bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved treatment of uncertainty in hydrologic modeling: Combining the strengths of global optimization and data assimilation
Authors: Vrugt Jasper A. , Diks Cees G. H. , Gupta Hoshin V. , Bouten Willem , Verstraten Jacobus M.
Published: January 2005

Hydrologic models use relatively simple mathematical equations to conceptualize and aggregate the complex, spatially distributed, and highly interrelated water, energy, and vegetation processes in a watershed. A consequence of process aggregation is that the model parameters often do not represent directly measurable entities and must therefore be estimated using measurements of the system inputs and outputs. During this process, known as model calibration, the parameters are adjusted so that the behavior of the model approximates, as closely and consistently as possible, the observed response of the hydrologic system over some historical period of time. In practice, however, because of errors in the model structure and the input (forcing) and output data, this has proven to be difficult, leading to considerable uncertainty in the model predictions. This paper surveys the limitations of current model calibration methodologies, which treat the uncertainty in the input‐output relationship as being primarily attributable to uncertainty in the parameters and presents a simultaneous optimization and data assimilation (SODA) method, which improves the treatment of uncertainty in hydrologic modeling. The usefulness and applicability of SODA is demonstrated by means of a pilot study using data from the Leaf River watershed in Mississippi and a simple hydrologic model with typical conceptual components.
Fatty acid synthesis: from CO2 to functional genomics
Authors: Ohlrogge J. , Pollard M. , Bao X. , Focke M. , Girke T. , Ruuska S. , Mekhedov S. , Benning C.
Published: January 2005

For over 25 years there has been uncertainty over the pathway from CO2, to acetyl-CoA in chloroplasts. On the one hand, free acetate is the most effective substrate for fatty acid synthesis by isolated chloroplasts, and free acetate concentrations reported in leaf tissue (0.1–1 mM) appear adequate to saturate fatty acid synthase. On the other hand, a clear mechanism to generate sufficient free acetate for fatty acid synthesis is not established and direct production of acetyl-CoA from pyruvate by a plastid pyruvate dehydrogenase seems a more simple and direct path. We have re-examined this question and attempted to distinguish between the alternatives. The kinetics of 13CO2 and 14CO2 movement into fatty acids and the absolute rate of fatty acid synthesis in leaves was determined in light and dark. Because administered 14C appears in fatty acids within < 2–3 min our results are inconsistent with a large pool of free acetate as an intermediate in leaf fatty acid synthesis. In addition, these studies provide an estimate of the turnover rate of fatty acid in leaves. Studies similar to the above are more complex in seeds, and some questions about the regulation of plant lipid metabolism seem difficult to solve using conventional biochemical or molecular approaches. For example, we have little understanding of why or how some seeds produce >50%, oil whereas other seeds store largely carbohydrate or protein. Major control over complex plant biochemical pathways may only become possible by understanding regulatory networks which provide ‘global’ control over these pathways. To begin to discover such networks and provide a broad analysis of gene expression in developing oilseeds, we have produced micro-arrays that display approx. 5000 seed-expressed Arabidopsis genes. Sensitivity of the arrays was 1–2 copies of mRNA/cell. The arrays have been hybridized with probes derived from seeds, leaves and roots, and analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approx. 10% of the genes were expressed at ratios ≥ 10-fold higher in seeds than in leaves or roots. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases and transcription factors. The arrays were also found to be useful for analysis of Brassica seeds.
Application of a new method for the sensitive detection and quantification of acyl-CoA esters in Arabidopsis thaliana seedlings and mature leaves
Authors: Larson T. R. , Graham I. A.
Published: January 2005

We report a novel, highly sensitive and selective method for the detection and quantification of acyl-CoA esters from Arabidopsis thaliana. Extracted acyl-CoA esters were derived to their fluorescent acyl-etheno-CoA esters, separated by ion-paired reversed-phase HPLC, and detected fluorometrically. We report the composition and concentration of the acyl-CoA pool in mature A. thaliana leaves, and during storage-lipid breakdown in 2-day-old seedlings. The concentrations measured were in the 1–4 μM range for both tissue types. To our knowledge, this is the first time that reliable estimates of acyl-CoA ester concentrations have been made for plants.
Litchi chinensis fatty acid diversity: occurrence of the unusual cyclopropanoic fatty acids
Authors: Gontier E. , Boussouel N. , Terrasse C. , Jannoyer M. , Ménard M. , Thomasset B. , Bourgaud F.
Published: January 2005

Litchi chinensis (Sapindaceae) is a tree that originates from China and is cultivated for its sweet fruits all over the world in warm climates. Unusual fatty acids such as cyclopropanoic fatty acids have been identified in the seeds of Litchi. Because of their potential value for industry (as inks, cosmetics, detergents, lubricants, etc.), the variability in the relative levels of unusual fatty acids in the seeds of 28 different Litchi varieties was analysed at two locations (on Ræunion Island in the Indian Ocean) and on two different harvest dates. Except for one variety, all the seeds contained cis-9, 10-methylene-octadecanoic acid (C19CA) at a relative level of 35–48%. The only variety that contained no or only traces of C19CA was Groff, seeds of which were significantly much smaller than those from all other varieties.
Glycosylphosphatidylinositol-anchored proteins found in the moss Marchantia polymorpha
Authors: Okuyama H. , Hagiwara S. , Okamoto T. , Kato K.
Published: January 2005

A suspension culture of the moss Marchantia polymorpha was incubated with either [3H]-ethanolamine or [3H]myristic acid, precursors of a glycosylphosphatidylinositol (GPI) anchor. By both of these a secretory protein of 47 kDa was consistently labelled. Amino acid composition analysis of the chromatographically purified 47-kDa protein exhibited the occurrence of ethanolamine and glucosamine at approximate molar ratios to protein of 1 and 4, respectively. Thus the secretory 47-kDa protein of M. polymorpha was concluded to be a GPI-anchored protein.
Biotin carboxyl carrier protein isoforms in Brassicaceae oilseeds
Authors: Thelen J. J. , Mekhedov S. , Ohlrogge J. B.
Published: January 2005

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389–401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.
Modulation of fatty acid biosynthesis by antisense β-keto reductase expression
Authors: O'Hara P. , Slabas A. R. , Fawcett T.
Published: January 2005

Plants with an antisense construct to β-ketoacyl-acyl carrier protein reductase have reduced seed and leaf fatty acid contents and show distinct phenotypes, including leaf curl, stunted growth and wrinkled seeds. The carbon resource allocation is altered in both of these tissues. In leaves there is an inability to synthesize transient starch and in seeds embryogenesis is foreshortened and storage proteins accumulate prematurely.
Expression of fatty acid and lipid biosynthetic genes
Authors: O'Hara P. , Slabas A. R. , Fawcett T.
Published: January 2005

An assessment of the transcript abundance of several fatty acid and lipid biosynthetic genes has been made. Total RNA was extracted from embryos at different stages of development and mRNA was analysed by quantitative Northern blots and used to probe cDNA arrays. Central fatty acid synthase components were present at different steady-state levels, but at constant ratios throughout development; ancillary components were generally expressed at higher levels, but at constant ratios over the period of maximum fatty acid biosynthesis.
Molecular properties of the oleoyl-phosphatidylcholine desaturase from Arachis hypogaea L.
Authors: Powell G. L. , Jung S. , Bruner A. C. , Swartzbeck J. L. , Abbott A. G.
Published: January 2005

Deficiencies in the activity of the microsomal oleoyl phosphatidylcholine (oleoyl-PC) desaturase from peanuts are the basis of the high oleate oil. Mutation of aspartate-150 to asparagine and the attendant decrease in activity, together with the loss in expression of the higher activity transcript, was the molecular basis of the high oleate trait. The ability of oleoyl-PC desaturase to desaturate palmitoleate, oleate and 10(Z) nonadecenoate to methylene-interrupted products was not consistent with description of this activity as a Δ12 or ω-6 desaturase. Electrospray MS was used to examine the intact phospholipid products of desaturation by the oleoyl-PC desaturase. PC and phosphatidylinositol containing unsaturated moieties could be desaturated. The enzyme can act on either sn-1 or sn-2 moieties. Phosphatidylethanolamine was a poor substrate.
Perception and transduction of low-temperature signals to induce desaturation of fatty acids
Authors: Suzuki I. , Los D. A. , Murata N.
Published: January 2005

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to a low temperature, genes for fatty acid desaturases are expressed with resultant increases in the degree of unsaturation of fatty acids in membrane lipids. However, the sensor and transducers of low-temperature signals had not yet been identified. In order to identify these components we applied to the cyanobacterium Synechocystis sp. PCC 6803 the systematic disruption of all 43 putative genes for histidine kinases and random mutagenesis of the whole genome in conjunction with screening by the transcriptional activity of the promoter of the desB gene for the ω 3 desaturase. This allowed us to identify two histidine kinases and a response regulator as components of the perception and transduction of low-temperature signals for the expression of genes for fatty acid desaturases.
Further characterization of Δ8-sphingolipid desaturases from higher plants
Authors: Sperling P. , Blume A. , Zähringer U. , Heinz E.
Published: January 2005

A previously cloned cDNA from Helianthus annuus codes for a fusion protein composed of an N-terminal cytochrome b5, and a C-terminal desaturase domain. For a functional identification, this cDNA was expressed in Saccharomyces cerevisiae and the structures of sphingolipid longchain bases were analysed. The expression of this sunflower enzyme resulted in the formation of new Δ8-trans/cis-phytosphingenine from C18- and C20-phytosphinganine present in wild-type yeast cells. To elucidate the substrate specificity, the recently cloned Δ8-sphingolipid desaturases from Arabidopsis thaliana and Brassica napus were expressed in the yeast mutant sur2Δ that lacked the sphinganine C4-hydroxylase and was thus unable to form phytosphinganine. Long-chain base analysis of the transformed mutant cells did not show any conversion of C18- or C20-sphinganine into Δ8-sphingenine, whereas exogenously added C18-phytosphinganine was desaturated to Δ8-trans/cis-phytosphingenine. Furthermore, GLC-MS analysis did not reveal the presence of any Δ9-regioisomers as reported before. These results show that the sunflower gene codes for a Δ8-sphingolipid desaturase which accepts C18- and C20-phytosphinganine. The absence of Δ8-sphingenine as desaturation product in the transformed mutant suggests that C4-hydroxylation of sphinganine precedes Δ8-desaturation. Therefore, in yeast, the substrate for the plant Δ8-sphingolipid desaturase seems to be the phytosphinganine residue.
Biochemical and molecular characterization of corn (Zeo mays L.) root elongases
Authors: Schreiber L , Skrabs M. , Hartmann K. , Becker D. , Cassagne C. , Lessire R.
Published: January 2005

Root surfaces are protected against the soil environment by the deposition of lignin and suberin. In order to obtain more insight into the regulation of root suberin biosynthesis, elongases from primary roots of corn (Zea mays L.) seedlings were characterized. Elongase activities (acyl-CoA and ATP-dependent) were located in the microsomal fraction of the root cells. C20, C22 and C24 fatty acids were detected as primary products of elongases. Preferred substrates of the acyl-CoA elongases were C18.0-CoA and C20.0-CoA. Applying a molecular approach, using PCR and degenerate primers derived from the sequences of known leaf and seed 3-ketoacyl-CoA synthases (KCSs), catalysing the first step of very-longchain fatty acid synthesis, the cDNA of a putative root KCS was obtained showing high homology to known leaf and seed KCSs at the DNA and amino acid levels. Thus, our approach provides the first direct evidence for the presence and the activity of root elongases in Z. mays. Ongoing research is focusing on the molecular analysis and the regulation of KCS expression in roots in reaction to different environmental stimuli.
Expression of the wax-specific condensing enzyme CUTI in Arabidopsis
Authors: Kunst L. , Clemens S. , Hooker T.
Published: January 2005

The Arabidopsis thaliana gene CUT1 encodes a very-long-chain fatty acid-condensing enzyme required for the production of epicuticular wax in bolting stems. We have examined the expression pattern of CUT1 in Arabidopsis at different developmental stages and under different environmental conditions. RNA blot analysis showed that CUT1 was highly expressed in shoots, but not in roots. CUT1 expression was detectable throughout development. Light was required for CUT1 expression, and expression was increased by salt and drought treatments. The promoter region of the CUT1 gene was cloned, and 1.2 kb of the sequence 5′ to the translation start codon was used to direct β-glucuronidase (GUS) expression in transgenic plants. Histochemical and fluorometric (quantitative) GUS assays confirmed that the CUT1 promoter directed epidermal-specific expression and was highly active in Arabidopsis and in tobacco. A construct using the CUT1 promoter to drive CUT1 expression (CUT1p-CUT1) was used to transform Arabidopsis. Transgenic plants which had somewhat increased (overexpression) or greatly reduced (co-suppression) wax loads were recovered. Thus, the CUT1 promoter should be useful for genetic engineering applications that require epidermis-specific expression of genes.
Production of C20 polyunsaturated fatty acids (PUFAs) by pathway engineering: identification of a PUFA elongase component from Caenorhabditis elegans
Authors: Beaudoin F. , Michaelson L. V. , Lewis M. J. , Shewry P. R. , Sayanova O. , Napier J. A.
Published: January 2005

Using a combination of database-mining and functional characterization, we have identified a component of the polyunsaturated fatty acid (PUFA) elongase. Co-expression of this elongating activitiy with fatty acid desaturases has allowed us to heterologously reconstitute the PUFA biosynthetic pathway. Both these enzymes (desaturases and elongase components) have undergone gene-duplication events which provide a paradigm for the diverged nature of PUFA biosynthetic activities.
Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity
Authors: Hayman M. W. , Fawcett T. , Schierer T. F. , Simon J. W. , Kroon J. T. M. , Gilroy J. S. , Rice D. W. , Rafferty J. , Turnbull A. P. , Sedelnikova S. E. , Slabas A. R.
Published: January 2005

In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the PET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.
Morphological and metabolic changes in transgenic wheat with altered glycerol-3-phosphate acyltransferase or acyl-acyl carrier protein (ACP) thioesterase activities
Authors: Edlin D. A. N. , Kille P. , Wilkinson M. D. , Jones H. D. , Harwood J. L.
Published: January 2005

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.
Expression and characterization of diacylglycerol acyltransferase from Arabidopsis thaliana in insect cell cultures
Authors: Hobbs D. H. , Hills M. J.
Published: January 2005

Diacylglycerol acyltransferase (DGAT) catalyses the acylation of the sn-3 hydroxy group of sn-1,2-diacylglycerol using acyl-CoA. The gene encoding DGAT from Arabidopsis thaliana has been cloned and the function of the enzyme proved by expression of the coding sequence using a bacculovirus expression system in insect cell cultures. The expressed protein catalysed the synthesis of [14C]triacylglycerol from [14C]diacylglycerol and oleoyl-CoA. The heterologously expressed DGAT activity was found mostly associated with the 100000g pellet. The optimum activity was achieved at a neutral pH, in the presence of Mg2+, and at an optimum oleoyl-CoA concentration of 20 μM. The DGAT used the substrates palmitoyl-CoA and oleoyl-CoA equally effectively. In these experiments, the inclusion of recombinant acyl-CoA binding protein had a relatively small effect upon DGAT activity.
Expression in yeast of an acyl-CoA:diacylglycerol acyltransferase cDNA from Caenorhabditis elegans
Authors: Bouvier-Navé P. , Benveniste P. , Noiriel A. , Schaller H.
Published: January 2005

we have identified a cDNA from the nematode worm Caenorhabditis elegans that encodes an acyl-CoA: diacylglycerol acyltransferase (DGAT). Its expression in Saccharomyces cerevisiae resulted in an increase both in triacylglycerol content and in microsomal oleyl-CoA: diacylglycerol acyltransferase activity. Such effects were similar to those of characterized plant DGAT genes. This is the first DGAT gene isolated from an invertebrate. The phylogenetic relationships between DGATs and animal and yeast acyl-CoA: sterol acyltransferases are illustrated.
The involvement of phospholipid:diacylglycerol acyltransferases in triacylglycerol production
Authors: Banaś A. , Dahlqvist A. , Ståhl U. , Lenman M. , Stymne S.
Published: January 2005

We have characterized three CoA-independent types of enzyme, phospholipases, phospholipid: diacylglycerol acyltransferases (PDATs) and cholinephosphotransferases, responsible for the removal of unusual fatty acids from phosphatidylcholine (PC) in microsomal preparations from developing oil seeds. The metabolism of sn-2-[14C]acyl-PC was monitored in microsomal preparations from various oilseeds having either medium-chain, acetylenic, epoxy or hydroxy fatty acids as their major fatty acids in the oil. The results indicate that PDAT plays a major role in removing ricinoleic acid and vernolic acid from phospholipids in Ricinus communis and Crepis palaestina seeds, respectively. However, vernolic, crepenynic and capric acids are primarily removed from phospholipids by phospholipases in Euphorbia lagascae, Crepis rubra and elm seeds, respectively. Further, we show that significant PDAT activity is also present in vegetative tissues of Arabidopsis thaliana.
Partial purification and photoaffinity labelling of sunflower acyl-CoA:lysophosphatidyl-choline acyltransferase
Authors: Fraser T. , Stobart K.
Published: January 2005

Previous attempts to purify acyl-CoA: 1-acyl-lysophosphatidylcholine acyltransferase (EC 2.3.1.23) have been frustrated by difficulties in solubilizing the enzyme without inactivation. Microsomal preparations, from the developing cotyledons of sunflower, in high concentrations of urea retain activity. Gel-filtration liquid chromatography followed by trypsin treatment (minus urea) resulted in the removal of many contaminating proteins without loss of enzyme activity. SDS/PAGE showed the presence of two major peptides with apparent molecular masses of 52 and 59 kDa. These polypeptides cross-reacted with the radiolabelled photoreactive substrate 1-azido-oleoyl-sn-lysophosphatidyl-[N-methyl-3H]choline.
Sesamin inhibits lysophosphatidyl-choline acyltransferase in Mortierella alpina
Authors: Chatrattanakunchai S. , Fraser T. , Stobart K.
Published: January 2005

The filamentous fungus, Mortierella alpina accumulates complex lipids relatively rich in arachidonic acid (C20 4 Δ5.8.11.14). The lignan, sesamin, has been used to reduce arachidonic acid production by specifically inhibiting Δ5-desaturation [Shimizu, Akimoto, Shinmen, Kawashima, Sugano and Yamada (1991) Lipids 26, 512–516]. Microsomal membrane preparations from M. alpina exhibit acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase (LPCAT) activity. LPCAT is an enzyme involved in channelling fatty acid substrates to phosphatidylcholine for subsequent desaturation. Sesamin was found to inhibit this enzyme as measured in both spectrophotometric and radioactive assays. The inhibitory effect of sesamin on LPCAT was only evident in species of Mortierella and could not be demonstrated in other organisms.
Pea choline kinase: purification, properties and isolation of a cDNA
Authors: AI-Malki A. , Morby A. , Harwood J. L.
Published: January 2005

Choline kinase has been partially purified from pea seedlings and its properties studied. Using sequence information from soya bean and other choline kinases, we have also isolated a cDNA encoding the enzyme. It encodes a protein of 343 amino acids (calculated molecular mass of 39785 Da), which shows 82% homology with the soya bean choline kinase. The protein has been expressed in Eschericiha coli with very good activity and high expression levels.
Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies: use of fluorescent lipids to study lipid-body formation in an oleaginous fungus
Authors: Kamisaka Y. , Noda N.
Published: January 2005

Fluorescent phosphatidic acid and phosphatidylcholine were used to characterize lipid-transport pathways into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Several characteristics of the lipid transport such as temperature dependence and ATP dependence were evaluated. The transport depicted by these fluorescent lipids was consistent with metabolism of radiolabelled lipids, indicating that fluorescent lipids are useful to study lipid-body formation in this fungus. The results dissect lipid transport of phosphatidic acid and phosphatidylcholine into lipid bodies and reveal regulatory steps for lipidbody formation in this fungus.
Metabolism of glycosylphosphatidylinositol-anchored proteins in Arabidopsis
Authors: Darjania L , Ichise N. , Ichikawa S. , Okamoto T. , Okuyama H. , Thompson G. A.
Published: January 2005

Although glycosylphosphatidylinositol (GPI)-anchored proteins have now been found in several plants, very little is known regarding their metabolism there. This report describes studies of the biosynthesis and turnover of arabinogalactan proteins, a class of abundant GPI-anchored proteins secreted by cultured Arabidopsis cells.
The role of triacylglycerol as a reservoir of polyunsaturated fatty acids for the rapid production of chloroplastic lipids in certain microalgae
Authors: Cohen Z. , Khozin-Goldberg I. , Adlerstein D. , Bigogno C.
Published: January 2005

Many microalgae are known to accumulate triacylglycerols (TAGs), especially under nitrate starvation. Generally, these TAGs are predominantly constructed of saturated and monounsaturated fatty acids. In contrast, the TAGs of the red microalga Porphyridium cruentum are rich in arachidonic acid (AA, 20: 4 ω 6) and eicosapentaenoic acid (EPA, 20: 5 ω 3). A mutant of this alga, impaired of growth at suboptimal temperatures, was shown to have reduced levels of EPA and of the eukaryotic molecular species of monogalactosyldiacylglycerol (MGDG) and an elevated level of TAG. Labelling experiments have shown that labelling of wild-type TAGs decreased, whereas that of the mutant remained high. Contemporarily, eukaryotic MGDG of the mutant was less labelled. Similarly, TAGs of the green alga T12, which can grow at a low temperature, are extremely rich in AA. We have labelled exponentially growing cultures of T12 kept at 25 °C with radioactive AA and cultivated the cultures for a further 12 h at 25 °C, 12 °C or 4 °C. At low temperatures, labelled AA was transferred from TAGs to polar lipids. These findings may indicate that polyunsaturated fatty acids that can be incorporated into the membranes, enabling the organism to quickly respond to low-temperature-induced stress.
Compartmentation and topology of glucosylceramide synthesis
Authors: Cantatore J. L. , Murphy S. M. , Lynch D. V.
Published: January 2005

Evidence is presented supporting a model for glucosylceramide formation on the apoplastic side of the plasma membrane in plants. Glucosylceramide synthase and sterol glucosyltransferase were both localized to the plasma membrane. Whereas sterol glucosylation was sensitive to proteolytic enzymes, ceramide glucosylation was not. These results are consistent with our model in which steryl glucoside is synthesized on the cytosolic side of the membrane and then translocated across the membrane where it donates glucose to ceramide.
Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms
Authors: Leipelt M. , Warnecke D. C. , Hube B. , Zähringer U. , Heinz E.
Published: January 2005

Cerebrosides are typical membrane lipids of many organisms. They occur in plants, fungi, animals, humans and some prokaryotes. Almost all of our knowledge on the physiological functions of cerebrosides results from experimental data obtained with mammalian cells. However, very little is known about the roles played by these lipids in plants and fungi. To initiate such investigations we have cloned and characterized a ceramide glucosyltransferase from the yeast Candida albicans. Functional expression of this gene in Saccharomyces cerevisiae led to the accumulation of new glycolipids which were not present in wildtype baker's yeast. They were identified by MS and NMR spectroscopy as β-D-glucopyranosyl ceramides. The ceramide moieties of these cerebrosides comprised phytosphinganine and mainly long-chain (C26) α-hydroxy fatty acids in amide linkage. We also generated a ceramide glucosyltransferase-knock-out strain of C. albicans which was devoid of cerebrosides. The viability of this mutant showed that for this organism glucosyl ceramides are not essential for vegetative growth on complete or minimal media. In addition, we have cloned and functionally expressed one of the three putative glucosylceramide synthases from Caenorhabditis elegans as well as a corresponding enzyme from Pichia pastoris.
Assaying Arabidopsis lipase activity
Authors: Beisson F. , Arondel V. , Verger R.
Published: January 2005

A low lipase activity from a crude extract of Arabidopsis seedlings was assayed using three sensitive methods (radiolabelled triacylglycerols, commercial resorufin ester and triacylglycerols containing the naturally fluorescent parinaric acid as substrates). The specific activity of the extract was found to be similar using the three methods. However, the plant lipase activity measured using the radioactivity and the fluorescence assays could be abolished by heating the extract, contrary to the apparent activity measured using the commercial colorimetric assay. Unlike the radioactivity assay, the fluorescence assay can be monitored continuously. The parinaric acid-based method is therefore the only one to provide a sensitive, specific and continuous assay.
Altered membrane lipase expression delays leaf senescence
Authors: Thompson J. , Taylor C. , Wang T.-W.
Published: January 2005

A cDNA clone encoding a lipase that is upregulated in senescing leaves and flower petals has been isolated by screening an expression library. The abundance of the lipase mRNA increases as flowers and leaves begin to senesce, and expression of the gene is also induced by treatment with ethylene. Transgenic Arabidopsis plants in which levels of the senescence-induced lipase protein have been reduced show delayed leaf senescence.
A patatin-like protein with galactolipase activity is induced by drought stress in Vigna unguiculata leaves
Authors: Matos A. R. , d'Arcy-Lameta A. , França M. , Zuily-Fodil Y. , Pham-Thi A. T.
Published: January 2005

This paper reports the cloning of a cDNA (Vupat 1) expressed in Vigna unguiculata leaves coding for a protein with 48% sequence homology to patatin, the major protein from potato tuber which has lipolytic acylhydrolase activity. Two cultivars differing in drought tolerance were ex-amined in Northern-blot analyses. Expression of Vupat 1 is stimulated by drought stress, especially in the drought-sensitive cultivar. Vupat 1 was expressed in the baculovirus system as a fusion protein secreted in the culture medium. The recombinant protein displays lipolytic activity towards monogalactosyldiacylglycerol, digalactosyldiacylglycerol and sulphoquinovosyldiacylglycerols.
Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin
Authors: Hartmann M.-A. , Wentzinger L. , Hemmerlin A. , Bach T. J.
Published: January 2005

Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G1 phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q10. In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.
Biochemistry and site-directed mutational analysis of Δ7-sterol-C5(6)-desaturase
Authors: Rahier A. , Benveniste P. , Husselstein T. , Taton M.
Published: January 2005

This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Δ7-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.
Molecular evolution of lycopene cyclases involved in the formation of carotenoids with ionone end groups
Authors: Krubasik P. , Sandmann G.
Published: January 2005

A survey is given of the lycopene cyclase genes present in bacteria, fungi and plants where two completely unrelated types exist. One is the classical monomeric bacterial β-cyclase gene, crt Y which may be an ancestor of crtL the gene for a β-cyclase in cyanobacteria. From crtL a line of evolution can be drawn to plant β- and ε-cyclase genes and to the gene of capsanthin/capsorubin synthase. In Gram-positive bacteria two genes crtYc and crtYd are present. They encode two proteins which have to interact as a heterodimer for lycopene β-cyclization. From this type of lycopene cyclase gene the fungal lycopene cyclase/phytoene synthase fusion gene evolved.
Study of phospholipases D and C in maturing and germinating seeds of Brassica napus
Authors: Novotna Z. , Valentova O. , Martinec J. , Feltl T. , Nokhrina K.
Published: January 2005

Different forms of phospholipase D (dependent on and independent of the presence of phospha-tidylinositol 4,5-bisphosphate, PIP2) were identified in maturing and germinating seeds of Brassica napus. Both forms were present in cytosolic and membrane fractions of maturing seeds. PIP2-dependent activity increased continuously during seed germination, while PIP2-independent activity appeared mostly at the very beginning of seed maturation. PIP2-dependent activity was detected mainly in the plasma-membrane fraction. Phosphatidylinositol-specific phospholipase C (PI-PLC) was found only in membrane fractions of both types of developing rape seed tissues. The increasing activities of PLC and PIP2-dependent PLD were mainly detected in hypocotyls of seedlings. Some biochemical characteristics of both described enzymes are also presented.
Role of the phosphoinositide pathway in the light-dependent C4 phosphoeno/pyruvate carboxylase phosphorylation cascade in Digitaria sanguinalis protoplasts
Authors: Coursol S. , Pierre J.-N. , Vidal J.
Published: January 2005

Stimulus-response coupling in animal cells frequently involves the hydrolysis of PtdIns(4,5)P2 which is catalysed by phosphoinositide-specific phospholipase C (PI-PLC). There is an increasing body of evidence for PI-PLC-based signalling in plant cells; however, the physiological role of this system remains poorly documented in plants. Our data provide the first evidence that a PI-PLC-based signalling system is a committed step in the transduction chain controlling the phosphorylation state of C4 phosphoenolpyruvate carboxylase (PEPC), the regulation of which is central to the assimilation of atmospheric CO2 in C4 plants.
14-3-3 Proteins and a 13-lipoxygenase form associations in a phosphorylation-dependent manner
Authors: Holtman W. L. , Roberts M. R. , Wang M.
Published: January 2005

Recently, we have demonstrated by two different methods that lipoxgenases (LOXs) and 14-3-3 proteins form interactions in barley embryos [Holtman, Roberts, Oppedijk, Testerink, van Zeij] and Wang (2000) FEBS Lett. 474, 48–52]. It was shown by both co-immunoprecipitations and surface-plasmon resonance experiments that 13-LOX, but not 9-LOX, forms interactions with 14-3-3 proteins. In the present report we show that the presence of 13-LOX and 14-3-3 proteins was established in high-molecular-mass complexes. Amounts of 13-LOX and 14-3-3 proteins in high-molecular-mass fractions increased during germination, but were reduced after dephosphorylation of protein extracts or competition with the 14-3-3-binding peptide P-Raf-259, indicating that 13-LOX and 14-3-3 proteins interact in a phosphorylation-dependent manner.
Regulation of oxylipin synthesis
Authors: Hildebrand D. F. , Afitlhile M. , Fukushige H.
Published: January 2005

Two very common groups of oxylipins formed in plants involve the conversion of fatty acid hydroperoxides, such as hydroperoxy-octadecatrienoic acid, into further metabolites by allene oxide synthase and hydroperoxide lyase. Both of these oxylipin branch pathways appear to be ubiquitous or nearly so in plants, but the relative activities of these two branches vary among plant species. In most plants examined, including Arabidopsis product formation from either of these pathways is minimal until elicited by wounding or some other means, upon which products from both pathways, such as jasmonic acid and C6 aldehydes and alcohols, can increase by orders of magnitude. In some plant species such as Artemisia and Jasminum spp. oxylipin product formation is heavily skewed towards allene oxide synthase products. Others such as watermelon (Citrullus lanatus) produce 10-fold higher amounts or more of hydroperoxide lyase than allene oxide synthase products. Arabidopsis and tobacco are intermediate between these extremes. Artemisia and Jasminum are also unusual in that they do not require wounding or other types of induction for high oxylipin product formation. Release of non-esterified fatty acids appears to be correlated with oxylipin formation, but phospholipase A2 appears not to be involved with oxylipin production, at least in the case of Artemisia leaves.
Formation of 4-hydroxy-2-alkenals in barley leaves
Authors: Weichert H. , Kolbe A. , Wasternack C. , Feussner I.
Published: January 2005

In barley leaves 13-lipoxygenases are induced by jasmonates. This leads to induction of lipid peroxidation. Here we show by in vitro studies that these processes may further lead to autoxidative formation of (2E)-4-hydroxy-2-hexenal from (3Z)-hexenal.
A lipid-hydrolysing activity involved in hexenal formation
Authors: Matsui K. , Kurishita S. , Hisamitsu A. , Kajiwara T.
Published: January 2005

Short-chain aldehydes such as (3Z)-hexenal and n-hexanal are formed from lipids through sequential actions of lipid-hydrolysing, lipoxygenase and fatty acid hydroperoxide lyase activities. The aldehydes are formed upon wounding of plant tissues, and are reported to have bactericidal and fungicidal activities. Furthermore, it has been reported that the aldehydes can induce expression of a subset of genes involved in disease resistance and that they are involved in a defence response against insect herbivores. Although several genes encoding lipoxygenases and the lyases have been isolated, and characterized to some extent, only little is known about the enzyme accountable for the lipid-hydrolysing step. In this study, we tried to characterize the lipid-hydrolysing activity involved in the short-chain aldehyde formation in Arabidopsis. When Arabidopsis leaves were homogenized, (3Z)-hexenal was formed rapidly within a few minutes. During this time period, the amount of α-linolenic acid and C16:3 rapidly decreased. Such a rapid increase of the aldehyde was repressed almost completely when the leaves were homogenized under a nitrogen stream, and instead free trienoic acids accumulated. A lipase inhibitor, quinacrine, successfully repressed the hydrolysis. It was revealed that trienoic acids in monogalactosyldiacylglycerol were predominantly hydrolysed during the formation of short-chain aldehydes. Collectively, it is suggested that the lipolytic enzyme involved in the short-chain aldehyde formation is a galactolipid-specific lipase.
Influence of (9Z)-12-hydroxy-9-dodecenoic acid and methyl jasmonate on plant protein phosphorylation
Authors: Tarchevsky I. A. , Karimova F. G. , Grechkin A. N. , Moukhametchina N. U.
Published: January 2005

The products of the lipoxygenase pathway, methyl jasmonic acid (MeJA) and (9Z)-12-hydroxy-9-dodecenoic acid (HDA), hardly changed the relative level of phosphorylated polypeptides (RLPPs) during 2 h of incubation: 15 and 17 kDa RLPPs were enhanced by HDA, but decreased by MeJA. RLPPs of 73 and 82 kDa were increased by both compounds. MeJA and HDA treatment induced specific and unspecific effects in some RLPPs. It was shown that HDA and MeJA increased protein kinase activity in the presence of 1 μM cAMP.
Seasonal variations of diacylglyceryl-N,N,N-trimethylhomoserine content in Polypodiophyta
Authors: Rozentsvet O. A. , Saksonov S. V.
Published: January 2005

Diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) belongs to a family of three known betaine lipids. It is synthesized by a number of bacteria, fungi, brown, red and green algae, in addition to lichens, and is also found in higher plants Bryophyta, Equisetophyta and Polydiophyta. It has been determined that DGTS plants can be DGTS-positive or DGTS-negative. The purpose of the present study was to examine the leaves of the ferns Drytopteris filix-mas (L.) Tod. and Pteridium aquilinum (L.) Kuhn, collected at different time periods during a single vegetation season for their DGTS contents. The results obtained show that amounts of DGTS in all of the species examined varies relative to the time periods in which they were collected.
Effect of age on the fatty acid content of Blumeria graminis conidia
Authors: Muchembled J. , Sahraoui A. Lounes-Hadj , Grandmougin-Ferjani A. , Sancholle M.
Published: January 2005

Blumeria (=Erysiphe) graminis f.sp. tritici (Bgt), the causal agent of wheat powdery mildew, is responsible for an important disease leading to considerable yield reductions in wheat worldwide. Conidia of the obligate plant pathogen Bgt were analysed for their total fatty acid (FA) composition as a function of their ontogeny. A total of 17 FAs were detected (C12–C24 saturated and unsaturated ones), including the presence of unusual longchain monoenoic FAs. In young conidia, the major FAs were C18:2 (23%), C16:0 (16%), C18:0 (15.2%) and C18:1 (14.3%). In old conidia, the main FAs were C24:1 (20.7%), C22:0 (15%), C22:1 (13.5%) and C24:0 (9.7%). The amount of total FA was about 39 μg.mg of dry weight-1 in young conidia and decreased clearly to 18 μg.mg of dry weight-1 in older conidia. For the first time, we have demonstrated that the FA composition of conidia changes greatly with age. Medium-chain FAs (C12–C18) are predominant in very young conidia (75%), whereas long-chain FAs (C22–C24) are the major compounds in old conidia (74%). This study showed a significant elongation of FAs and a drastic decrease in the total FA amount during the ontogeny of conidia.
Aging in Pinus sylvestris L. seeds: changes in viability and lipids
Authors: Tammela P. , Hopia A. , Hiltunen R. , Vuorela H. , Nygren M.
Published: January 2005

Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seedquality parameters, such as germinability and electrolyte leakage.
Variations in fatty acid composition of neem seeds collected from the Rajasthan state of India
Authors: Kaushik N. , Vir S.
Published: January 2005

Neem (Azadirachta indica) is a multipurpose tree native to the Indian subcontinent and South-East Asian countries. Products derived from neem have been used for centuries, particularly in India, for medicinal and pest-management purposes. Azadirachtin and neem oil are the two major commercially important products derived from the tree. The oil contains palmitic, stearic, oleic and linoleic acids in good proportion. Although there is growing demand for quality planting material for plantation of neem, efforts are lacking for the selection of neem trees based on their biochemical composition. In the present study, 60 Neem seed samples were collected from different provinances of the Rajasthan state in India. These samples were analysed by GLC to study the variability of fatty acid composition. Significant variability in individual fatty acids was observed. The palmitic acid ranged from 16 to 34%, stearic acid from 6 to 24%, oleic acid from 25 to 58% and linoleic acid from 6 to 17%. This variability can be exploited for selection of trees and for studying the genetic variability in neem. These selections can also be utilized for genetic improvement of the tree.
Influence of UV-B irradiation on the carotenoid content of Vitis vinifera tissues
Authors: Steel C. C. , Keller M.
Published: January 2005

The carotenoid content was examined in leaf and berry tissues of grapevines (cv. Cabernet Sauvignon) grown either under ambient conditions or under a polyester film to reduce UV light by 98%. Total carotenoids in leaves were less in vines grown under the UV screen. Levels of β-carotene decreased with berry development around veraison. This effect was more pronounced in vines grown under reduced UV light. The lutein content of berries appeared to remain relatively constant with berry development, but levels were decreased under the UV screen. These observations are important for the wine industry because of the biosynthetic link between carotenoids and wine flavour and aroma compounds.
Temperature and oxygen regulation of microsomal oleate desaturase (FAD2) from sunflower
Authors: Martínez-Rivas J. M. , García-Díaz M. T. , Mancha M.
Published: January 2005

The effect of temperature and oxygen on the in vivo oleate desaturation and microsomal oleate desaturase (FAD2) activity was studied in peeled developing sunflower seeds. Using an oxygen concentration that was saturating for FAD2 enzyme, the amount of linoleic acid increased for all studied temperatures, being maximal at 20°C. Under these conditions, FAD2 activity increased at the beginning of the incubation, remaining constant for the rest of the time, but reaching a lower level at 30°C. Anoxia brought about a decrease in the FAD2 activity for all studied temperatures, becoming faster as the temperature increased. All these data suggest that temperature and oxygen control the level of FAD2 activity by separate mechanisms.