compounds, set off by the activation of a phospholipase A and further processing by lipoxygenase activity. The alamethicin-induced signal-transduction pathway interferes with the octadecanoid cascade, probably due to increased salicylic acid levels, resulting in an inhibition of the typical jasmonic acid-induced volatile profile.
Role of lipids in molecular thermoadaptation mechanisms of seagrass Zostera marina
Authors: Goncharova S. N. , Sanina N. M. , Kostetsky E. Y.
Published: January 2005

By using the combined approaches of microcalorimetric analysis and polarizing microscopy, the crystal-liquid crystal-isotropic melt phase transition of major phospholipids (phosphatidylcholine and phosphatidylethanolamine) and glycolipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol) isolated from marine seagrass Zostera marina were investigated. Here we discuss the results in view of the fatty acid structures of the lipids studied.
Thermotropic behaviour of membrane lipids from brown marine alga Laminaria japonica
Authors: Sanina N. M. , Kostetsky E. Y. , Goncharova S. N.
Published: January 2005

Microcalorimetry was used to show that transition-temperature ranges of phospho- and glycolipids of the brown marine alga Laminaria japonica were similar (from –62 to –47°C up to 62– 65°C), except for monogalactosyldiacylglycerol, the low-temperature limit of which was shifted to –78°C. As was shown by polarizing microscopy, the low-enthalpy peaks at temperatures of approx. 30–45°C corresponded to isotropic melting of galactolipids and coincided with the hightemperature limit for photosynthetic and mitochondrial activity of the algae. As a whole, a classical interrelation was observed between thermotropic behaviour and the fatty acid unsaturation of lipids.
Sodium chloride effects on lipase activity in germinating rape seeds
Authors: Miled D. D. Ben , Zarrouk M. , Chérif A.
Published: January 2005

Seeds of rape (Brassica napus L.) were germinated at various NaCl concentrations up to 200 mM. Germinating levels, seedling growth, triacylglycerol mobilization and lipase activity were investigated. High salt concentrations resulted in retardation of seed germination. Seedling growth as measured by radicle length was severely reduced by NaCl doses higher than 50 mM. Moreover, the mobilization of storage oil in control rapeseed seedlings, started about 24 h after imbibition. As for germination and growth, elevated salt concentrations are found to delay triacylglycerol degradation. Experiments using triolein as substrate indicated clearly that lipase activity was inhibited by salt treatment.
Two novel plant cDNAs homologous to animal type-2 phosphatidate phosphatase are expressed in cowpea leaves and are differently regulated by water deficits
Authors: Marcel G. C. F. , Matos A. , d'Arcy-Lameta A. , Kader J-C. , Zuily-Fodil J. Y. , Pham-Thi A.
Published: January 2005

Two cDNAs encoding putative phosphatidate phosphatases (PAPs) designated VuPAP-α and VuPAP-β were cloned in cowpea (Vigna unguiculata L.) leaves. The predicted proteins have six membrane-spanning regions in common with animal type-2 PAPs. Unlike VuPAP-β, VuPAP-α has an N-terminal transit peptide and is targeted in vitro to the chloroplasts. Gene expression of VuPAP-β is stimulated by rapid air-desiccation of leaves and VuPAP-α transcripts increase during rehydration of plants exposed to drought-like conditions.
Effect of fungal infection on the composition of acyl lipids in wheat seedlings
Authors: Vereshchagin A. G. , Zhukov A. V.
Published: January 2005

Infection of etiolated wheat seedlings with a root rot fungus Bipolaris sorokiniana caused a strong deviation in the fatty acid composition of their total lipids from the control. The deviation occurred at the expense of that lipid group, which predominates in a given plant organ (shoots or roots), and peak deviation coincided with the onset of a severe inhibition of growth.