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PLENARY LECTURES

Wednesday, June 6th 8:45 PM

Sterol and Ceramide Glucosides: Cloning of Enzymes contributing to their Biosynthesis inPlants and Fungi Ernst Heinz Martina Leipelt, Philipp Ternes, Dirk Warnecke, Petra Sperling andin collaboration with Ulrich Zähringer, Forschungsinstitut Borstel, and Stephan Franke, Institut fürOrganische Chemie, Universität Hamburg Institut für Allgemeine Botanik Universität HamburgOhnhorststr. 18 22609 Hamburg Germany

Sterol glucosides and ceramide glucosides are the predominant (easily extractable) glycolipids inmembranes of the nucleocytoplasmic compartment of plant cells. Apart from their contribution to lowtemperature adaptation, hardly any precise functional implication can be ascribed to thesecompounds. For a functional assignment via a genetic approach, we have started to clone enzymesinvolved in their biosynthesis.In this context we have cloned UDP-glucose:sterol glucosyltransferases from various organisms,including plants, fungi and yeasts. Knock-out mutants of microbial organisms had lost the capacity tosynthesize these glycolipids, but otherwise did not show any phenotype under normal growthconditions.Efforts in our and other labs have resulted in cloning of most of the enzymes catalyzing thebiosynthesis of the predominating species of cerebrosides in plants, which are composed of the ß-glucosyl residue, a 4,8-sphingadienine or phytosphinganine and a N-linked 2-hydroxy-acyl group.The cloned enzymes include the serine palmitoyltransferase, various desaturases and hydroxylasesacting on sphinganine and N-acyl moieties as well as ceramide glucosyltransferases. In plants, thisglucosyltransferase has been suggested to use sterol glucoside as donor, which can be checked inappropriately engineered yeasts expressing the plant enzyme in the absence of sterol glucoside.Overexpression of ceramide glucosyltransferases from various organisms in yeasts results in theappearance of several new molecular species of cerebrosides normally not found in these hosts.Detailed structural analysis reveals unexpected complications, which necessitate additionalassumptions regarding subcellular targetting of enzymes or membrane-bound substrates involved insphingolipid biosynthesis.

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Friday, June 8th 8:00 PM

The biosynthesis of polyunsaturated fatty acids Johnathan A. Napier, Frédéric Beaudoin,Mervyn J. Lewis, Louise V. Michaelson & Olga Sayanova.IACR-Long Ashton Research Station, University of Bristol, Long Ashton, Bristol BS41 9AF, UK.

We have been studying the biosynthesis of C20 polyunsaturated fatty acids with the long-term aimof reconstituting this metabolic pathway in transgenic plants. Characterisation of the “front-end” fattyacid desaturases involved in this pathway has identified a class of cytochrome b5-fusion enzymeswhich are distinct from the more prevalent (in plants) methyl-directed desaturases. It is also clearthat these b5-fusion fatty acid desaturases share a related ancestry with sphingolipid long chainbase desaturases. We have examined this relationship using a number of techniques with the aimof understanding the molecular determinants of substrate- and regio-specificity.We have also recently characterised a component of the C18 to C20 PUFA elongase. Whenexpressed in yeast, this polypeptide is capable of directing the elongation of ∆6-desaturated C18fatty acids, and may act as a condensing enzyme in the elongase. In conjunction with the ∆5- and∆6- (front-end) fatty acid desaturases, we have used this elongating activity to reconstitute the C20PUFA biosynthetic pathway by co-expression in a heterologous host. We have also soughtadditional enzymatic components of this PUFA elongase, using yeast as a model system.Candidate genes for these additional components have been identified and their implications forfatty acid elongation will be discussed. The long-term prospects for the production of C20 and C20+PUFAs in transgenic plants will also be considered.

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Saturday, June 9th 8:00 PM

Genetic and Genomic Approaches towards Understanding the Regulation of Plant LipidMetabolism Christoph Benning, Dept. of Biochemistry and Molecular Biology, Michigan StateUniversity, East Lansing, MI 48824

Lipid metabolism in plants generally serves the maintenance and building of functional membranes.In addition, lipid metabolism is dedicated to the biosynthesis of storage lipids during a short period ofthe life cycle in seed oil plants. Nearly 80% of membranes in photosynthetic tissues are found in thechloroplast where they are important for the proper function of the photosynthetic membranes. Of allpossible environmental factors considered thus far, only phosphate deficiency affects the complexlipid composition of plants in drastic ways. In general, phospholipids are replaced by glycolipidsunder these conditions. Particularly intriguing is the sulfolipid/phospholipid substitution hypothesis.We have began to use sulfolipid- and phosphatidylglycerol-deficient mutants of Arabidopsis to testthis hypothesis. Furthermore, we designed a high-throughput screen to identify suppressors ofexisting leaf lipid mutants, which are expected to be deficient in the phosphate-control of leaf lipidmetabolism.Storage lipid accumulation in developing seeds is under developmental control. We are pursuing avariety of approaches to understand the underlying regulatory mechanisms. The goal is to identifyfactors which may be limiting to oil biosynthesis. Traditionally, the focus has been on the regulationof carbon flow from photosynthate to triacylglycerols. We have began to refocus our attention onreducing equivalents which may represent an impasse for fatty acid biosynthesis in developingseeds. I will discuss approaches which address the question, whether reducing equivalents mayactually be limiting to oil biosynthesis in developing oil seeds.

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ORAL PRESENTATIONS

A1 Molecular biology of acetyl-CoA metabolism; the role of ATP-citrate lyase. Basil J.Nikolau, David J. Oliver and Eve Syrkin Wurtele. Iowa State University, Ames, Iowa

Acetyl-CoA is a pivotal intermediate in metabolism, occurring at the juxtaposition of catabolicand anabolic processes. The generation of acetyl-CoA is of primary importance for thebiosynthesis of a large number of phytochemicals (e.g., fats, oils, waxes, isoprenoids, aminoacids, flavonoids, stilbenoids, etc.). Despite the fundamentally crucial metabolicrequirements for acetyl-CoA, the regulation of its biogenesis and subsequent metabolism indifferent spatial and temporal compartments is poorly understood. We are characterizingthe role of three enzyme systems (acetyl- CoA synthetase, plastidic pyruvatedehydrogenase complex, and ATP-citrate lyase) in generating acetyl-CoA, and how theyeach juxtapose with two acetyl-CoA-metabolizing enzymes (heteromeric and homomericacetyl-CoA carboxylase). These characterizations have lead to the development of testablehypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. We willpresent data from combined biochemical, genetic and molecular biological experiments thattest these hypotheses; with a particular emphasis on the role of ATP-citrate lyase.

A2 Structure-Function of Biotin Carboxyl Carrier Protein (BCCP). John E. Cronan, Jr.Depts of Microbiology and Biochemistry, Univ. of Illinois, Urbana, IL 61801.

BCCP is an essential subunit of the multiprotein acetyl-CoA carboxylases (ACCs) of bacteriaand chloroplasts. These BCCPs differ from biotinylated proteins that catalyze carboxylation(or decarboxylation) of molecules other than acetyl-CoA by a seven residue insertion intothe conserved sequence. This insertion is seen as a protruding "thumb" in the structures ofE. coli BCCP biotin domain that disrupts the strong symmetry of the molecule. I report thatexpression of BCCP species having deletions of the thumb residues fail to complement anE. coli strain that encodes a temperature-sensitive BCCP (the mutant BCCP is rapidlydegraded at non-permissive temperatures). Although the thumb-minus BCCPs aremetabolically stable and efficiently biotinylated, both constructs fail to restore fatty acidsynthesis to the mutant E. coli strain. Point mutants of conserved thumb residues will bereported. Two chimeric BCCPs have also been constructed. In the first construct the biotindomain of E. coli BCCP was replaced with that of transcarboxylase whereas in the secondthe putative hinge region of BCCP was replaced with the known hinge region of E. colipyruvate dehydrogenase. Although efficiently biotinylated, the first protein is completelyinactive in vivo whereas the second has high activity. Finally I report that mutant BCCPslacking the lysine residue modified by biotinylation partially complement the temperature-sensitive BCCP mutant, although no biotinylation occurs. Complementation requires anotherwise native BCCP structure. Complementation fails to stabilize the temperature-sensitive BCCP of the mutant host strain and appears to result from a protein-proteininteraction between the two mutant BCCPs during the ACC reaction. These results indicatethat the BCCP biotin domains form a functional dimer in the active ACC, although there isno indication of biotin domain dimerization in solution even at mM protein concentrations.

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A3 Structure Based Targeted Mutagenesis to Alter the substrate specificity ofcondensing enzymes Katayoon Dehesh Calgene, 1920 Fifth street, Davis CA 95616

β-ketoacyl-ACP synthases (KASs) belong to an important family of structurally andfunctionally related condensing enzymes that play critical roles in the biosynthesis of avariety of natural products, including fatty acids, the polyketide precursors of commercially-important pharmacological agents, and the mycolic acid precursor for the cell wall of diseasecausing mycobacteria. These enzymes have been recent targets for engineering of novelplant seed oils, and development of new drugs for the treatment of cancer and tuberculosis.In the type II fatty acid synthesis systems of plants and the majority of prokaryotes, multipleforms of KASs (I-III) have been identified that have different substrate specificities. KASIIIcatalyzes the elongation of an acetyl-CoA primer by malonyl-ACP, whereas KASI and KASII,which have overlapping specificities, utilize only acyl-ACP for elongation by malonyl-ACP.Both active KAS I and II are homodimers that efficiently elongate 6:0-ACP through 14:0 -ACP substrates. Previuosly we had solved the crystal structure of KASII from E. coli aloneor complexed with cerulenin. Recently we obtained the crystal structure of KASII fromSynechocystis sp. PCC 6803. Structural comparison of these and other availablecondensing enzymes enable us to identify residues that when mutated changed thesubstrate specificity of the Synechocystis KASII. The details of these experiments will bediscussed.

A4 Acyl CoA synthetases - subcellular localization of members of a large protein familyMartin Fulda, Ernst Heinz*, John Browse Institute of Biological Chemistry, Washington StateUniversity, Pullman, WA 99164-6340 *Institut fuer Allgemeine Botanik, Ohnhorststr. 18,22609 Hamburg, Germany

Acyl CoA synthetases (ACS) provide the cell with precursors for glycerolipid synthesis,protein acylation, fatty acid elongation, and beta oxidation. They are also involved in fattyacid transport through membranes and thus may control the acyl CoA pools in differentsubcellular compartments.In Arabidopsis a family of 11 ACS genes have been identified and cloned. Based onsequence similarities and comparison to ACS proteins from other plant species, wesubdivided the family into six groups and introduced a new nomenclature for plant ACS. Inorder to gain a better understanding of the role of specific ACS proteins in lipid metabolismwe tried to elucidate their subcellular localization. This was accomplished by generatingfusion proteins containing ACS proteins and color varients of green flourescent protein,followed by microscopic analysis of the fusion proteins in transient expression systems. Bythis method it was possible to localize peroxisomal as well as plastidial ACS isoforms. Forperoxisomes, two ACS isoforms were found which share about 72 % identity at the proteinlevel. Enzyme assays and northern analysis of these two genes suggest an important roleduring the early stages of seed germination. These data might indicate an involvement inglyoxysomal �-oxidation and the utilization of storage lipids.

Currently we are screening Arabidopsis knock out populations for mutant lines of specificACS genes and possible phenotypes will be discussed.

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B1 A fatty acid desaturase modulates the activation of defense signaling pathways inArabidopsis. John Shanklin1, Pradeep Kachroo2, Jyoti Shah2, Ed Whittle1 and DanKlessig2. 1Department of Biology, Brookhaven National Laboratory, Upton, NY 11973.2 Boyce Thompson Institute for Plant Research, Tower Rd., Ithaca, NY 14853.

Several years ago, a novel soluble fatty acid desaturase was shown to be involved in plantdefense against insects in geranium trichomes. This study extends the involvement ofsoluble fatty acid desaturase in plant defense to include response to pathogen attack. AnArabidopsis mutant ssi2-1 was identified based on its ability to constitutively activate theNPR1-independent pathway leading to pathogenesis related (PR) gene expression andresistance to Peronospora parasitica. In the same plants, induction of some jasmonic acid-dependent defense responses was impaired. The SSI2 gene was isolated by chromosomewalking; it encodes a isoform of the stearoyl-ACP desaturase. The mutant form of thedesaturase has a point mutation that decreases its activity 10-fold with respect to wild-typeenzyme. In ssi2 plants 18:0 fatty acid levels were elevated. The mutant phenotype could bereversed by injection of 18:1. Since a defect in the fatty acid desaturation pathway leads toactivation of certain defense responses and inhibition of others, it appears that a fatty acid-derived signal modulates cross-talk between the different defense pathways.

B2 A Tale of Two Subunits: Satyam Subrahmanyam and John Shanklin, Brookhaven National Labs, Upton, NY 11973.

The castor stearoyl-ACP desaturase is a soluble homodimeric enzyme oxidizing thesaturated fatty acid stearate into the monounsaturated fatty acid oleate while reducing amolecule of oxygen to water. A functional desaturase dimer is redundant in its catalyticcapabilities in having two diiron centers (the reaction centers) and two acyl-ACP bindingsites per homodimer. In this study we address the catalytic competency of the each half ofthe homodimer when functionally decoupled from its dimer partner by the use ofheterodimers carrying wildtype and mutant subunits. Two classes of mutants eitherdefective in substrate binding (Sub) or competent in substrate binding but catalytically dead(Cad) were generated. The effect of these mutants in heterodimer complexes with thewildtype enzyme will be presented.

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B3 STRUCTURE AND FUNCTION OF ACYL-CoA DESATURASES OF MOTHS AND THEFLY DROSOPHILA MELANOGASTER Douglas C. Knipple1, Claire-Lise Rosenfield1,Seong Eun Jeong2, Kyung Man You1, Wendell L. Roelofs1, Renaud Dallerac3, and ClaudeWicker-Thomas3 1 Cornell University, Department of Entomology, New York StateAgricultural Experiment Station, Geneva, NY 14456, USA; 2 Hannam University, Departmentof Life Sciences, 133 Ojung-Dong, Taeduk-Ku, Taejon 300-791, Korea; 3 Université Paris-Sud, UMR 8620, NAMC, Bat. 446, 91405 ORSAY Cédex, France

Lepidopteran insects, comprising maore than 150,000 species, use fatty acid derived sexpheromones for their species-specific mate recognition systems. Elucidation of the chemicalstructures of pheromone components and the pheromone biosynthetic pathways of manymoth species has revealed thousands of unique structures, which are produced by differentcombinations, temporal orders, and substrate specificities of a relatively small number ofbiosynthetic enzymes mediating desaturation, limited chain-shortening by β-oxidation, andvarious terminal functional group modifications. It is clear that the evolution of diversefunctional properties of desaturases has played a major role in the generation of structuraldiversity of moth sex pheromone constituents, since several regio- and stereo-selectivedesaturation mechanisms have been described along with a wide range of substratespecificities. Recent molecular cloning and functional expression work by my group and ourassociated collaborators has confirmed the prediction of early biochemical studiessuggesting that pheromone biosynthetic desaturases are homologues of the membrane-associated acyl-CoA �9 desaturases that occur ubiquitously in animal and fungal cells. Inthis presentation, I will describe some of our ongoing work in this area focusing on thestructural and functional conservation and divergence among acyl-CoA desaturases ofseveral moth species and the fly Drosophila melanogaster. Towards the goal of identifyingdiscrete structural elements that are correlated with specific functional subtypes, we haveisolated and performed comparative sequence analysis of all or most of the desaturase-encoding sequences present in the genomes of several moth species and Drosophila; wehave quantified desaturase mRNA levels corresponding to the several desaturase-encodingsequences of a couple of moth species; we have established the identities of several mothdesaturases and two from Drosophila by analyzing the structures of the unsaturated fattyacid products formed in vivo following functional expression of the encoding cDNAs in adesaturase-deficient ole1 mutant strain of the yeast Saccharomyces cereviseae.

B4 Deciphering the catalytic specificity of membrane-bound diiron desaturases andhydroxylases John Broadwater, Edward Whittle, and John Shanklin, Biology Department,Brookhaven National Laboratory, Upton, NY 11973

Hydroxy fatty acids are unusual fatty acids that are incorporated into seed triacylglycerols ina few species of plants, notably Ricinus communis (Castor) and Lesquerella fendleri. Inboth of these plants, an oleate hydroxylase enzyme catalyzes the hydroxylation chemistry toconvert oleate (cis-9 octadecenoic acid, or 18:1 ∆9) to ricinoleate (D-12-hydroxyoctadec-cis-9-enoic acid, or 12-OH 18:1∆9). Amino acid sequence alignments of these two oleatehydroxylases with several oleate desaturases indicated that there are only a few conserveddesaturase residues that are not conserved in the hydroxylases. The substitution of theseresidues with the corresponding residues of the Lesquerella hydroxylase was found to besufficient to convert the desaturase into a bifunctional desaturase/hydroxylase (Science(1998) 282, 1315-1317). In this study, we report that incorporating the Castor oleatehydroxylase residues into the Arabidopsis oleate desaturase produces a bifunctionalenzyme with improved specificity for hydroxylation. Furthermore, the contributions of theindividual residues in determining product outcome have been assessed by analyzing aseries of single, double, and triple mutants of oleate desaturase through expression in bothArabidopsis thaliana and Saccharomyces cerevisiae. Finally, we present evidence that thebifunctional nature of the Lesquerella hydroxylase/desaturase is a general phenomenonamong membrane-bound desaturase enzymes.

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B5 Expression and Structural Studies of Transmembrane Domains of a Diverged FAD2Desaturase R.E. Minto W.J. Gibbons, Jr., B.M. Hardesty, A.E. Schloemer, J.P. Esposito,and G.A. Lorigan. Department of Chemistry and Biochemistry, Hughes Laboratory, MiamiUniversity, Oxford, OH 45056

Enzymes of the integral membrane fatty acid desaturase family oxidatively modify acylgroups resulting in hydroxy, epoxy, acetylenic, and (poly)unsaturated fatty acids. Chemicalcontrol of desaturase-like enzymes will be aided by a more detailed structural model and bythe definition of their interactions with lipid bilayers and auxillary electron-transport proteins.The diverged FAD2 desaturase isolated from the seeds of Crepis alpina is an importantmember of this family of enzymes. It is one of the few known acetylene-forming enzymesand it is capable of both acetylenase and desaturase activities. We have developed a novel,inexpensive fusion protein method to overexpress hydrophobic segments of theacetylenase. Circular dichroism spectroscopy and solid-state NMR studies of the amino-terminal putative transmembrane domain have provided strong evidence for an α-helicalconformation and a transmembrane orientation with respect to the lipid bilayer. Preliminaryresults obtained from larger regions of the protein will be discussed.

C1 Increased production of epicuticular wax mediated by a transcription factor inArabidopsis Pierre Broun, Robert A. Creelman and Jose Luis Riechmann

In the course of our functional characterization of Arabidopsis transcription factors, we haveidentified a novel gene that plays a central role in the control of epicuticular waxbiosynthesis. Constitutive expression of WAX1 in transgenic Arabidopsis plants results in astriking phenotype: levels of leaf alkanes are up to 10-fold higher in overexpressers than incontrol plants. In addition to these biochemical changes, ectopic WAX1 overexpressioncauses a stunted phenotype, and poor fertility. We are in the process of defining the cellularlocalization of wax in leaf cells of overexpressing plants, as well as the effect of WAX1 ongene expression.

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C2 Functional identification of a fatty acid elongase component specific forpolyunsaturated fatty acids by gene targeting Thorsten K. Zank, Hauke Holtorf, RalfReski, Jens Lerchl and Ernst Heinz Institutfür Allgemeine Botanik, Universitaet Hamburg,Ohnhorststrasse 18, D-22303 Hamburg, Germany Institut für Biologie II, UniversitaetFreiburg, Sonnenstrasse 5, D-79104 Freiburg, Germany BASF Plant Science, D-67056Ludwigshafen, Germany

We recently described the cloning and functional identification of a cDNA (PSE1) encoding afatty acid elongase from the moss Physcomitrella patens. By heterologous expression in theyeast Saccharomyces cerevisiae we showed that the encoded protein (Pse1p) is involved inthe elongation of polyunsaturated fatty acids. By feeding experiments we could show that ithas a selectivity for ∆6-C18 polyunsaturated fatty acids and discriminates ∆9-C18polyunsaturated fatty acids. We provide now further evidence for the function of Pse1p ascomponent of the fatty acid elongase complex. We disrupted the PSE1 gene in the moss byhomologous recombination. One mutant plant was obtained that contained significantreduced proportions of C20 polyunsaturated fatty acids (~10% of the wilde-type content) andincreasing proportions of -γ-linolenic acid, which acts as substrate for the elongase.However, no difference in the appearance of wild-type and mutant plants could be observed,so that possible functions of the polyunsaturated fatty acids arachidonic acid andeicosapentaenoic acid remain to be elucidated. The PSE1 sequence shares some limitedsimilarities to the ELO1 sequences. e.g. it contains a his box motif normally present indesaturases and related enzymes and a tyrosine box characteristic for the ELO proteinfamily. Interestingly, these sequences do not share any homology with the beta-ketoacyl-CoA synthases (KCS) isolated from various plant species. It is questionable, whether theELO protein family catalyzes the same reaction as the KCS protein family since it does notcontain a conserved cysteine, which is essential for this activity. Biochemical andphylogenetic implications will be discussed.

C3 Synthesis of Polyunsaturated Fatty Acids by a Polyketide Synthase in the Eukaryotic Microalga Schizochytrium. 3 Paul Roessler, Jim Metz,1 Franziska Dittrich,2 Frederic

Domergue,2 Jerry M. Kuner,1 Michael Lassner, 3 and John Browse.2 1Omegatech, Inc.,Boulder, CO 80301; 2Washington State University, Pullman, WA 99163; 3Monsanto Co.,Davis, CA 95616 and San Diego, CA 92121.

The thraustochytrid Schizochytrium produces very high levels of docosahexaenoic acid(22:6ω3) and docosapentaenoic acid (22:5ω6). A combination of biochemical studies andmolecular genetic analyses have provided evidence that these polyunsaturated fatty acids(PUFAs) are produced de novo by a polyketide synthase (PKS) complex. In vitro labelingstudies with ultracentrifuged cellular extracts indicated that [14C]malonyl-CoA wasincorporated into 22:6ω3 and 22:5ω6 in the absence of membrane-bound desaturases orelongases. Furthermore, in vivo labeling studies with 14C-labeled acetate, palmitate, oleate,or linolenate provided no evidence for precursor-product relationships between shorter chain(C16 - C20) fatty acids and the C22 PUFAs. In addition, sequence analysis of ~8500 randomlychosen clones from a Schizochytrium cDNA library indicated the presence of three separategenes that encode multi-domain enzymes having significant homology to various PKSenzymes from other organisms; these genes were expressed at moderate to highabundance (0.2 - 0.5% of the total clones). Conversely, only two fatty acid desaturasehomologs were observed in the EST dataset; one was present at moderate abundance(0.3%) and the other was present as a single copy (0.01%). At least five desaturases wouldbe necessary to produce 22:6ω3 via the typical elongation/desaturation route when startingwith fatty acid synthase-derived saturated fatty acids. These results strongly suggest thatthe primary route of PUFA biosynthesis in Schizochytrium is via the action of a soluble PKScomplex.

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C4 ENVIRONMENTAL EFFECTS ON EXPRESSION OF CER6 AND WAX ACCUMULATIONIN ARABIDOPSIS Tanya Hooker and Ljerka Kunst, Department of Botany, University ofBritish Columbia, Vancouver, BC, Canada. V6T 1Z4

In a number of species, epicuticular waxes are affected by environmental conditionsincluding light, water deficit, and cold. Furthermore, waxes are probably important in plantadaptations to at least some of these conditions. CER6 is a very long chain fatty acid(VLCFA) condensing enzyme required for epicuticular wax production in Arabidopsisthaliana. Its promoter contains several elements (including the DRE (Drought ResponseElement) and several ABA response elements) that mediate environmental responses.Therefore, we have examined the effects of dehydration, cold, and light on CER6 expressionand on wax accumulation in Arabidopsis. Northern blot analysis confirmed that light wasrequired for CER6 expression and that dehydration and cold treatments induced higherlevels of expression. Plants subjected to cold, as well as overexpressors of the CBF1transcription factor, which binds the DRE, accumulated more wax than the control plants.These results show a correlation of CER6 expression with increased wax production inplants subjected to environmental stresses. In addition, we have generated severaltransgenic lines overexpressing CER6 that accumulate up to 50% more wax thanuntransformed Arabidopsis plants. This indicates that expression levels of wax biosyntheticenzymes affect epicuticular wax accumulation. These results may be significant inadvancing our potential to engineer plants that are more stress tolerant.

C5 Ultrastructural Phenotypes of Arabidopsis waxless (eceriferum) mutants.Lacey Samuels and Ljerka Kunst. Department of Botany, UBC, 6270 University Blvd.,Vancouver, BC, Canada, V6S 1L3

Glossy mutants with reduced wax (eceriferum, or cer, mutants) in the model plantArabidopsis thaliana have been previously described in terms of their wax components.However, there have been no cell biological studies of these interesting mutants. Theobjective of this study is to correlate the subcellular phenotypes of the cer mutants withknown sequence and homology data, the chemical phenotype of the wax and fatty acids,and the alterations in wax bloom. We are using cryofixation (high pressure freezing/freezesubstitution) as well as conventional chemical fixation methods to prepare samples fortransmission electron microscopy. Striking changes have been observed in cell structure inthe elongating zone of the bolting stem of cer mutants, when compared to wild type plants.Mutants such as cer6, with the least wax as assayed by scanning EM, had epidermal cellswith abundant endomembranes and reduced vacuoles. Several mutant lines showedchanges in the underlying cortical cell plastids, either by changes to the membrane stacking(cer2) or in starch levels (cer3). By integrating the known biochemical phenotypes andavailable information on the cloned genes with the cell structure that we observe, we aspireto increasing our understanding of wax biosynthesis and secretion.

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D1 METABOLIC FLUX ANALYSIS IN DEVELOPING EMBRYOS OF BRASSICA NAPUS BYSTABLE ISOTOPE LABELING JOERG SCHWENDER AND JOHN OHLROGGE,DEPARTMENT OF BOTANY & PLANT PATHOLOGY, MICHIGAN STATE UNIVERSITY,EAST LANSING, MI 48824

During oil accumulation in developing rapeseed embryos, fatty acid biosynthesis is mainlyfed by sucrose, which is taken up from the liquid endosperm. To investigate the metabolicfluxes between sucrose and acetyl-CoA, 20d old embryos of Brassica napus were cultivatedon a liquid medium containing 13C-labeled sucrose and glucose. During a subsequent 14dthe embryos continued to accumulate TAG and protein. The isotopomer composition of thefatty acids and of 13 proteinogenic amino acids was analyzed by GC/MS of the methyl- andt-butyl-trimethylsilyl derivatives, respectively. Amino acids are derived from severalintermediary metabolites and thus their labeling pattern provides insight into metabolic fluxesin intermediary metabolism, leading to acetyl-CoA. In addition, the McLafferty ion in themass spectra of fatty acids gives information on the labeling of acetyl-CoA. Thus metabolicfluxes from intermediary metabolism to acetyl-CoA can be measured. Initial results will bepresented and it will be discussed, how these methods provide a valuable means to betterunderstand the pathways involved in TAG biosynthesis in oil seeds.

D2 Metabolic Profiling of Arabidopsis Membrane Lipids. Ruth Welti1, Maoyin Li1, YongmingSang2, Cunxi Wang2, Han-E Zhou3, Homigol Biesiada4, Channa B. Rajashekar3, Todd D.Williams4, Xuemin Wang2. 1Division of Biology, 2Dept. of Biochemistry, and 3Dept. ofHorticulture, Kansas State University, Manhattan, KS 66506, USA and 4Mass SpectrometryLaboratory, University of Kansas, Lawrence, KS 66045, USA.

A metabolomic approach is being developed to determine cellular membrane lipidcomposition and to understand the regulation and role of membrane lipid compositionaldynamics in plant responses to stresses. The highly sensitive approach, based onelectrospray ionization tandem mass spectrometry (ESI-MS/MS) of extracts from geneticallymanipulated plants, requires only simple sample preparation and small samples to fullycharacterize plant membrane lipid molecular species. ESI-MS/MS has been employed toprofile membrane lipid molecular species and to determine the compositional dynamics inArabidopsis plants undergoing stress treatments. Phosphatidylcholine,lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine,phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, monogalactosyldiacylglycerol,and digalactosyldiacylglycerol were quantified and speciated with respect to total carbonnumber and number of double bonds. The capability to combine full lipid profiling withcellular analysis of the machinery that generates compositional changes should yieldunprecedented information on how cellular machinery and metabolites interact in a dynamicmanner in the cellular response to changing environments.

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D3 Patterns of gene expression in developing Arabidopsis seeds during storage lipidsynthesis Sari Ruuska1, Christoph Benning2 and John B. Ohlrogge11Michigan StateUniversity, Department of Plant Biology, East Lansing MI 48824 USA, 2 Michigan StateUniversity, Department of Biochemistry

We have used microarrays to explore factors that regulate the overall partitioning of seedreserves into storage compounds, and particularly the accumulation of seed oil. Arabidopsiscan be used as a model to study lipid synthesis in oil crops, because triacylglycerides arethe major reserve in its seeds. To this end, we have produced microarrays containing about5000 clones selected from a cDNA library prepared from developing Arabidopsis seeds, andused them to study gene expression profiles during seed filling. This approach allowed us tosimultaneously compare mRNA levels of genes for fatty acid synthesis (FAS), storageproteins, glycolysis, etc. from the same developing seed material. Analysis of geneexpression profiles at different stages of development identified almost 2000 genes whoseexpression changed significantly during the accumulation of the storage reserves. Severalgenes encoding FAS enzymes (such as acetyl-CoA carboxylase subunits, KAS I, and acyl-carrier protein) showed coordinated expression. Typically, the highest gene expressioncoincided with the most active storage lipid accumulation period, and then decreasedrapidly. This pattern was distinctly different from that for some glycolytic enzymes, storageproteins and oleosins, as well as fatty acid modifying enzymes (FAD3 and FAE1). Theseobservations imply different regulatory controls over gene expression for different storagecomponents in seeds. We also identified several transcription factors, as well as kinasesand phosphatases, whose expression changed during the seed development, and whichmay be involved in the regulation of the storage compound synthesis. Moreover, clusteranalysis of the expression profiles established distinct groups of co-regulated genes.

D4 Genetic rigidification of membrane lipids enhances cold inducibility of heat shockgenes in Synechocystis. Masami INABA, Silvia FRANCESCHELLI1, Iwane SUZUKI,Balazs SZALONTAI3, Yu KANESAKI2, Dmitry A. LOS4, Bruno MARESCA5 and NorioMURATA; Natl. Inst. Basic Biol., Okazaki 444-8585; 1Univ. Salerno, Italy, 2School of LifeScience, Grad. Univ. Advanced Studies, Okazaki 444-8585, 3Inst. Biophys., Biol. Res.Center, Szeged, Hungary, 5Inst. Plant Physiol., 4Russian Academy of Sciences, Moscow,Russia, and 5Int. Inst. Genet. Biophys., Naples, Italy.

Poikilothermic organisms respond to a shift in the ambient temperature by changing patternof the gene expression. In Synechocystis sp. PCC 6803, expression of the desA, desB anddesD genes for acyl-lipid desaturases are induced at low temperatures. Targeted disruptionof the desA and desD genes replaced polyunsaturated lipid molecules by mono-unsaturatedones. The desA-desD- mutation rigidified fatty acyl chains of plasma membrane lipids,particularly at low temperatures. DNA microarray analysis revealed that the expression ofthe hspA, htpG, dnaK2, htrA and dnaJ4 genes was induced by cold shock in desA-desD-

cells, whereas the cold inducibility of these genes was negligible in wild-type cells. Thesefindings are in accord with the hypothesis that the rigidification of membrane lipids is aprimary signal in the perception of decreases in the ambient temperature.

Keywords: Cold inducibility, gene expression, acyl-lipid desaturase, heat-shock gene,membrane fluidity, Synechocystis

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D5 Elucidating the Greenhouse Effect on lipid metabolism using transgenic wheats.John L. HARWOOD Duncan. A.N. Edlin, M. Williams, M.D. Wilkinson, J. Richards, H. Jones,P. Kille. School of Biosciences Cardiff University PO Box 911 Cardiff CF10 3US U.K.

Work with wild-type wheat has shown that elevated temperature and/or carbon dioxide canchange lipid metabolism. The alterations can be explained by differing activities of glycerol3-phosphate acyltransferase and acyl-ACP thioesterase. We have used microparticlebombardment to manipulate wheat with genes coding these enzymes (obtained from peaand Arabidopsis, respectively). Transgenic plants were then examined for gene expression(including the endogenous wheat thioesterase), growth, morphology and lipid metabolism tolook for correlations with the 'Greenhouse Effect'.

E1 Requirement of phosphatidylglycerol in photosystem II of photosynthesisHajime Wada1, Miki Hagio1, Zoltán Gombos1,2, Zsuzsanna Várkonyi2, Lászlo Kovács2,Masayo Iwaki3, Shigeru Itoh3 1Fac. of Sci., Kyushu Univ., Ropponmatsu, Fukuoka 810-8560; 2Inst. of Plant Biol., Biol. Res. Cent. of Hung. Acad. of Sci., P. O. Box 521, H-6701Szeged; 3Natl. Inst. for Basic Biol., Okazaki 444-8585

Thylakoid membranes in chloroplasts and cyanobacterial cells provide the matrix for theprimary reactions of oxygenic photosynthesis that are performed in the pigment-proteincomplexes surrounded by the bilayers of glycerolipids. The lipid composition of thylakoidmembranes is highly conserved among oxygenic photosynthetic organisms.Phosphatidylglycerol (PG) is a ubiquitous lipid constituent of thylakoid membranes and isconsidered to play an important role in the ordered assembly and structural maintenance ofthe photosynthetic apparatus in the membranes. However, its function in photosynthesisremains poorly understood. In this study we have studied the function of PG in oxygenicphotosynthesis with a mutant of Synechocystis sp. PCC6803 incapable of synthesizing PG.The PG content in this mutant decreased as the deprivation of PG in the growth medium inparallel with the loss in the photosynthetic oxygen evolving activity. To understand thefunction of PG we investigated the phenotypes of the mutant using several techniques suchas fast fluorescence yield and thermoluminescense analyses that reflect the photosyntheticelectron transport and the energy transfer in thylakoid membranes. The obtained resultsclearly demonstrated that a loss of PG led to the suppression of the photosynthetic electrontransfer between QA and QB in photosystem II reaction center and also the turn over of watersplitting reaction. It is thus, concluded that PG molecules are indispensable components inthe maintenance of photosystem II reaction center, and the obtained findings provide thefirst firm identification of the functional site of PG in photosynthetic reaction.

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E2 MOLECULAR CLONING AND CHARACTERIZATION OF ARABIDOPSIS CDNASINVOLVED IN METABOLISM OF SPHINGOLIPIDS. Hiroyuki Imai, Yasuaki Morimoto,Junichro Mori, Hideki Nishiura and Kentaro Tamura, Department of Biology, KonanUniversity, Kobe 658-8501 Japan

Sphingolipids are constituents of the membrane lipids of fungi, animals, and some bacteria,and of plants, including the ferns and bryophytes. Sphingolipid biosynthesis is initiated by areaction catalyzed by serine palmitoyltransferase (SPT; EC 2.3.1.50) that condenses L-serine with palmitoyl-CoA to form 3-ketosphinganine. In this study, two Arabidopsis cDNAs,AtLCB1 and AtLCB2, which encode subunits of SPT have been cloned and characterized.AtLCB1 gene is present as a single-copy gene on chromosome IV, whereas at least twoDNA regions with high homology to the AtLCB2 gene are exist in the Arabidopsis genome.The distribution of SPT activity among subcellular fractions was characterized in squash fruit(Lynch and Fairfield, 1993) using differential centrifugation and marker enzymes, indicatingthat SPT activity was localized to ER. Thus, to examine whether the AtLCB2 protein islocalized in ER, tobacco BY-2 cells were transformed with a chimeric gene encoding thesynthetic green fluorescent protein (sGFP; S56T) and AtLCB2 protein to generate atransformant sGFP-AtLCB2/BY2. The transformant sGFP-AtLCB2/BY2 showed a strong andstable fluorescence on the ER-network and nuclear envelope, indicating that AtLCB2 wastranslocated to the ER. Finally, our current studies on Arabidopsis genes encodingsphinganine kinase and an enzyme involving ceramide synthase activity will talk in theMeeting.

E3 GLY1 encodes a glycerol-3-phosphate dehydrogenase that affects the level of 16:3fatty acids in leaves. Martine Miquel Seed Biology, INRA, 78026 Versailles, France

The Arabidopsis mutants designated gly1 exhibit a reduced carbon flux through theprokaryotic pathway that is compensated for by an increased carbon flux through theeukaryotic pathway. Biochemical approaches reveal that the gly1 phenotype cannot beexplained by a deficiency in the enzymes of the prokaryotic pathway. The chemicalcomplementation of the mutant phenotype by exogenous glycerol treatment of gly1 plantssuggests a lesion affecting the glycerol-3-phosphate (G3P) supply within the chloroplast. Asan alternative to the biochemical study of the gly1 mutants we set out to clone the GLY1gene. The gly1 mutant being an EMS (ethyl methane sulfonate) mutant we used a strategybased on the polymorphism existing between Arabidopsis ecotypes, here Columbia (gly1background) and Landsberg erecta. We first mapped gly1 on chromosome 2. The completesequence of chromosome 2 revealed that on chromosome 2 in a region spanning 3.7 Mband covering T7D17, two glycerol-3-phosphate dehydrogenase genes can be found. Wesequenced the two regions from Col, gly1 and an allele of gly1. A point mutation in gly1removes a splicing site leading to a premature stop codon whereas a point mutation in thegly1 allele changes a very conserved glutamic acid residue to a lysine residue. Further workis under progress to precise the localisation of this isoform and to confirm its function.

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E4 Four genes are involved in the triacylglycerol and steryl ester synthesis inSaccharomyces cerevisiae Maria H Gustavsson1*, Line Sandager2*, Anders Dahlqvist3,Ulf Ståhl4, Eva Wiberg3, Antoni Banas3, Marit Lenman2, Hans Ronne4, Sten Stymne2

1Department of Plant Biochemistry, Lund University, PO Box 117, S-221 00 Lund, Sweden 2

Department of Crop Science, SLU, PO Box 44, S-230 53 Alnarp, Sweden 3 ScandinavianBiotechnology Research AB, PO Box 116, S-230 53 Alnarp, Sweden 4 Department of PlantBiology, SLU, PO Box 7080, S-750 07 Uppsala, Sweden *Both authors contributed equallyto this work

Triacylglycerol and steryl esters are important storage compounds in eukaryotic cells, wherethey accumulate as well-defined lipid bodies. We have used single and multiple genedisruptions to study storage lipid synthesis in the yeast Saccharomyces cerevisiae. Fourenzymes were found to contribute to triacylglycerol synthesis, while two of them also areinvolved in steryl ester synthesis. A yeast strain that lacks all four enzymes is viable andshows no growth defects under standard conditions. Significantly, this strain is devoid ofboth triacylglycerol and steryl esters, which shows that storage lipid synthesis is notessential for vegetative growth. Further, fluorescence microscopy revealed that lipid bodiesare totally absent from this strain.

E5 Seed-expressed fluorescent proteins as versatile tools for easy co-transformationand high-throughput functional genomics in ArabidopsisA. R. Stuitje(1), E. C. Verbree(1), K. H. van der Linden(1), E. Mietkiewska(2,3), J.P. Nap(2) andT.J.A. Kneppers(1). (1)Department of Genetics, Vrije Universiteit, de Boelelaan 1087,1081HV, Amsterdam, The Netherlands. (2)BU Genomics, Plant Research International, P.O.Box 16, NL-6700 AA Wageningen, The Netherlands. (3)Plant Breeding and AcclimatizationInstitute, Mlochow Research Center, Poland

We demonstrate that seed-expressed fluorescent proteins can be used as efficient visualselection markers in the transformation of Arabidopsis thaliana by the floral dip method.Seed-specific expression of green fluorescent protein (GFP) variants as well as DsRedallows identifying mature transformed seeds efficiently in a large background ofuntransformed seeds by fluorescence microscopy. It also allows for the easy identification ofdeveloping transformed seeds in siliques. In planta visualization of transformed maturingseeds in siliques shows that susceptibility for flower dip transformation is limited to a small,defined window in flower development. In the competent stage, up to 25% of the seedswithin a single silique become transformed. Fluorescent proteins with different spectralcharacteristics can be used in combinations that allow each protein to be visualizedindependently. This greatly simplifies the identification and genetic analysis of seeds thathave received multiple genes-of-interest in co-transformation experiments. The resultsindicate that Agrobacterium tumefaciens-mediated co-transformation is an essentiallyrandom process. The visualization of transformation by fluorescence circumvents the use ofantibiotics and other positive selection markers. Moreover, it allows rapid identification ofpotential problems with the expression of transgenes that affect seed development and/orgermination. Visualization of transformation is likely to provide a powerful high-throughputsystem for transformation in functional genomics. It may also contribute to the developmentof a universal plant transformation procedure using flowers and seeds, rather than tissueculture and plant regeneration.

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F1 The Phospholipase D Gene Family of Arabidopsis thaliana and Surface DilutionKinetic Analysis of the Plasma Membrane-Associated Phospholipase Dδδ Chunbo Qin,Cunxi Wang, and Xuemin Wang, Department of Biochemistry, Kansas State University,Manhattan, KS 66506

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellularprocesses such as signal transduction, membrane remodeling, and lipid degradation.Totally 12 PLD genes have been identified in the Arabidopsis thaliana genome, and they aredivided into six classes according to their gene structures and catalytic properties, includingcalcium dependency, phophoinositide requirements, and fatty acid activation. In order toinvestigate the kinetic property of PLD isoforms and their regulation mechanism by calciumand PIP2, surface-dilution studies were carried out on the newly-characterized isoform PLDδ,which is associated primarily with the plasma membrane. PLDδ activity was dependent onboth bulk concentration and surface concentration of substrate phospholipid in the micelles.Vmax, Ks, and Km were calculated for PLDδ on PC/Triton or PE/Triton, and PE was foundto be a preferred substrate. PC-hydrolyzing activity of PLDδ in this Triton mixed micellesystem was activated by PIP2, but didn’t require it (in contrast to PLDβ and γ), and the curveof activation versus PIP2 molar ratio fitted a Michaelis-Menton equation with maximalactivation observed at a PIP2 molar ratio around 0.01. The presence of PIP2 decreased theinterfacial Michaelis constant Km, and PIP2 may activate PLD by promoting substratebinding to the enzyme. Calcium also significantly reduced the Km, indicating that it mayhave an effect similar to that of PIP2 in terms of activation mechanism.

F2 The many changing colors of fatty acid biosynthesis. Gert-Jan de Boer and ChrisSomerville. Carnegie Institution of Washington, Department of Plant Biology 260 PanamaStreet, Stanford Ca 94305, USA

In recent years live cell imaging has opened up a wealth of new information about organellestructure and dynamics. This has primarily been due to the availability of fluorescent dyes to“paint” different organelles and the use of fluorescent proteins such as GFP. In addition, tostudying organelle structure and dynamics, GFP also promises to be an interesting tool tostudy protein turnover and protein interactions invivo. Previously we have used GFP to studythe regulation and localization of integral membrane fatty acid desaturases. Our dataconfirmed that these enzymes are localized in the membrane of the Endoplasmic Reticulum(ER) and the use of GFP has enabled us to identify sequences in the protein of the delta-15desaturase that are involved in ER-retention but more importantly, these results show thatthe delta-15 desaturase is post-transcriptionally regulated. Currently GFP-taggedmicrosomal desaturases are employed in mutant screens to help identify the factorsinvolved in this process. Another component of plant fatty acid biosynthesis underinvestigation is oleosin. Constitutive expression of oleosin fused to GFP in transgenicArabidopsis shows that the fusion protein is integrated into the oil bodies of storage lipidsynthesizing cells. Interestingly in transgenic Arabidopsis plants, in which the expression ofthe oleosin-GFP fusion protein is controlled by the CaMV-35S promoter, a high level offluorescence is only observed in tissues that contain oilbodies such as the developingembryo or in the cotyledons of seedlings shortly after germination. Current experiments arefocused on 1) co-localization of oleosin and the delta-12 desaturase using different spectralvariants of GFP to visualize oilbody biogenesis and 2) to assess the feasibility of using theoleosin-GFP fusion protein to isolate Arabidopsis mutants with defects in storage lipidsynthesis or degradation.

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F3 Biosynthesis and Function of Divinyl Ether Oxylipins Aya Itoh and Gregg A. HoweDOE-PRL, Michigan State University, East Lansing, Michigan 48824

Oxylipins comprise a group of bioactive compounds that are derived from oxidativemetabolism of polyunsaturated fatty acids. Recent research indicates that a plant-specificfamily (CYP74) of cytochrome P450 enzymes plays a central role in oxylipin biosynthesis,namely in the metabolism of hydroperoxy fatty acid products of lipoxygenase. Includedamong CYP74 P450s are allene oxide synthase (CYP74A) of the jasmonic acid biosyntheticpathway and hydroperoxide lyase (CYP74B) of the C6 aldehyde/traumatin pathway. Usingexpressed sequence tag information as a starting point to characterize new members of theCYP74 gene family in tomato, we discovered a novel gene (designated CYP74D1) whosesequence defines a previously unidentified P450 subfamily (CYP74D). RecombinantCYP74D1 efficiently metabolize 9- but not 13-hydroperoxides of linoleic and linolenic acid.Incubation of CYP74D1 with 9-hydroperoxides of linoleic and linolenic acid yielded divinylether fatty acids called colneleic acid (CA) and colnelenic acid (CnA), respectively. Thesefindings represent the first identification of a gene encoding a divinyl ether synthase.Characterization of transgenic plants that are engineered for increased or decreasedsynthesis of CA/CnA is currently in progress. This research was supported by grants fromthe USDA/NRI and the DOE Biosciences.

F4 Plasma Membrane Phosphatidylinositol 4,5-bisphosphate Levels Decrease with Timein Culture Ingo Heilmann, Imara Y. Perera, Wolfgang Gross, and Wendy F. Boss.Department of Botany, North Carolina State University, Box 7612, Raleigh, NC 27695-7612

During the stationary phase of growth, after 7 to12 days in culture, the levels of phos-phatidylinositol 4,5-bisphosphate (PtdInsP2) decreased by 75% in plasma membranes of thered alga Galdieria sulphuraria. Concomitant with the decrease in PtdInsP2 levels in plasmamembranes, there was an increase in PtdInsP2 in microsomes, suggesting that the levels ofplasma membrane PtdInsP2 are regulated differentially. The decline of PtdInsP2 in plasmamembranes was accompanied by a 70%-decrease in the specific activity of PtdInsP kinaseand by reduced levels of protein cross-reacting with antisera against a conserved PtdInsPkinase domain. Upon osmotic stimulation, the loss of PtdInsP2 from the plasma membraneincreased from 10% in 7-day-old cells to 60% in 12-day-old cells, although the levels ofinositol 1,4,5-trisphosphate (InsP3) produced in whole cells were roughly equal at both times.When cells from different time points during the stationary phase were osmoticallystimulated, a mild osmotic stress (12.5 mM KCl) activated PtdInsP kinase prior to InsP3

production in cells with low plasma membrane PtdInsP2 levels, whereas in cells with highplasma membrane PtdInsP2 more severe stress (250 mM KCl) was required to induce anincrease in PtdInsP kinase activity. The differential regulation of a plasma membranesignaling pool of PtdInsP2 is of great importance for understanding changes in theresponsive state of cells.

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F5 ß-OXIDATION DURING GERMINATION AND SEEDLING DEVELOPMENT IN NON-GLUCONEOGENIC PLANTS: I. MITOCHONDRIAL AND PEROXISOMAL ß-OXIDATIONCAPACITIES OF ORGANS FROM DEVELOPING PEA SEEDLINGS. Christine Mastersonand Clifford Wood. Biological & Nutritional Sciences,University of Newcastle upon Tyne,NE1 7RU, UK

Following a recent review highlighting the evidence for mitochondrial ß-oxidation in higherplants1 two obvious questions requiring immediate answer are: What is the physiological roleof two ß-oxidation sites?; and What is the flux to these two sites? This report addressesthese two questions, comparing the dual mitochondrial and peroxisomal ß-oxidationcapacities of higher plant organs. Oxidation of [1-14C]palmitate was measured in thecotyledons, plumules and radicles of Pisum sativum, a starchy seed, over a 14-day periodfrom the commencement of imbibition. Respiratory chain inhibitors were used in order todifferentiate between mitochondrial and peroxisomal ß-oxidation. Peroxisomal ß-oxidationgave a steady, baseline rate and in the early stages of seedling development accounted for70-100% of the ß-oxidation observed. Mitochondrial ß-oxidation gave peaks of activity atdays 7 and 10-11, which accounted for up to 82% of the total ß-oxidation activity at thesetimes. These peaks coincide with key stages of seedling development, such as greening,leaf development and secondary root formation, and were not observed when normaldevelopment was disrupted by growth in the dark. Peroxisomal ß-oxidation was unaffectedby etiolation. Since mitochondrial ß-oxidation was overt only during times of intensebiosynthetic activity it might be switched on or off during seedling development.Peroxisomes, in contrast, maintained a continuous, low ß-oxidation activity that could beessential to remove harmful free fatty acids e.g. those produced by protein and lipidturnover.

1. Masterson, C. and Wood, C. (2000) Mitochondrial ß-oxidation of fatty acids in higherplants. Physiologia Plantarum 109, 217-224.

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G1 Increasing the seed oil content by modulating the ABA level during seed developmentIvo Feussner1, Uta zur Nieden2, Michael Leps1, Udo Conrad1 1Institute of Plant Genetics andCrop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany 2Leibniz-Institute ofPlant Biochemistry, Weinberg 3, D-06120 Halle/Saale, Germany

The phytohormone abscisic acid (ABA) is an important regulator during seed development.ABA is directly involved in the development of seed dormancy, in regulating biosynthesis ofstorage proteins and in development of desiccation tolerance. In order to study thesephenomenons we generated transgenic plants, which specifically accumulated a single-chain antibody (scFv) against ABA within the endoplasmic reticulum of developing seeds.Here, the antibody was expressed to high amounts even before the deposition of storagecompounds started and was kept at that level till seed ripening processes are coming to anend. To achieve this goal, the scFv coding sequences were expressed under the control ofthe USP promotor from Vicia faba. Seeds who accumulated high levels of more than 1 % oftotal seed protein of the antibody turned out to be viviparous, storage globulins were nearthe detection limit in the embryo and the number of oil bodies was dramatically reduced.Moreover, oil bodies turned out to fuse to one large vacuole-type organell. Analysis of theseed oil content of controls, accumulating an anti-herbicide scFv, of wild type plants, andthese transgenic lines showed no differences in the seed oil accumulation of about 40 % oftotal seed weight. However, when we analyzed 60 independent lines of the T2 generationand of about 30 lines of their T3 progenies in more detail, it turned out that intermediatephenotypes expressing the anti-ABA-scFv to levels of below 1 % of total seed protein,showed a dramatic increase within the fatty acid content of up to 80 % of total seed weight.Moreover, these lines were not viviparous and the embryos did not exhibit a viviparousphenotype. However, when we analyzed the ultra structure of these embryos, it turned outthat within these transgenes many of the embryonic cells were devoid of protein bodies andthe remaining space was filled of with oil bodies. We conclude from these results thatimmunmodulation of the ABA functions beginning at early stages of seed developmentcauses different influences to single elements of the ripening process depending on thedifferent levels of ABA not bound by the antibody. This may result in a loss of protein bodyformation and in an increase of the biosynthesis of storage lipids in seeds exhibiting aspecific relation of ABA to antibody concentration.

G2 Distribution of fatty acids in polar and neutral lipids during seed development inArabidopsis thaliana genetically engineered to produce acetylenic, epoxy andhydroxy fatty acids. Anders S. Carlsson Stefan Thomæus, and Sten Stymne Departmentof Crop Science, Swedish University of Agricultural Science, Växtskyddsvägen 1, 23053Alnarp Sweden.

Genetically engineered plants of Arabidopsis thaliana containing either the Crepis palaestinalinoleate delta 12-epoxygenase gene, the Crepis alpina linoleate delta 12-acetylenase geneunder transcriptional control of a napin promoter or a Lesquerella fendleri oleate hydroxylaseunder the transcriptional control of the endogenous promoter were analysed for thedistribution of fatty acids in phosphatidylcholine (PC) and neutral lipids during seeddevelopment. Seed samples were analysed from 9 to 23 days after pollination (DAP) as wellas mature seeds. The unusual fatty acids (acetylenic, hydroxy and epoxy fatty acids)produced in the transgenic A. thaliana plants accumulated in significant amount in seedphospholipids during seed development. Despite that the percentage of the unusual fattyacids accumulated in neutral lipids in the transgenic plants were 5 to 35 times less than inthe wild plant species accumulating these acids, the relative levels of these acids in PC wereabout the same (for epoxy and hydroxy fatty acids) or much higher (for acetylenic fattyacids) than in that lipid in the wild plants. Results are discussed as regards to possiblebottlenecks for increasing the content of unusual fatty acids in the seed oil.

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G3 Production of conjugated fatty acids in transgenic oilseeds Edgar B Cahoon, Kevin GRipp, Sarah E Hall, Anthony J Kinney and Sean J. Coughlan DuPont Nutrition and Health,Experimental Station, Wilmington, DE 19880-0402

One of our targets in the DuPont oils group has been to engineer crop plants to produceseed oils that contain fatty acids with conjugated double bonds. Oils rich in these fatty acidsare valuable drying oils (e.g., tung oil) and may potentially be used as feeding supplementsto enhance the lean mass of livestock. Using a genomics based approach, we haveidentified cDNAs for fatty acid desaturase-related enzymes that catalyze conjugated doublebond synthesis in various non-agronomic species including Momordica charantia (bittermelon),Impatiens balsamina (impatiens), and Calendula officinalis Seed oils of these plants

are enriched in α-eleostearic acid (18:3∆9cis,11trans,13trans) and α-parinaric acid(18:4∆9cis,11trans,13trans,15cis), and calendic acid (18:4 d ) which contain three and fourdouble bonds in conjugation, respectively. We obtained our first results from the transgenicexpression of the Momordica and Impatiens cDNAs in the soybean embryo model systemand in soy seeds In these experiments, expression of transgenes was driven by the strong,seed-specific promoter -conglycinin. In soybean embryos expressing the Momordica cDNA,fatty acids with conjugated double bonds accumulated to amounts of 18% of the total fattyacids. This consisted of 16% eleostearic acid and 2% parinaric acid. These are some of thebest results, to date, for the accumulation of unusual fatty acids in transgenic plant tissues.Interestingly, production of fatty acids with conjugated double bonds was accompanied bylarge increases in the oleic acid content of soybean embryos. In the most extreme cases,amounts of oleic acid increased from the 10% found in the oil of wild-type embryos to morethan 40% in lines producing the highest amounts of fatty acids with conjugated doublebonds. The basis for this increase in oleic acid is not known, but similar phenotypes havealso been observed with the expression of other fatty acid desaturase-related enzymes(e.g., epoxidases and hydroxylases) in transgenic plants. In addition, expression of theImpatiens cDNA in soybean embryos resulted in the accumulation of eleostearic andparinaric acids to amounts of 3% and 2%, respectively of the total fatty acids. Similaramounts of eleostearic and parinaric acids were also obtained with the expression of theImpatiens cDNA in Arabidopsis seeds. Expression of a Calendula officinalis cDNA in bothsoy embryos and soy seeds gave calendic acid levels of between 20-25% in both neutraland polar lipids.

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G4 Characterization and application of fatty acid hydroperoxide lyase; a biocatalyst forthe production of food flavors Minke A. Noordermeer, Gerrit A. Veldink and JohannesF.G. Vliegenthart Bijvoet Center for Biomolecular Research, Department of Bio-organicChemistry, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

Volatile C6- and C9-aldehydes and alcohols are important contributors to the characteristicodor of plants. They are widely used as food flavors to reconstitute the 'fresh green' smell offruit and vegetables that is lost during processing. The low abundance of these compoundsin plants and the preference for natural food additives have urged the industry to developbiocatalytic processes to produce these compounds on a large scale. Plants produce C6-and C9-aldehydes and alcohols in response to wounding, and these compounds are knownto have anti-fungal and anti-bacterial activity. They are formed by the subsequent actions oflipoxygenase and hydroperoxide lyase on linoleic and linolenic acid. To improve theavailability of hydroperoxide lyase for characterization studies and biocatalysis, three full-length cDNAs from alfalfa seedlings coding for fatty acid hydroperoxide lyases were clonedand expressed in Escherichia coli. The isoenzymes are specific for the production of C6-aldehydes and do not form C9-aldehydes. The optimal expression and reaction conditionswere determined. One enzyme molecule can convert about 1.6·105 molecules ofhydroperoxy-linoleic acid or 0.9·105 molecules of hydroperoxy-linolenic acid, with initialturnover rates of 330 and 750 s-1, before it is inactivated. Inactivation is independent of thesubstrate or product concentration. The amount of hydroperoxide lyase obtained from 1 literof E. coli culture can convert up to 24 mmol hydroperoxy-linoleic or 15 mmol hydroperoxy-linolenic acid. The Km and Vmax at optimal pH (pH 8.25) are ~40 µM and ~650 µmol·min-1·mg-

1 for hydroperoxy-linolenic acid. Alfalfa hydroperoxide lyase was characterized as acytochrome P450 enzyme by UV/Vis and EPR. The enzyme contains Fe(III) in the low spinstate. In the presence of Triton X-100 however, the Fe(III) appeared to be in the high spinstate and the enzymatic activity increased twofold. EPR and CD spectra showed thatdetergents induce a subtle conformational change, which might result in improved substratebinding. A biocatalytic process is developed based on safflour oil as substrate, and soybeanmeal and the recombinant E. coli strain as lipoxygenase and hydroperoxide lyase sources,respectively.

G5 MAXIMIZING THE PRODUCTION OF MEDIUM-CHAIN FATTY ACIDS IN TRANSGENICBRASSICA NAPUS Kathryn Lardizabal, Deborah Hawkins, James Byrne, and KatayoonDehesh Calgene Campus/Monsanto, 1920 Fifth Street, Davis, CA 95616

The Mexican shrub Cuphea hookeriana (Ch) accumulates up to 75 mol % caprylate (8:0)and caprate (10:0) in its seed oil. We have identified and cloned a medium chain specificthioesterase (Ch FatB2) and a medium chain specific β-ketoacyl-ACP synthase (Ch KAS 4)cDNA from C. hookeriana and generated transgenic Brassica plants over-expressing theseenzymes, singly or in combination, in a seed specific manner. Oil analysis data indicate thatonly concomitant expression of Ch FatB2 and Ch KAS IV leads to accumulation of highlevels of 8:0 and 10:0 in seeds that otherwise do not accumulate any of these fatty acids.However, the best transgenic line accumulates up to only 50% of the levels measured in thenative plant. To identify the limiting factors preventing higher accumulation of 8:0 and 10:0,we have carried out a series of analyses and identified a new strategy that enabled us toproduce higher levels of these fatty acids.

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POSTER PRESENTATIONS

P1 Effects of manipulating expression of acetyl-CoA carboxylase I in Brassica napusembryos Chloe Sellwood, A. R. Slabas* and S. Rawsthorne Department of MetabolicBiology, John Innes Centre, Norwich, Norfolk, NR4 7UH, UK *Department of BiologicalSciences, University of Durham, South Road, Durham, DH1 3LE, UK

Acetyl-CoA carboxylase (ACCase) catalyses the first committed step in fatty acidbiosynthesis [1]. There are two isoforms identified in higher plants. ACCase I is apredominantly cytosolic, multifunctional homodimer capable of carboxylating acetyl-CoA andpropionyl-CoA (i.e. it also has a propionyl-CoA carboxylase (PCCase) activity) [2]. ACCaseII is a plastidial heterotetramer, which is capable of the acetyl-CoA carboxylation only. Indicotyledonous plants ACCase II is responsible for de novo fatty acid biosynthesis. Brassicanapus is the only species reported to date that also has an isozyme of ACCase I in theplastid as well as ACCase II [3]. Transgenic B. napus plants have been made in which apartial antisense cDNA of cytosolic ACCase I has been expressed under the control of anembryo specific acyl carrier protein promoter. This has led to a reduction in propionyl-CoAcarboxylase (PCCase) activity in the embryo and, surprisingly, changes in carbonpartitioning in the seed. These plants have a wrinkled seed phenotype and a dramaticallyreduced lipid content in the mature seed [4]. Analyses of storage product accumulation(lipid, protein, starch and soluble carbohydrate) in developing and mature embryos of thewild type B. napus c.v. Westar Double Haploid and two transgenic lines (C4S6-16 andC7S1-2) have been carried out. The activity of PCCase, catalysed by ACCase I, has beenused to monitor changes in the ACCase I distribution and amount in the embryo. We havealso analysed the de novo incorporation of 14C-labelled metabolites into fatty acids. Wehave demonstrated that there is significantly less lipid in mature seeds of the transgenicplants and that there are changes in other seed constituents. Effects on embryo metabolismwill also be described.1. Harwood, J. L. (1988) Ann. Rev. Plant Physiol. Plant Mol. Bio. 39, 101–1382. Ashton, A. R. et al. (1994) Plant Mol. Bio. 24, 35–493. Schulte, W. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3456–34704. White, A. J. et al. (1998) in Advances in Plant Lipid Research (Sánchez, J. et al., eds.),pp. 63-66, Universidad de Sevilla, Secretariado de Publicaciones

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P2 Expression of a cotton (Gossypium hirsutum L.) cDNA encoding a FatB palmitoyl-acylcarrier protein thioesterase in Eschericia coli and in transgenic cotton. Tu T.Huynh,Shea Austin-Brown, Robert M. Pirtle, and Kent D. Chapman Department of BiologicalSciences, Division of Biochemistry and Molecular Biology, University of North Texas,Denton, TX, 76203.

In addition to producing spinnable fibers, cottonseed is about 16% oil by weight and ranksthird behind soy and canola in world oilseed crushings (Inform 11:820, 2000). Thesubstantial percentage of palmitic acid (~25 mol%) in cottonseed oil may be due to theactivity of a palmitoyl-acyl carrier protein (ACP) thioesterase expressed in developing seeds.The isolation and characterization of a cotton FatB cDNA (Genbank #AF034266) encoding apalmitoyl-ACP thioesterase revealed that its expression was highest during seed oilaccumulation. Here we expressed this cDNA in various E.coli strains and examined severalbiochemical parameters. Transcription and translation in vitro in a coupled system showedtwo [35S-Met]-labeled translation products; a 46kDa polypeptide likely represented thetranslated preprotein while a 35kDa polypeptide likely represented a translation productinitiated at an alternative, internal in-frame initiation codon. An immunoreactive 35kDa wasrecognized in transformed E. coli cell lysates by polyclonal antibodies directed toward an 18-amino acid sequence of the deduced cotton FatB protein. When acyl-CoA synthetase-minus E.coli mutants (K27 fadD88 mutant, CGSC #5478) were transformed with anexpression construct of the cotton FatB cDNA, an increase in 16:0 free fatty acid contentwas measured in the culture medium (compared with controls). Acyl-ACP thioesteraseactivity assays in these E.coli lysates revealed that there was a clear preference forpalmitoyl-ACP over oleoy-ACP. Furthermore, overexpression of this cotton FatB cDNA(under control of the CaMV35S promoter) in transgenic cotton embryos resulted in higherlevels of immunoreactive FatB protein and increased levels of palmitic acid (approximately70 mol%), compared with controls. Collectively, our results indicate that the cotton FatBcDNA encodes a functional acylACP thioesterase with a preference for saturated acyl-ACPs(FatB) over unsaturated acyl-ACPs (FatA), and may provide a molecular target for themanipulation of palmitic acid content in cottonseed oil.This research was supported by USDA-NRICGP #98-355503-6339.

P3 THE SUBSTRATE FOR THE DESATURASE FROM ENDOPLASMIC RETICULUM INDEVELOPING PEANUTS IS PHOSPHATIDYL CHOLINE. G.L. Powell, J. Parrish and A.G. Abbott. Department of Genetics and Biochemistry, Clemson University, Clemson, SC29634-0326

We have cloned the most active ahFAD2B from the developing seeds of peanut (Arachishypogaea L.) and expressed the cDNA in yeast. The reading frame was inserted intopYES2 under the control of the GAL1 gene in S. cerevisae. This system has proven to bequite useful in elucidating the molecular basis of the high oleate phenotype (Jung, et al.,2001; Bruner, et al., 2001) and for describing the specificity of this enzyme. When the abilityto desaturate fatty acyl groups was tested, the products indicated that the primary referencewas the existing double bond rather than counting from the carboxyl (∆12) or omega (ω-6)ends (Swartzbeck, et al., 2001). By inducing the enzyme at time zero and sampling theintact lipids after six and twelve hours, we can directly observe the lipid species desaturatedby the desaturase using electrospray mass spectrometry (ESI/MS). The mass of eachspecies desaturated was reduced by two for each double bond that was formed. Thespectra for lipids observed after twelve hours showed that phosphatidyl choline (PC)containing palmitoleate and oleate were desaturated in one or both acyls. Thus PC was agood substrate. Phosphatidyl inositol was also desaturated but phosphatidyl ethanolaminewas not. This observation was verified using thin-layer and gas-liquid chromatography.Because certain intact lipid species could be desaturated twice, the enzyme must becapable of desaturating at either the sn-1 or sn-2 positions (Powell, et al., 2000).(Support provided by the SC Experiment Station and AgraTech Seeds Inc, GA)

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P4 Function of phosphatidylglycerol in photosynthesis Miki Hagio, Isamu Sakurai1, ZoltanGombos2 and Hajime Wada2, Grad. Sch. Sci., Undergrad. Sch. Sci., 2Fac. Sci., KyushuUniv., Fukuoka 810-8560

Thylakoid membranes are the site of photochemical reactions of photosynthesis, and arecomposed of proteins and glycerolipids. Although they contain galactolipid, sulfolipid andphospholipid as major glycerolipids, the specific function of each lipid in photosyntheticprocesses has not been understood. In this study in order to understand the function ofphosphatidylglycerol (PG), which is only phospholipid in thylakoid membrane, we identified apgsA gene of Synechocystis sp. PCC6803 that encodes a phosphatidylglycerol phosphate(PGP) synthase involved in the biosynthesis of PG. We inactivated the pgsA gene ofSynechocystis and made a mutant (pgsA mutant) that could not synthesize PG, and studiedthe function of PG in photosynthesis by comparing the phenotype of mutant to that of wildtype. The obtained pgsA mutant could grow only in the medium containing PG, and thephotosynthetic activity of the pgsA mutant dramatically decreased with a concomitantdecrease of PG content in thylakoid membrane when the cells grown in the presence of PGwere transferred to the medium without PG. This decrease of photosynthetic activity wasattributed to the decrease of photosystem (PS) II activity, but not to the decrease in PS Iactivity. These findings demonstrate that PG is essential for growth of Synechocystis andthat PG plays an important role in PS II. To understand the function of PG in higher plants itis important to identify the genes involved in the biosynthesis of PG. We searched thedatabase of the genomic sequence of Arabidopsis thaliana with an amino acid sequence ofPGP synthase of Synechocystis, and found two genes encoding polypeptides homologousto PGP synthase of Synechocystis. To confirm that the genes, designated PGS1 and PGS2,of Arabidopsis encode functional PGP synthases we expressed cDNAs corresponding tothese genes in the E. coli mutant YA5512, which is deficient in PGP synthase. The relativecontent of PG was 2 mol% in each of YA5512 and the transformant with pKK233-2, whereasthat of PG in the transformant with pKK-PGS1 and pKK-PGS2 were markedly increased to41 and 21 mol%. This result demonstrates that the PGS1 and PGS2 cDNAs encode PGPsynthases. Furthermore, in order to investigate the localization of PGS1 protein weexpressed PGS1 fused to green fluorescent protein (GFP). Transient expression of GFPfusion protein showed that PGP synthase encoded by PGS1 was targeted into chloroplastsand the other organelle, which might be mitochondrion. This finding suggests that PGPsynthase encoded by PGS1 is located in chloroplasts and mitochondria.

P5 PLASTID-LOCATED GLYCErol-3-PHOSPHATE ACYLTRANS-FERASE ISOZYMES INSQUASH EXPRESS DIFFERENTIALLY DURING LEAF DEVELOPMENT. Ikuo NishidaDepartment of Biological Sciences, Graduate School of Science, The University ofTokyo, Hongo, Bunkyoku, Tokyo, 113-0033, Japan

Expression of plastid-located glycerol-3-phosphate acyltransferase (GPAT) isozymes duringleaf development in squash was investigated using leaves developed along the squash vine.I recently reported that squash (Cucurbita moschata cv. Shirogikuza) contained twoisogenes for GPAT, which I designated CmATS1;1 and CmATS1;2. RNA gel blot analysisshowed that the level of CmATS1;2 transcripts increased in older leaves developed alongthe vine, whereas that of CmATS1;1 transcripts remained constant. These patterns oftranscript accumulation were consistent with the result of immunoblot analysis with antiseraprepared with specific oligopeptides, that the polypeptide level of CmATS1;2 but not that ofCmATS1;1 increased with leaf age along the vine. The level of CmATS1;2 polypeptidereached the maximum earlier than chlorophyll and RuBisco large subunit levels reached themaximum. All the results suggest that CmATS1;2 levels in squash leaves may betranscriptionally regulated in association with chloroplast development and that CmATS1;2play a major role in the lipid synthesis for chloroplast development. However, the role ofCmATS1;1 still remains unknown.

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P6 Analysis of polyunsaturated fatty acid elongation in yeast (Saccharomyces cerevisiae).Frédéric Beaudoin, Alison Lovegrove and Johnathan Napier. IACR-Long Ashton ResearchStation, Long Ashton, Bristol BS41 9AF, UK.

We have previously identified and characterized functionally in yeast a C. elegans ORFencoding the presumptive condensing enzyme activity of a fatty acid elongase (Beaudoin etal., 2000). This ORF (F56H11.4) showed a preference for ∆6-desaturated C18polyunsaturated fatty acid (PUFA) substrates converting γ-linolenic acid (C18:3 ∆6,9,12) intodi-homo-γ-linolenic acid (C20:3 ∆8,11,14) and stearidonic acid (C18:4 ∆6,9,12,15) intoeicosatetraenoic acid (C20:4 ∆8,11,14,17). We speculated that the heterologous C. elegansORF is likely to interact with endogenous components of a yeast elongation system causinga redirection of enzymatic activity toward these C18 PUFA substrates. Searching for suchcomponents we have recently identified a yeast mutant in which the activity of F56H11.4 isspecifically disrupted whereas other enzymes such as borage ∆6-desaturase are notaffected. We report here the characterization of this mutant which, although not lethal,exhibit a reduced growth rate in complete medium and a pronounced hyphal phenotype.

Frédéric Beaudoin, Louise V. Michaelson, Sandra J. Hey, Mervin J. Lewis, Peter R. Shewry,Olga Saynova and Johnathan A. Napier (2000). Proc. Natl. Acad. Sci. USA 97, 6421-6426.

P7 Substrate Specificity of Arabidopsis 3-Ketoacyl-CoA Synthases Brenda Blacklock,Mahin Ghanevati, and Jan Jaworski, Department of Chemistry and Biochemistry, MiamiUniversity, Oxford, OH 45056

The recent publication of the complete sequence of the Arabidopsis genome represents anunprecedented resource for the study of metabolism in plants. We are interested in theelongation of long chain fatty acids to very long chain fatty acids by 3-ketoacyl CoAsynthases (KCS) and their incorporation into plant lipids and waxes. Recent work in ourlaboratory has demonstrated the isolation of (His)6 FAE1KCS from yeast microsomes. Thisadvance has allowed us to carefully address the substrate specificity of FAE1KCS sincesubstrate fatty acyl CoAs can be presented directly to the enzyme. Recent mining of theArabidopsis genome sequence database has revealed at least 12 genes with homology toknown KCS genes. We are investigating the substrate specificity of these potential KCSs byexpression and isolation of (His)6 fusion proteins and assay of a battery of possible acyl CoAsubstrates. Progress toward identifying the substrate specificity of a number of predictedKCSs will be presented.

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P8 Condensing Enzymes Involved in the Production of Very Long Chain Fatty Acids inLesquerella fendleri Gangamma Chowrira. Hangsik Moon, Mark Smith, and Ljerka KunstDepartment of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.

Lesquerella fendleri seed oil contains up to 60% hydroxy fatty acids, nearly all of which isthe 20-carbon hydroxy fatty acid lesquerolic acid (D-14-hydroxyeicos-cis-11-enoic acid).Previous work suggested that lesquerolic acid in L. fendleri was formed by the elongation ofthe 18-carbon hydroxy fatty acid, ricinoleic acid. To identify a gene encoding the enzymeinvolved in hydroxy fatty acid elongation, a L. fendleri genomic DNA library was screenedusing the coding region of the Arabidopsis FAE1 condensing enzyme as a probe. Twogenes with high sequence similarity to known very-long-chain fatty acid (VLCFA)condensing enzymes, were isolated. RNA blot analysis indicated that one of the genes,LfKCS3, was expressed only in the embryos of L. fendleri and first appeared in the earlystages of development. Seeds of Arabidopsis plants transformed with LfKCS3 showed nochange in their VLCFA content. However, when these Arabidopsis plants were crossed withthe transgenic plants expressing the castor oleate 12-hydroxylase, significant amounts of20-carbon hydroxy fatty acids accumulated in the seed, indicating that the LfKCS3condensing enzyme specifically catalyzes elongation of 18-carbon hydroxy fatty acids.Fusion of the LfKCS3 promoter to the uidA reporter gene and expression in transgenicArabidopsis resulted in a high level of β-glucuronidase activity exclusively in developingembryos. The promoter of the second gene isolated, LfKCS45, when fused with the gusreporter gene and expressed in transgenic Arabidopsis, exhibited a root-specific expressionpattern. The function of LfKCS45 is currently being studied.

P9 Analysis of isomers of very long chain unsaturated fatty acids in transgenic yeast byGC/MS J. HAN and J. Jaworski Department of Chemistry and Biochemistry, MiamiUniversity, Oxford, OH 45056

As a comparison, we have expressed the Arabidopsis FAE1 (James et al., 1995) intransgenic yeast (Saccharomyces cerevisiae). Surprisingly, around 55% of very long chainfatty acids (VLCFAs, >C18) was found in the total fatty acids. Since the wild type yeastproduces both saturated and unsaturated fatty acids up to C18 in which it contains mainly16:1∆9 and 18:1∆9, there may be a series of unsaturated isomers of VLCFAs in thetransgenic cells. Thus, we have used the dimethyl disulfide derivatives (DMDS) to determinethe position of double bonds in these isomers by GC-MS. The resulting data showed that theArabidopsis FAE1, like the Brassica KCS (Han et al., submitted), is able to elongate not only18:1∆9 to 26:1∆17 but also 16:1∆9 to 26:1∆19. However, the Arabidopsis KCS prefers touse 18:1∆9 and 18:1∆11 to produce 20:1∆11 and 20:1∆13, repectively, while the BrassicaKCS tends to use 20:1∆11 and 20:1∆13 to make 22:1∆13 and 22:1∆15. In fact, expressionof the Arabidopsis KCS resulted in the accumulation of 4.0% of 20:1∆11 and 15.3% of20:1∆13 while the Brassica KCS produced 6.0% of 22:1∆13 and 9.2% of 22:1∆15.

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P10 Profiling the chemical composition of an extensive new collection of maize glossymutants reveals novel aspects of epicuticular wax biosynthesis. M. Ann D.N. Perera,Charles R. Dietrich, Patrick S. Schnable, Basil J. Nikolau Iowa State University, Ames, IA50011.

The aerial organs of plants are coated with a complex mixture of acyl-derived lipids, thecuticle. The cuticle is composed of the polymer, cutin, which is immersed and coated by aclass of lipids call the epicuticlar waxes. The biosynthetic origin of the epicuticular waxes isbeing elucidated via a combination of molecular and biochemical genetic approaches.Mutations in genes involved in the production of these waxes (glossy genes) affect theamount and/or compositions of the waxes present on leaf surfaces. A collection of over 150independently isolated maize glossy mutants has been genetically characterized by placingthe alleles into 26 complementation groups, nine of which define previously undefinedglossy loci. The reference mutant allele of each mutant locus has been backcrossed into aconstant genetic background (the maize inbred B73). The chemical compositions of theepicuticular waxes produced by these near-isogenic stocks are being compared to that ofthe wild-type B73 inbred. These chemical compositions are being determined via acombination of HPLC and GC-MS analyses and have revealed that the epicuticular waxes ofmaize seedling are more complex than previously reported. Furthermore, comparisons ofthe compositions between wild-type and mutant seedlings provide valuable clues as to thebiochemical function encoded by the glossy loci.

P11 Novel fatty-acid esters of p-coumaryl alcohol in epicuticular wax of apple fruitBruce D. Whitaker, Produce Quality & Safety Laboratory, and Walter F. Schmidt,Environmental Quality Laboratory, Beltsville Agricultural Research Center, AgriculturalResearch Service, USDA, 10300 Baltimore Avenue, Beltsville, MD 20705-2350

During a study of α-farnesene accumulation and oxidation in the skin of cold-stored applefruit, hexane extracts of epicuticular wax from 'Gala' apples were noted to have an unusual,broad absorbance maximum at about 258 nm, which led us to isolate and identify theprimary UV- absorbing compounds. Column and thin-layer chromatography steps yielded afraction that gave a series of paired, 260-nm-absorbing peaks on C18-HPLC. These wereshown to be a family of phenolic fatty-acid esters, for which retention times increased withincreasing fatty-acid chain length and paired peaks were esters of two related phenolics withthe same fatty-acid moiety. Alkaline hydrolysis of the esters released two water-solublephenolics separable by C18-HPLC. Electrospray MS gave a molecular mass of 150 for both,and proton-NMR plus UV absorbance spectra identified them as E and Z isomers of p-coumaryl alcohol (p-CA). Gradient HMQC-NMR of the HPLC-purified stearate ester of E-p-CA indicated that fatty-acid esterification occurs at the gamma-OH rather than at the 4-OHon the phenyl ring. To our knowledge, this is the first report of fatty-acid esters ofmonolignols as a natural plant product. The mechanism of fatty-acid esterification and thephysiological role of these compounds are presently unknown. They did, however, showmoderate antioxidant activity in two different assays, which suggests that they may bebeneficial in the human diet.

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P12 Characteristics of High αα-Linolenic Acid Accumulation in Seed OilsMohammed Abdel-Reheem, Resham Bhella, and David Hildebrand N-103 AGN Dept.Agronomy Lexington, KY 40546 USA.

In order to investigate the molecular basis of the high accumulation of the ω-3 fatty acid (α-linolenic acid, 18:3) in seed oil triglyceride (TAG) of certain plants, cotyledons of 5-6 differentdevelopmental stages were taken from three different 18:3 high accumulator plants, flax(Linum usitatissimum), dragonshead (Dracocephalum moldavica), and perilla (Perillafrutescens). In all high 18:3 accumulators, the linolenic acid content started at moderatelevels. The 18: 3 content was found to be higher at the very early cotyledon developmentalstage (first stage) of perilla (35%) compared with flax (25%) and dragonshead (15%). α-Linolenic acid gradually accumulated in developing cotyledons of all three plants. However,the 18:3 content was found to be higher at the late mature developmental stage ofdragonshead (68%) compared with perilla (59%) and flax (45%) cotyledons. The 18:3 and18:2 contents in both PC and TAG were determined during various stages of seeddevelopment for flax and perilla plants. Slightly higher 18:3 was present in PC of perilla seedin the first three stages of cotyledon development compared to the amount of 18:3 in PC offlax seeds. Increase of 18:3 content was observed in TAG of developing seeds in both flaxand perilla, however flax seeds showed gradual increase of 18:3 in TAG throughout allstages of development, on the other hand a dramatic increase of 18:3 in TAG of perillaseeds was observed during the later stages of development (from 15 mg/mg seed at thefourth stage up to 380 mg/mg seed at seed maturation). Northern bolt analysis data of threedifferent stages of dragonshead, flax, compared with moderately low 18:3 producers,soybean (Glycine max), Arabidopsis thaliana and Brassica napus, (8 - 10%) at mature stageof zygotic embryo development showed that ω-3 desaturase mRNA levels were higher in allthree high 18:3 producers, flax, dragonshead and perilla as might be expected. Thisindicates that the high level of α-linolenic acid in TAG may be largely controlled by the levelof ω-3-desaturase gene expression. However, the PC vs. TAG fatty acid composition datasuggested that ω-3-desaturase is not the only key factor in 18:3 accumulation in TAG, andthe high accumulators have a selective transfer of α-linolenic acid into TAG which in turnsuggested that DGAT or other acyltransferases may play a key role in 18:3 accumulation intriacylglycerols.

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P13 Expression of Yeast sn-2 Acyltransferase Improves Oil Content, Erucic Acid Contentand Seed Yield in Field-Tested Transgenic Rapeseed Vesna Katavic1, Jitao Zou2,Dennis L. Barton2, Jing An1, Winnie Friesen1, Yan Ge2, Kalie K. Gossen2, Michael E. Giblin2,Daryl Males1, Samuel L. MacKenzie2, David C. Taylor2 1 Saskatchewan Wheat PoolAgricultural Research and Development, 201-407 Downey Road, Saskatoon, SK S7N 4L8,Canada;

A major goal of our research is to produce, by genetic manipulation, Brassica napus L.cultivars with higher amounts of 22:1 in their seed oil than in present Canadian HEAcultivars developed through traditional breeding. Previously, we reported that transgenicexpression of a mutated yeast sn-2 acyltransferase (SLC1-1) in industrial rapeseed resultedin increased seed oil content, increased proportions of erucic acid and increased averageseed weight (Zou et al., (1997), Plant Cell. 9: 909-923.). Those results were reported onlyfor plants grown in a controlled greenhouse setting. Here we report a summary of theresults from two successive years of field trials with T4 and T5 generations of B. napus cvHero transformed with the SLC1-1 gene. These trials, conducted at Rosthern,Saskatchewan in two very different growing seasons, show that the SLC1-1 transgenicsclearly and consistently out-performed controls, exhibiting much improved 22:1 and oilcontent, as well as yield, under varying field conditions.

P14 Variation in seed oil content and fatty acid composition of Sesamum indicum L. andits wild relatives in Kenya B. A. Were1,2

, M. Lee3 and S. Stymne2 1Department of Botany,Moi University, P. O. Box 1125, Eldoret, Kenya; 2 Department of Crop Science, The SwedishUniversity of Agricultural Sciences, P.O. Box 44, S-23053 Alnarp, Sweden. 3ScandinavianBiotechnology Research AB, P.O. Box 166, S-23053 Alnarp, Sweden

Seeds from 29 market accessions of Sesamum indicum L. and 3 wild species of Sesamumwere analysed gas liquid chromatography to compare their fatty acid composition and oilcontent. The four species had similar fatty acid profiles with some variation in the amount ofindividual fatty acids. The observed ranges for the main fatty acids in the seed oil were 6.4-11.8% palmitic, 4.4-10.6% stearic, 33.8-41.0% oleic and 38.5-50.1% linoleic acids. Most ofthe seeds also contained up to 1% of palmitoleic, linolenic and C-20 acids. Seeds from thewild species had less oil (26.9-36.2%) than those from cultivated sesame (36.3-57.6%). Inabout one third of the collection, oil made up for at least 50% of the seed weight. From thisstudy, it is evident that there is sufficient variation in oil content among cultivars of Kenyansesame that could be useful for improvement of the crop by line selection and breeding. Butother methods like mutation breeding or genetic transformation need to be employed for thediversification of the fatty acid composition of sesame oil.

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P15 Evolution of lipoxygenase activity during storage of potato tubers Fauconnier M.-L.1,Rojas- Beltran J.2, Hoyaux P.1, Delcarte J.1, du Jardin P.2 and Marlier M.1 FacultéUniversitaire des Sciences Agronomiques, 1 Unité de Chimie Générale et Organique, 2 Unitéde Biologie Végétale 2, Passage des déportés, B-5030 Gembloux, Belgium

The lipoxygenase pathway is a cascade of enzymatic reactions that catalyses thetransformation of fatty acids into a wide range of compounds involved in essentialphysiological processes in plants. Lipase hydrolyses lipids furnishing free fatty acidsafterwards, lipoxygenase (E.C. 1.13.11.12) catalyses the addition of molecular oxygen onpolyunsaturated fatty acids containing a (Z)-1, (Z)-4-pentadiene structure, mainly linoleic andlinolenic acids in plants. Depending on botanical origin and on reaction conditions, variableamounts of 13 and or 9-hydroperoxides of fatty acids are formed by lipoxyganase. Thehydroperoxides can be transformed enzymatically or not in a variety of molecules (e.g.jasmonic acid, traumatic acid, green note aldehydes and alcohols, colneleic and colnelenicacids). The fatty acids hydroperoxides can also be reintegrated in the membranesdecreasing their flexibility. In potato tubers, the main lipoxygenase isoform is Lox-1 whichforms mainly 9-hydroperoxides. Lipoxygenase, a key enzyme in lipid peroxidation, has beenextensively studied but little is known about its implication during storage of potato tubers. Inour study, we particularly focussed on the determination of lipoxygenase activity duringstorage of potato tubers (Solanum tuberosum L) . cv. Bintje and Désirée in three storageconditions :

- 20 °C without anti-sprouting treatment- 20 °C with anti-sprouting treatment- 4 °C without anti-sprouting treatment

The potato tubers were stored during one year. Samples were taken regularly: each twoweeks during two months and each two months after. The lipoxygenase activity wasevaluated by different methods:

- by studying the substrate of the lipoxygenase (determination of the double boundindex by GC analysis of fatty acid)- by measuring lipoxygenase activity of crude extract (UV measurment)- by measuring gene expression (lox-1) (northern blot)- by measuring the content of product accumulated in situ (fatty acid hydroperoxide)

(colorimetric method)The study was completed by the determination of the proportion of 13 and 9-fatty acidhydroperoxides formed by the lipoxygenase (HPLC determination). To evaluate themembrane integrity of the potato tubers during the storage, electrolyte leakage wasmeasured on all the samples. Sugar content (glucose, fructose, sucrose) was alsodetermined (HPLC) becasues the increase sugar content can be considered as a Secondaryeffect of the loss of membrane integrity.

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P16 Early reactions in the lipoxygenase dependent degradation of storage lipids MartinaKoerner, Armin Schlereth, Ekkehart Berndt, Ivo Feussner Department of Molecular CellBiology, Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben,Corrensstr.3, Germany

The mobilization of storage lipid is an essential process during germination of oilseeds.Despite the physiological importance of lipid mobilization its mechanism is only partiallyunderstood. While the common hypothesis claims that the mobilization of storage lipid isinitiated by an unspecific TAG-lipase, previous data suggest a new alternative degradationmechanism that is dependent on a lipid body trilinoleate 13-lipoxygease (13-LOX). Thisenzyme is induced during germination and uses esterified fatty acids as substrates. Thelipoxygenase reaction leads to a transient accumulation of ester lipid hydroperoxides (13-HPODE) which are preferentially liberated from the glycerol backbone by specifictriacylglycerol lipases. The free hydroperoxy fatty acids are subsequently reduced to theirhydroxy derivates, which in turn may serve as substrates for �-oxidation. Here, we willpresent recent data on early enzymatic reactions, like degradation of the lipid bodymembranes which led to the availability of triacylglycerols as substrate for a lipid bodytrilinoleate 13-LOX and an involvement of proteases and phopholipases therein.

P17 The GPI-anchored purple acid phosphatase of Spirodela oligorrhiza and itspreferential localization in the cell wall Miwa Nishikooria, Akira Haseb, and HidetoshiOkuyamaa aLaboratory of Environmental Molecular Biology, Graduate School ofEnvironmental Earth Science, Hokkaido University, Kita-ku, Sapporo 060-0810, JapanbBiological Laboratory, Hakodate College, Hokkaido University of Education, Hachiman-cho,Hakodate 040-8567, Japan

The Spirodela oligorrhiza purple acid phosphatase (PAP) inducibly synthesized in plants (–Pplants) grown under phosphate-deficient conditions was proved to be aglycosylphosphatidylinositol (GPI)-anchored protein found in higher plants. One of the mostsignificant properties of the S. oligorrhiza GPI-anchored PAP is that more than 90% of itsactivity can be recovered in the soluble fraction, suggesting that PAP must have lost itsmembrane-anchoring lipid moiety probably by phospholipase cleavage. Immuno-histochemical results using the anti-S. oligorrhiza PAP antibody and –P plant rootsdemonstrate that PAP is preferentially distributed in the outer-most cortical cells of roots butnot in their epidermis, suggesting its role for acquiring inorganic phosphate from mediumunder phosphate-deficient conditions. PAP was released by washing the cell wall with 1.0 MNaCl and by digesting it with cellulase. The predicted amino acid sequence of the PAPcDNA showed the absence of a dibasic motif, which is regarded as a signal targeting GPI-anchored proteins to the cell wall in yeast, upstream of the putative GPI-anchoring site. Allthese results imply that the PAP is synthesized as a cell wall protein. Furthermore weconsider that GPI anchor itself may function as a signal for transporting PAP to the cell wallin S. oligorrhiza. Northern blot analysis using the PAP cDNA of S. oligorrhiza as a probedemonstrates that expression of the PAP gene increased during growth of –P plants andthis time-dependent occurrence in mRNA levels of the PAP in –P plants was observed alsoin their protein and activity levels.

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P18 MEASUREMENT OF IN VIVO PHOSPHOLIPASE ACTIVITY IN LEAF TISSUES. Mike Pollard, Troy Paddock and John Ohlrogge. Department of Botany and PlantPathology, Michigan State University, East Lansing, MI 48824.

By incubating [13C218O2]acetate with spinach leaf discs and analyzing the carbonyl 18O

content of fatty acids in different lipid classes we previously demonstrated that the export ofthe fatty acyl groups from the plastid occurred with a 50% loss of 18O content. Thus weconcluded that fatty acyl export occurs via a free carboxylate anion, that is, through ahydrolytic step. These experiments also show that acyl groups in phosphatidylcholine (PC)undergo deacylation-reacylation reactions through a hydrolytic mechanism. We areinvestigating this latter phenomenon using a variety of time course and pulse chaseexperiments with [13C2

18O2]acetate and [18O2]heptadec-9cis-enoic acid as substrates.These studies show that acyl groups in PC undergo hydrolytic deacylation-reacylationreactions at 5-10% per hour, with most if not all acyl groups in PC susceptible to thisturnover. The continuous duration of the turnover and other experiments suggest that thisturnover is basal and not a wound response. The turnover has been measured in spinachand pea leaves, is higher in younger leaves, and is seen with palmitoyl, oleoyl, linoleoyl andlinolenoyl groups in PC and PE. These hydrolytic acyl deacylation-reacylation rates havebeen measured for the first time in plant tissues, and their flux is at least equivalent to theflux of acyl groups entering PC from de novo fatty acid biosynthesis. Controls with[18O2]heptadec-9cis-enoic acid also suggest that in vivo there is no significant futile cycleinvolving acyl-CoA hydrolysis then acyl-CoA resynthesis.

P19 Fatty Acid Breakdown in Developing Embryos of Brassica napus L. Tansy Chia andSteve Rawsthorne Department of Metabolic Biology, John Innes Centre, Colney, Norwich,NR4 7UH England

Lipid in the form of triacylglycerol is a major component of oilseed storage reserves.Complete breakdown involves the process of lipolysis, β-oxidation, glyoxylate cycle andgluconeogenesis. Studies thus far have relied on detecting the presence of key enzymesassociated with these pathways such as malate synthase (MS) and isocitrate lyase (ICL)and expression of the genes that encode them. Studies of the glyoxylate cycle have shownthat lipid breakdown is essential to post-germinative growth [1]. The role of the glyoxylatecycle in providing carbon, energy and respiration substrates from fatty acid breakdown isalso suggested during senescence of green tissues [2], carbohydrate starvation [3] andembryo development [4]. The possibility that lipid catabolism may be occurring indeveloping embryos that are primarily concerned with lipid accumulation is investigated.Study of the activity and expression of key enzymes of lipid catabolic pathways duringembryo development was carried out. Significant levels of protein and activity of MS, ICL,phosphoenolpyruvate carboxykinase and the multifunctional protein were detected towardslate-staged embryo development. The integrated in vivo function of these enzymes wasinvestigated by feeding whole-isolated embryos with [1-14C] acetate as a carbon tracer. Thisshowed i) an increase in the relative (organic versus aqueous extraction phases) andabsolute fluxes of label into products present in the aqueous fraction as the embryomatured, ii) 60% recovery of aqueous phase as glucose and 15% as malate and iii)respiration of 50% of label. This result suggests an anaplerotic and gluconeogenic functionfor the glyoxylate cycle. When embryos were fed with [1-14C] decanoic acid, a more directcarbon tracer, newly labelled glucose and malate were detected. This confirms that lipidcatabolic pathways are functional and fully active at late-staged embryos.

1. Eastmond, P.J., Germain, V., Lange, P. R., Bryce, J. H., Smith, S.M. & Graham, I. A.(2000). PNAS 97 (10): 669-56742. Wanner, L., Keller, F. & Matile, Ph. (1991) Plant Science 78: 199-2063. Graham, I.A., Denby, K.J. & Leaver, C.J. (1994) The Plant Cell 6: 761-7724. Ettinger, E. F. & Harada, J.J. (1990). Arch Biochem Biophys 281: 139-143

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P20 ß-OXIDATION DURING GERMINATION AND SEEDLING DEVELOPMENT IN NON-GLUCONEOGENIC PLANTS: II. ACYL COA DEHYDROGENASE ACTIVITY INCOTYLEDONS FROM DEVELOPING PEA SEEDLINGS. Christine Masterson, AdrianBlackburn and Clifford Wood. Biological & Nutritional Sciences,University of Newcastle uponTyne, NE1 7RU, UK

Acyl CoA dehydrogenase, the first enzyme of mitochondrial ß-oxidation, was assayed in thecotyledons of Pisum sativum over a 14-day period from the commencement of imbibitionusing C4, C12 and C16 acyl CoA substrates. There was a post-imbibition surge of activity atday 1 with short- and mid-chain length substrates, whilst activity with the C16 substrate firstpeaked at days 3-4, coinciding with the onset of plumule unfurling and greening. Furtherpeaks were observed with all three substrates, coinciding with secondary root formation andleaf enlargement. As the activity peaks for the different chain-lengths did not transpose at allpoints and the ratios of the chain-length activities were not constant, it has to be concludedthat more than one acyl CoA dehydrogenase is present in pea cotyledon mitochondria.Variation in acyl CoA dehydrogenase activity with seedling development follows a similarpattern to that reported for overall mitochondrial ß-oxidation, suggesting that mitochondrialß-oxidation has a role to play in seedling development, perhaps providing acetyl CoA and/orATP for biosynthesis.

P21 BIOSYNTHESIS OF 3-ACETYL-1,2-DIACYL-sn-GLYCEROLS IN DEVELOPINGEUONYMUS ALATUS SEEDS. Anne Milcamps, Troy Paddock, Mike Pollard and John B.Ohlrogge. Department of Botany and Plant Pathology, Michigan State University, EastLansing, MI 48824.

The major neutral lipid from the seed oil of Euonymus alatus is 3-acetyl-1,2-long-chaindiacyl-sn-glycerol. This lipid accumulates over seed development at a rate of about 0.5µmoles/hr/gram fresh weight and is readily labeled by incubating developing seed tissuewith exogenous [14C]acetate. In such experiments [14Cacetyl] 3-acetyl-1,2-diacyl-sn-glycerolis highly labeled. [14C]Acetyl groups are not found associated with intermediates of the lipidbiosynthetic pathway, particularly phosphatidylcholine or 1,2-diacyl-sn-glycerol. Thisobservation, along with the fact that [14Cacetyl] labeling of 3-acetyl-1,2-diacyl-sn-glycerolexhibits no lag phase is consistent with biosynthesis via direct acetylation at the sn-3position of a 1,2-diacyl-sn-glycerol. Label from [14C]acetate is also found in long-chain fattyacyl groups esterified to triacylglycerol, 3-acetyl-1,2-diacyl-sn-glycerol, 1,2-diacyl-sn-glyceroland phospholipids. However, exogenous acetate is preferentially used in the presumablycytosolic sn-3 acetylation reaction rather than for plastidial fatty acid synthesis. Cell freeextracts from developing seeds will convert [14C]acetyl-CoA to [14Cacetyl] 3-acetyl-1,2-diacyl-sn-glycerol at moderate rates, but in these extracts there is a very high acetyl-CoAhydrolase activity. The evidence to date suggests that Euonymus alatus seed has DAGATactivity which can accommodate acetyl groups.

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P22 ON THE EXPORT OF FATTY ACIDS FROM THE CHLOROPLAST Abraham JK Koo, JohnOhlrogge and Mike Pollard Department of Plant Biology Michigan State University, East Lansing,MI 48824

Transport systems control the flux of metabolites between organelles, and are often highlyregulated. The current model for the export of fatty acids from plastid proposes that acyl-ACP hydrolysis occurs in the stroma by acyl-ACP thioesterases and that the free fatty acid(FFA) released is transferred to the outer envelope of the plastid where it is reactivated toacyl-CoA for utilization in cytosolic glycerolipid synthesis. However the mechanism todeliver nascent FFA from stroma to the acyl-CoA synthetase (ACS) remains unclear. The invivo FFA pool size was estimated using kinetic labeling experiments with spinach leaves.Our results indicate that the maximum residence time for FFA in the export pool is 1 secondor less. To better understand the export process, a series of [14C]fatty acid labeling assayswere designed using isolated intact chloroplasts. (1) There is a linear accumulation of FFA inthe presence of ATP alone, and when CoA is also added, there is also a linear accumulationof acyl-CoA thioesters (plus derived polar lipids). Since the product accumulation reaches itssteady state very rapidly, the FFA supplying pool should be filled rapidly, which also impliesa low Km for ACS. This could be explained by either fast diffusion rate of FFA within thechloroplast or by channeled delivery of FFA from the thioesterase reaction to ACS. (2) An insitu FFA pool was generated by incubating chloroplasts in the light with ATP but withoutCoA. On transfer to the dark with the addition of CoA the ACS reaction was very efficient,being at least 5-7 fold faster than the rate of de novo FAS. (4) This in situ FFA pool couldalso be rapidly removed from the chloroplast at rates comparable to ACS reaction rate byadding BSA into the reaction mixture. However, once bound to BSA, FFA was a muchpoorer substrate for the ACS reaction. (5) The BSA competed with ACS for pre-existing, insitu generated FFA substrate at ratio of BSA:ACS = 1:2.3 in the dark. However for the“nascent” FFA substrate produced in the light, BSA competed much less efficiently(BSA:ACS = 1:8). Possible mechanistic interpretations of the above observations include afacilitated export mechanism or channeling of “nascent” FFA from the plastid to the ACS.

P23 Cloning of Multiple Amplified DNA Hermann Schmidt DNA Cloning Service,Klaus-Nanne-Straße 48, 22457 Hamburg, Germany

In the last years different organismn have been sequenced and the genes of interest aremostly available. Cloning of genes behind different promotors, with different leader peptidesor with different markers is a laborious task. Often this part of the scientific work consumesmuch time due to the fact that existing expression cassettes don´t fit. We have optimisedcloning of multiple gene fragments by two methods. In both of them different DNA fragmentswill be amplified in the first step with primers containing the necessary restriction sites. Thenthe amplified DNA fragments will be connected by restriction and ligation or overlappingprimers. In the second step the ligated construct – consisting of up to 3 fragments – will beamplified with the outer primers. The full length construct is then restricted and cloned intothe vector of choice, and finally sequenced. With this methods we have cloned a largenumber of genes. We have opitimised this method for several years and recommend it onlyfor very experienced scientist. To make this technique available for all scientist we haveestablished a cloning service.

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P24 LIPID AND LIPID CONJUGATES PRESENT IN OLIVES J. Milosevic, M. Cocksedge, M.Zloh and W.A. Gibbons Department of Pharmaceutical and Biological Chemistry, TheSchool of Pharmacy, 29-39 Brunswick Square, London WC1N 1AN

Extracts from plant and fruit sources contain a rich and varied selection of lipids, of potentialimportance in medicine and food science (1, 2). In this context olive fruit, olive pulp and oliveskins were examined by gas chromatography-mass spectrometry (GC-MS), liquidchromatography-mass spectrometry (HPLC-MS) and by nuclear magnetic resonancespectroscopy (NMR), for the presence of lipids and lipoconjugates. Compounds found inolive pulp have previously been found in studies on whole olives [3] with the exception of 1,4dimethyl-3-ethyl-2 naptholglycoside not previously identified. The analysis of olive skinextracts showed the presence of 14 different fatty acids in the non-saponified sample andfive additional acids in the saponified sample. A comparison of the fatty acids present inolive skin with those present in olive pulp indicated that olive skin contains a more complexpattern of fatty acids both in the non-saponified and in the saponified samples. Thepresence of C (26:0) and C (28:0) acids in the saponified sample indicated the presence ofthese chains in the non-saponified conjugates. This is the first reported finding of suchsubstituent chains. The presence of 9-oxo-nonanoic acid is a novel finding and is of interestin understanding both the structure and role of the conjugate molecules present in olive skin.Conjugates such as the phospholipids, phosphatidylcholine (PC) andphosphatidylethanolamine (PE), di and triglycerides and sterols have been identified inwhole olives.

References:1. Farnsworth, N. R. The role of etnopharmacology in drug development; In: Bioactivecompounds from plants; Wiely, West Sussex, England, 3-13 (1990).

2. Harwood, L. J. Plant lipid metabolism; In: Plant Biochemistry, edited by Day, P. M. & Harborne, J. B.; Academic Press Ltd., London, 237-272 (1997).

3. Macheix, J. J., Fleuriet, A., Billot, J. Fruit Phenolics; In: Fruit Phenolics; CRC Press, Inc., Bocaraton, Florida, (1990).

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P25 A NOVEL MEMBER OF THE PATATIN-LIKE GENE FAMILY IN COWPEA. Matos A R.a,Diop N. N., d'Arcy-Lameta A.a, M.a, Zuily-Fodil Y.a,b, Pham-Thi A.T.a,b aLaboratoire deBiochimie et Physiologie de l'Adaptation Végétale, Université Paris 7 Denis Diderot, 2, PlaceJussieu, 75251 Paris cedex 05, France; bUMR 7632 CNRS

Galactolipids are found in plastid membranes and their hydrolysis is observed when plantsundergo stress conditions like drought, cold and pathogen attack. Recently we have cloneda patatin-like gene designated Vupat1 induced by drought stress in cowpea (Vignaunguiculata) leaves. The recombinant protein expressed in the baculovirus systemhydrolyses preferentially galactolipids. Vupat1 gene is composed of seven exons and seemsto be a low copy gene. In Arabidopsis thaliana there are nine patatin-like genes that can bedivided in two main groups accordingly to sequence homology. One of those groups iscomposed of five genes with 6 or 7 exons presenting homology to Vupat1 and patatin-likegenes from other plants studied so far (potato, tobacco, cucumber and latex). The secondgroup consists of four genes with a single intron. An exhaustive screening of all the ESTsequences published in sequence databases allowed us to identify a partial cDNA sequencefrom soybean hypocotyls presenting high homology to one member of the “single intron”group. Primers selected from the alignment of A. thaliana (accession number T48109) andsoybean (BE473424) sequences were used in RT-PCR reactions using as template cowpeaRNAs. A 452 bp RT-PCR product presenting high homology to soybean and A. thalianagenes and less homology to Vupat1 was obtained. This gene designated Vupat2 isexpressed mainly in cowpea roots and also in leaves and stems. Furthermore its expressionin response to water deficit in two cowpea cultivars (EPACE-1 tolerant and 1183 sensitive)seems to differ from that of Vupat1. The cloning of the full-length Vupat2 cDNA and theexpression of the recombinant protein is currently underway and should contribute topropose a possible in vivo role.

P26 MOLECULAR CLONING AND FUNCTIONAL ANALYSIS OF ARABIDOPSIS cDNA FORMITOCHONDRIAL FATTY ACID SYNTHASE Rie YASUNO, Hajime WADA; Dept. Biol.,Grad. Sch. Sci., Kyushu-Univ., Fukuoka 810-8560, Japan

In plant cells, fatty acids are mainly synthesized in plastids, but we recently foundthat fatty acids are also synthesized in mitochondria although the amount of fatty acids ismuch less than that of fatty acids synthesized in plastids. All of the enzymes involved in thebiosynthesis of fatty acids in plastids were identified and their biochemical properties havebeen well studied. By contrast, none of the enzymes involved in mitochondrial fatty acidsynthesis has been identified. In this study, we have isolated and characterized anArabidopsis cDNA encoding a mitochondrial 3-ketoacyl-acyl carrier protein synthase(mtKAS). KAS is one of the components of fatty acid synthase and catalyzes the condensingreaction in the fatty acid synthesis. DNA-sequence analysis of the mtKAS CDNA revealedan open reading frame predicting a protein of 461 amino acids with a molecular mass of49.4 kDa. Southern hybridization analysis suggested that mtKAS is encoded by a single-copy gene in Arabidopsis genome. Comparison of the deduced amino acid sequence ofmtKAS with those of KAS I and KAS II homologs of E. coli and plastids shows high degreeof sequence identity and the presence of a leader sequence presumably required for importinto mitochondria. mtKAS cDNA was shown to code for a protein, which has KAS activity,by its ability to complement a E. coli mutant (CY244) defective in KAS I and KAS IIactivities. mtKAS has a characteristic cysteine residue, which is well conserved amongpreviously identified KAS homologs as an active site. To determine the subcellularlocalization of mtKAS, a fusion protein in which a part of N-terminal guard region of mtKASwas fused to green fluorescent protein (GFP) was expressed in tobacco BY-2 cells and cellsof Vicia faba. The green fluorescence was detected only in mitochondria, suggesting that theN-terminal region of mtKAS contains a mitochondrial targeting signal and that mtKAS islocated in mitochondria.

37

LIST OF PARTICIPANTS

Mohammed Abdel-ReheemN-103 AGN Dept. AgronomyLexington, KY 40546Phone: 859-257-7319Fax: 859-257-7874mtabd@pop.uky.edu

John BroadwaterBrookhaven National LaboratoryBiology, Bldg. 463Upton, NY 11973Phone: 631-344-5360Fax: 631-344-3407broadwater@bnl.gov

Kirk E. AptMartek Biosciences Corp6480 Dobbin RdColumbia, MD 21045Phone: 410-740-0081Fax: 410-740-2985kirkapt@martekbio.com

Pierre BrounMendel Biotechnology21375 Cabot BoulevardHayward CA 94545Phone: 510-264-0280Fax: 510-264-0254pbroun@MendelBio.com

Frederic BeaudoinIACR-Long Asbton Research StationLong Ashton, BristolBS419AF, UKPhone: 00 44 (0) 1275 549218Fax: 00 44 (0) 1275 394281frederic.beaudoin@bbsrc.ac.uk

Anders CarlssonDepartment of Crop ScienceSwedish University of Agricultural SciencesP.O.Box 44, SE-230 53 Alnarp, SWEDENPhone: +46 40 415561Fax: +46 40 415519anders.carlsson@vv.slu.se

Frederic BeissonDepartment of Plant BiologyMichigan State Univ,East Lansing, MI 48824-1312Phone: 517-353-0611Fax: 517-353-1926beisson@pilot.msu.edu

Kent D. ChapmanDept Biol SciencesUniv. of North TexasDenton, TX 76203Phone: 940-565-2969Fax: 940-565-4136Chapman@unt.edu

Christoph BenningDept. of Biochemistry and Molecular BiologyMichigan State UniversityEast Lansing, MI 48824-1319Phone: (517) 355-1609Fax: (517)-353-9334

benning@pilot.msu.edu

Tansy ChiaDepartment of Metabolic BiologyJohn Innes CentreColney, Norwich.NR4 7UHEnglandPhone: 01603-450543Fax: 01603-450014Tansy.Chia@bbsrc.ac.uk

Brenda BlacklockDepartment of Chemistry and BiochemistryHughes HallMiami UniversityOxford, OH 45056Phone: 513-529-1641Fax: 513-529-5715

blacklb@muohio.edu

Gangamma M ChowriraDept. of Botany, University of BritishColumbia,6270 University Blvd.,Vancouver, B.C. V6T 1Z4, CanadaPhone: (604) 822-2370Fax: (604) 822 6089

sunita_chowrira@yahoo.com

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Sean CoughlanDuPont Ag products,E402/4251 Experimental StationWilmington DE 19880-0402Phone: 9302)-695-8328Fax: (302)-695-9149

sean.j.coughlan-1@usa.dupont.com

Ivo FeussnerInstitute for Plant Genetics and Crop PlantResearch (IPK)Corrensstraße 3D-06466 GaterslebenGermanyPhone: +49-39482-5-547Fax: +49-39482-5-548

feussner@ipk-gatersleben

John Cronan Jr.Department of Microbiology University ofIllinoisB103 Chemical and Life Sciences Laboratory601 S. Goodwin AveUrbana, IL 61801Phone: 217 333-7919Fax: 217 244-6697

j-cronan@life.uiuc.edu

JoAnne FillattiMonsanto, Calgene Campus1910 Fifth St.Davis, CA 95616Phone: 530-753-6313Fax: 530-792-2453

joAnne.fillatti@monsanto.com

Gert-Jan de BoerCarnegie Institution of WashingtonDepartment of Plant Biology260 Panama StreetStanford, Ca 94305Phone: (650) 3251521 ext 428Fax: (650) 3256857

geejay@andrew2.stanford.edu

Masako Fukuchi-Mizutani1-1-1 WakayamadaiShimamoto-cho, Mishima-gun, Osaka,Japan 618-0024Phone: 075-962-8807Fax: 075-962-8262

Masako_Mizutani@suntory.co.jp

Katayoon DeheshCalgene1920 Fifth StreetDavis, CA 95616Phone: 530.792-2279Fax: 530.792-2453

katie.dehesh@monsanto.com

Martin Fulda Institue of Biological Chemsitry Washington State University Pullman, WA 99164 Phone: 509- 335-2337 Fax: 509-335-7643 Fulda@wsu.edu

Marie-Laure FauconnierFaculté Universitaire des SciencesAgronomiques de Gembloux,Unité de Chimie Générale et OrganiquePassage des Déportés, 2B-5030 Gembloux, BelgiumPhone: 0032.81.62.22.92Fax: 0032.81.62.22.27

fauconnier.ml@fsagx.ac.be

Martijn GipmansBASF Plant Science GmbHCarl-Bosch-Str. 38BPS - A 03067056 LudwigshafenGermanyPhone: 0049-6216041330Fax: 0049-6216021156

martijn.gipmans@basf-ag.de

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Maria GustavssonDepartment of Biochemistrylund University,Box 117,Iund 221 00, SwedenPhone: +46-40-415544Fax: +46-46-2224116

maria.g@plantbio.lu.se

Okuyama HidetoshiLaboratory of Environmental MolecularBiology,Graduate School of Environmental EarthScience,Hokkaido University,Sapporo 060-0810, JapanPhone: 81-11-706-4523Fax: 81-11-706-2347

hoku@ees.hokudai.ac.jp

Miki Hagio Department of Biology, Graduate School of Sciences, Kyushu university, 4-2-1 Ropponmatsu, Fukuoka 810-8560, Japan Phone: 81-92-726-4761 Fax: 81-92-726-4761 mikircb@mbox.nc.kyushu-u.ac.jp

Tanya HookerDepartment of Botany, UBC3529-6270 University BoulevardVancouver, BC V6T 1Z4 CanadaPhone: (604)-822-2370Fax: (604)-822-6089

tshooker@mail.botany.ubc.ca

Jixiang HanDepartment of Chemistry and BiochemistryHughes LaboratoryMiami UniversityOxford, OH 45056Phone: 513-529-8267Fax: 513-529-5715

hanj1@muohio.edu

Hiroyuki ImaiDepartment of Biology, Konan University8-9-1 Okamoto, Higashinada-ku,Kobe 658-8501 JapanPhone: +81-78-4352513Fax: +81-78-4352539

imai@konan-u.ac.jp

John HarwoodSchool of Biosciences Cardiff UniversityPO Box 911Cardiff CF10 3US U.K.Phone: +44 (0)29 2087 4108Fax: +44 (0)29 2087 4116

Harwood@cardiffac.uk

Masami InabaNational Institute for Basic Biology,Myodaiji-cho, Okazaki 444-8585,JapanPhone: +81-564-55-7602Fax: +81-564-54-4866

Fmasami@nibb.ac.jp ax

Ingo HeilmannDepartment of Botany,North Carolina State University,Box 7612,Raleigh, NC 27695-7612phone: (919) 515-6043fax: (919) 515-3436

ingo_heilmann@ncsu.edu

Aya ItohS-208 Plant Biology Building Michigan StateUniversityEast Lansing, MI48824Phone: 517-355-5197fax: 517-353-9168

itoh@pilot.msu.edu

Ernst HeinzInstitute fir Allgemeine BotanikOhnhorststr 1822609 Hamburg, GermanyPhone: 49 40 42816 369Fax: 49 40 42816 254

eheinz@botanik.uni-hamburg.de

Jan JaworskiMaimi UniversityDepartment of Chemistry & Biochemistry211 Hugehs HallOxford Ohio 45056Phone : (513)529-2094FAX: (513)529-5715

jaworsjg@muohio.edu

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Abraham Jeong-Kyu Koo1451-C Spartan vlg.E.Lansing, MI, 48823Phone: 517-355-1258

koojeon1@msu.edu

Ljerka KunstDepartment of Botany,University of British Columbia,6270 University Blvd.,Vancouver, BC,V6T 1Z4, CanadaPhone: (604)822-2351Fax: (604)822-6089

kunst@interchange.ubc.ca

Vesna KatavicNRC/PBI110 Gymnasium Place,Saskatoon SK, S7N0W9, CanadaPhone: (306) 975-5273Fax: (306) 975-4839

vkatavic@pbi.nrc.ca

Kathy LardizabalCalgene Campus / Monsanto1920 Fifth Street Davis, CA 95616Phone: (530) 792-2295Fax: (530) 792-2453

kathy.lardizabal@monsanto.com

Doug KnippleCornell University,Entomology, NYSAES,Geneva, NY 14456 USAPhone: 315.787.2363Fax: 315.787.2326

dck2@cornell.edu

Maoyin Li1012 Fremont, Apt3Manhattan, KS 66502Phone: 785-323-0306

maoyin@ksu.edu

Martina KoernerInstitute for Plant Genetics and Crop PlantResearch (IPK)Corrensstraße 3D-06466 GaterslebenGermanyPhone: +49-39482-5-556Fax: +49-39482-5-548

koerner@ipk-gatersleben.de

Jonathan LightnerExilixis Plant Sciences16160 SW Upper Boones Ferry RdPortland, OR 9704216160Phone: 503-670-7702Fax: 503-670-7703

jlightner@exelixis.com

Jean KridlRenessen1920 5th StreetDavis, CA 95616Phone: 530-792-2307Fax: 530-792-2453

Jean.Kridl@renessen.com

J. Casey LippmeierMartek Biosciences6480 Dobbin Rd.Columbia, MD 21045Phone: 4107400081Fax: 4107402985

casey@martekbio.com

Jerry KunerOmegaTech, Inc.,4909 Nautilus Court NorthSuite 208Boulder, CO 80301Phone: (303) 381-6229Fax: (303) 381-8181

jkuner@omegadha.com

Alison LovegroveIACR-Long Ashton Research Station,Department of Agricultural Sciences,University of Bristol,Long Ashton, Bristol.BS41 9AF, U.K.Phone: +44(0)1275 392181Fax: +44(0)1275 549225

Alison.Lovegrove@BBSRC.ac.uk

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Zenon LysenkoThe Dow Chemical Co1702 BuildingMidland MI 48674-1702Phone: 517-636-3903Fax: 517-636-4019

zlysenko@DOW.COM

Ela MietkiewskaNational Research Council/Plant BiotechnologyInstitute110 Gymnasium PlaceSaskatoon, SKS7N 0W9Phone: (306) 975-5273fax: (306) 975-4839

Ela.Mietkiewska@nrc.ca

Christine MastersonBiological & Nuritional SciencesAgriculture Building Kings RoadNewcastle UniversityNewcastle Upon TyneNE1 &RU.UKPhone: +44(0) 191 222 7881Fax: +44 (0) 191 222 8684

masterson@ncl.ac.uk

Anne MilcampsMichigan State UniversityDepartment of Botany and Plant PathologyWilson Road, Plant Biology building, room 366East Lansing, MI 48824Phone: 517 353 9399

milcamps@msu.edu

Ana Rita MatosLaboratoire BPAVUniversité Paris 72 Place Jussieu, case 701975251 Paris cedex 05FrancePhone: Tel: +33 (0)144276065Fax: fax: +33 (0)144276068

matos@ccr.jussieu.fr

Rob MintoDepartment of Chemistry and BiochemistryHughes LaboratoryMiami UniversityOxford, OH 45056Phone: 513-529-8267Fax: 513-529-5715

mintore@muohio.edu

Per-Johan MeijerCarlsberg Research CenterGamle Carlsberg Vej 10DK-2500 VALBYDENMARKPhone: +45 3327 5398fax: +45 3327 4764

meijer@crc.dk

Martine MiquelSeed Biology, INRA Rte de St-Cyr,F-78026 Versailles CedexFrancePhone: 33 1 30 83 30 49Fax: 33 1 30 83 30 99

Martine.Miquel@versailles.inra.fr

Margareta MelanderPlant Science Sweden ABHerman-Ehles väg 2-4SE-268 31 SvalövSwedenPhone: +46 418 667072Fax: +46 418 667081

margareta.melander@plantscience.se

Volker MittendorfBASF Plant Science26 Davis DriveResearch Triangle ParkNorth Carolina 27709Phone: 919-5472345Fax: 919-5472423

mittenv@basf.com

Jim MetzOmegaTech, Inc.4909 Nautilus Court North, Suite 208Boulder, CO 80301Phone: (303) 381-6228Fax: (303) 381-8181

jmetz@omegadha.com

Junichiro MoriDepartment of Biology,Konan University, 8-9-1 Okamoto,Higashinada-ku, Kobe, 658-8501JapanPhone: +81-78-4352513Fax: +81-78-4352539

mn023009@center.konan-u.ac.jp

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Johnathan A. NapierCell BiologyIACR Long Ashton Research StationDepartment of Agricultural SciencesUniversity of BristolLong AshtonBristol BS41 9AF, UKPhone : 01275 549424Fax : 01275 549225

jon.napier@bbsrc.ac.uk

Ann Perera2254 MBBDepartment of Biochemistry Biophysics andMolecular BiologyIowa State UniversityAmes, IA 50011Phone: 515-294-0347 or 515-294-3177Fax: 515-294-0453

mperera@iastate.edu

Basil NikolauIowa State University,2210 Molecular Biology Building,Ames, IA 50011Phone: 515-294-9423Fax: 515-294-0453

dimmas@iastate.edu

Mike PollardDepartment of Botany and Plant PathologyPlant Biology Building, Room 366Michigan State UniversityEast Lansing, MI 48824-1312Phone: 517-355-5237Fax: 517-353-1926

pollard9@pilot.msu.edu

Ikuo NishidaDepartment of Biological SciencesGraduate School of ScienceThe Uniersity of TokyoHongo 7-3-1, BunkyokuTokyo 113-0033, JapanPhone: +81-3-5841-4476Fax: +81-3-3814-1728

nishida@biol.s.u-tokyo.ac.jp

Gary L. PowellBiological Sciences, Clemson University,Clemson, SC 29634-0326Phone: 864 656-3593Fax: 864 656-0435

glpwl@clemson.edu

Miwa NishikooriLaboratory of Environmental Molecular BiologyGraduate School of Environmental EarthScience,Hokkaido UniversitySapporo 060-0810, JapanPhone: 81-11-706-4523Fax: 81-11-706-2347

mnishi@ees.hokudai.ac.jp

Chunbo QinDepartment of Biochemistry104 Willard Hall,Kansas State University,Manhattan, KS 66506Phone: (785)532-6422Fax: (785)532-7278

cbqin@ksu.edu

Minke NoordermeerBio-organic chemistryPadualaan 83584 CH UtrechtThe NetherlandsPhone: +31 30 2533064Fax: +31 30 2540980

minke@boc.chem.uu.nl

Andreas RenzBASF Plant Science GmbHCarl-Bosch-Str. 38BPS - A 03067056 LudwigshafenGermanyPhone: 0049/6216079507Fax: 0049/6216021156

andreas.renz@basf-ag.de

John OhlroggeDepartment of Plant BiologyMichigan State Univ,East Lansing, MI 48824-1312Phone: 517-353-0611Fax: 517-353-1926

ohlrogge@pilot.msu.ed

Paul Roessler Dow Chemical Company5501 Oberlin DriveSan Diego, CA 92121Phone: 858-352-4430Fax: 858-952-4557

pgroessler@dow.com

43

Jeff RouschBASF Plant Science26 Davis DriveResearch Triangle Park, NC 27709-3528Phone: (919) 547-2334Fax: (919) 547-2423

rouschj@basf.com

Joerg SchwenderMichigan State University166 Plant BiologyEast Lansing, MI 48824Phone: 517 355 5237

schwend2@pilot.msu.edu

Sari RuuskaMichigan State UniversityDept. of Botany and Plant Pathology166 Plant Biology BuildingEast Lansing, MI 48824 USAPhone: (517) 355 5237Fax: (517) 353 1926

ruuska@pilot.msu.edu

Chloe SellwoodDepartment of Metabolic BiologyJohn Innes CentreColney, Norwich.NR4 7UHEnglandPhone: 01603-450543Fax: 01603-450014

chloe.sellwood@bbsrc.ac.uk

Lacey SamuelsDept. of Botany, UBC6270 University Blvd.Vancouver,BC Canada V6T 1Z4Phone: 604-822-5469Fax: 604-822-6089

lsamuels@pop.interchange.ubc.ca

John ShanklinBiology, Bldg 463Brookhaven National Laboratory50 Bell Ave.Upton, New York, 11973Phone: 631 344 3414Fax: 631 344 3407

shanklin@bnl.gov

Tom SavageCalgene1920 Fifth StreetDavis, CA 95616Phone 530-792-2246Fax: 530-792-2453

thomasJ.savage@monsanto.com

Bo Shen7300 NW 62 nd AveJohnston, IA 50131-1004Phone: 515 270 3442Fax: 515 254 2619

shenbo@phibred.com

Kathy SchmidDept. of Biological SciencesButler University4600 Sunset Ave.Indianapolis IN 46208Phone: (317) 940-9957

kschmid@butler.edu

Basil Shorrosh2540 East Drake Road,Fort Collins, CO 80525Phone: 970-482-8818Fax: 970-482-3870

basil_shorrosh@cargill.com

Hermann SchmidtDNA Cloning ServiceKlaus-Nanne-Str. 4822457 Hamburgphone: 0049-40-55 983 944fax: 0049-40-55 983 945

heschmi@attglobal.net

Mark SmithDepartment of BotanyUniversity of British Columbia6270 University BoulevardVancouverBC V6T 1Z4 CanadaPhone: 604 822 2370Fax: 604 822 6089

msmith@mail.botany.ubc.ca

44

Patrik StoltScanBi Ltd.Box 166SE-230 53 AlnarpSwedenPhone: +46 70 67 214 69Fax: +46 40 41 55 35

Patrik.Stolt@scanbi.se

Dale L ValCalgene LLC.1920 Fifth St.Davis, CA 95616Phone: 530 792 2137Fax: 530 792 2453

dale.l.val@monsanto.com

Dr. A. R. StuitjeDepartment of GeneticsVrije UniversiteitDe Boelelaan 10871081HV AmsterdamThe NetherlandsPhone: +31.20.4447138Fax:: +31.20.4447155

stuitje@bio.vu.nl

Brady VickUSDA-ARSP.O. Box 5677Fargo, ND 58105Phone: 701-239-1322Fax: 701-239-1346

brady_vick@ndsu.nodak.edu

Sten StymneScanBi Ltd.Box 166SE-230 53 AlnarpSwedenPhone: +46 70 67 214 70Fax: +46 40 41 55 35

Sten.Stymne@scanbi.se

Toni VoelkerMonsanto CompanyCalgene Campus1920 Fifth StreetDavis, CA 95616Phone: 5307922200

toni.voelker@monsanto.com

Satyam SubrahmanyamBrookhaven National Laboratory50 Bell Avenue Bldg 463Upton, NY 11973Phone: 6313445360Fax: 6313443407

satyam@bnl.gov

Penny von Wettstein-KnowlesDepartment of GeneticsInstitute of Molecular BiologyUniversity of CopenhagenOester Farimagsgade 2ADK-1353 Copenhagen KDenmarkPhone+45 35 32 21 80Fax: + 45 35 32 21 13

knowles@biobase.dk

Martin TruksaNational Research Council/Plant BiotechnologyInstitute110 Gymnasium Place,Saskatoon, SK S7N 0W9phone: (306) 975-5330fax: (306) 975-4839

Martin.Truksa@nrc.ca

Hajime WadaDepartment of Biology,Faculty of Sciences, Kyushu University,Ropponmatsu, Fukuoka 810-8560,JapanPhone: 81-92-726-4761Fax: 81-92-726-4761

wadarcb@mbox.nc.kyushu-u.ac.jp

Virginia UrsinMonsanto-Davis1920 Fifth StreetDavis, CA 95616Phone: (530) 792-2394Fax: (530) 792-2453

virginia.ursin@monsanto.com

Ruth WeltiDivision of BiologyAckert HallKansas State UniversityManhattan, KS 66506-4901Phone: 785-532-6241Fax: 785-532-6653

welti@ksu.edu

45

Beatrice WereDepartment of Crop Science,Swedish University of Agricultural Sciences,P.O. Box 44,23053 AlnarpPhone: 040-415592Fax: 040-415519

wbeatrice@hotmail.com

Thorsten ZankInstitut fuer Allgemeine BotanikPhysiology / AG Prof. Dr. ErnstHeinzUniversitaet HamburgOhnhorststrasse 1822609 HamburgGermanyPhone: +49 40 42816 367Fax: +49 40 42816 254

fb8a001@botanik.uni-hamburg.de

Bruce WhitakerUSDA, ARS,PQSL, Bldg. 002, Rm. 117,BARC-W, 10300 Baltimore Avenue,Beltsville, MD 20705-2350Phone: 301-504-6984Fax: 301-504-5107

WhitakeB@ba.ars.usda.gov

Clifford WoodBiological & Nutritional Sciences,Agriculture Building,Kings Road,Newcastle University,Newcastle upon Tyne,NE1 7RU,UKPhone: + 44 (0) 191 222 7881fax: +44 (0) 191 222 8684

clifford.wood@ncl.ac.uk

Hui Xiong1920 Fifth St.Davis, CA 95616Phone: (530) 753-6313Fax: (530) 792-2453

hui.xiong@monsanto.com

Rie YasunoDepartment of Biology,Graduate School of Sciences,Kyushu university,4-2-1 Ropponmatsu,Fukuoka 810-8560, JapanPhone: 81-92-726-4761Fax: 81-92-726-4761

yasuorcb@mbox.nc.kyushu-u.ac.jp

Jelena YorkThe School of Pharmacy, University of London,29/39 Brunswick Sq., London WC1 1AX;Great BritainPhone: 44-0207-2660509

Jelena@chemb.ulsop.ac.uk

46

Index of Authors

Abbott, A. G. P3 de Boer, Gert-Jan F2

Abdel-Reheem, Mohammed P12 Dehesh, Katayoon A3, G5

An, Jing P13 Delcarte J. P15

Austin-Brown, Shea P2 Dietrich, Charles R. P10

Banas, Antoni E4 Diop N. N P25

Barton, Dennis L. P13 Dittrich, Franziska C3

Beaudoin, Frédéric Speaker, P6 Domergue,Frederic C3

Benning,Christoph Speaker, D3 du Jardin P. P15

Berndt, Ekkehart P16 Edlin, Duncan. A.N. D5

Bhella, Resham P12 Esposito, J.P. B5

Biesiada,Homigol D2 Fauconnier M.-L. P15

Blackburn, Adrian P20 Feussner, Ivo G1, P16

Blacklock, Brenda P7 Franceschelli, Silvia D4

Borstel, Forschungsinstitut speaker Friesen, Winnie P13

Boss, Wendy F. F4 Fulda, Martine A4

Broadwater, John B4 Ge, Yan P13

Broun, Pierre C1 Ghanevati, Mahin P7

Browse, John C3, A4 Gibbons, Jr, W.J. B5

Byrne, James G5 Gibbons, W.A. P24

Cahoon, Edgar B G3 Giblin, Michael E. P13

Carlsson, Anders S. G2 Gombos, Zoltan E1, P4

Chapman, Kent D. P2 Gossen, Kalie K. P13

Chia, Tansy P19 Gross, Wolfgang F4

Chloe Sellwood, P1 Gustavsson, Maria H E4

Chowrira, Gangamma P8, Hagio, Miki E1, P4

Cocksedge, M. P24 Hall, Sarah E G3

Coughlan, Sean J. G3 Han, J. P9

Creelman, Robert A. C1 Hardesty, B.M. B5

Cronan, Jr., John E. A2 Harwood, John L. D5

Dahlqvist, Anders E4 Haseb, Akira P17, P18

Dallerac, Renaud B3 Hawkins, Deborah G5

d'Arcy-Lameta A P25 Heilmann, Ingo F4

47

Heinz,Ernst Speaker, C2, A4 Leps, Michael G1

Hildebrand, David P12 Lerchl, Jens C2

Holtorf, Hauke C2 Lewis, Mervyn J. Speaker

Hooker, Tanya C4 Li, Maoyin D2

Howe, Gregg A. F3 Lorigan, G.A. B5

Hoyaux P P15 Los, Dmitry A. D4

Huynh, Tu T. P2 Lovegrove, Alison P6

Ima, Hiroyuki E2 MacKenzie, Samuel L. P13

Inaba, Masami D4 Males, Daryl P13

Itoh, Aya F3 Maresca, Bruno D4

Itoh, Shigeru E1 Marlier M. Faculté P15

Iwaki, Masayo E1 Masterson, Christine F5, P20

Jaworski, Jan P7, P9 Matos A R. P25

Jeong, Seong Eun B3 Metz, Jim C3

Jk Koo, Abraham P22 Michaelson, Louise V. Speaker

Jones, H. D5 Mietkiewska, E. E5

Kachroo, Pradeep B1 Milcamps, Anne P21

Kanesaki, Yu D4 Milosevic, J. P24

Katavic, Vesna P13 Minto, R.E. B5

Kille, P. D5 Miquel, Martine E3

Kinney, ,Anthony J G3 Moon, Hangsik P8

Klessig, Dan B1 Mori, Junichro E2

Kneppers, T.J.A. E5 Morimoto, Yasuaki E2

Knipple, Douglas C. B3 Murata, Norio D4

Koerner, Martina P16 Nap, J.P. E5

Kovács, Lászlo E1 Napier, Johnathan A. Speaker, P6

Kuner, Jerry M. C3 Nikolau, Basil J. A1, P10

Kunst, Ljerka C4,C5 Nishida, Ikuo P5

P8 Nishikooria, Miwa P17,

Lardizabal, Kathryn G5 Nishiura, Hideki E2

Lassner, Michael C3 Noordermeer, Minke A. G4

Lee, M. P14 Ohlrogge, John B. D1, D3, P18, P21, P22

Leipelt, Martina Speaker Okuyama, Hidetoshi P17,

Lenman, Marit E4 Oliver, David J. A1

48

Paddock, Troy P18, P21 Sperling, Petra speaker

Parrish, J. P3 Ståhl, Ulf E4

Perera, Imara Y. F4 Stephan, Franke Speaker

Perera, M. Ann D.N. P10 Stuitje, A. R. E5

Pirtle, Robert M. P2 Stymne, Sten E4, G2, P14

Pollard, Mike P18, P21, P22 Subrahmanyam,Satyam B2

Powell, G.L. P3 Suzuki, Iwane D4

Qin, Chunbo F1 Szalontai, Balazs D4

Rajashekar, Channa B. D2 Tamura, Kentaro E2

Rawsthorne, S. P1, P19 Taylor, David C. P13

Reski, Ralf C2 Ternes, Philipp speaker

Richards, J. D5 Thomæus, Stefan G2

Riechmann, Jose Luis C1 Udo Conrad G1

Ripp, Kevin G G3 van der Linden, K. H. E5

Roelofs, Wendell L. B3 Várkonyi, Zsuzsanna E1

Roessler, Paul C3 Veldink, Gerrit A. G4

Rojas- Beltran J P15 Verbree, E. C. E5

Ronne, Hans E4 Vliegenthart, Johannes G4

Rosenfield, Claire-Lise B3 Wada, Hajime E1, P26, P4

Ruuska, Sari D3 Wang, Cunxi D2,F1

Sakurai, Isamu P4 Wang, Xuemin F1

Samuels, Lacey C5 Warnecke, Dirk Speaker

Sandager, Line E4 Welti, Ruth D2

Sang, Yongming D2 Were, B. A. P14

Sayanova, Olga Speaker Whitaker Bruce D. P11

Schlereth, Armin P16 Whittle,Edward B1, B4

Schloemer, A.E. B5 Wiberg, Eva E4

Schmidt, Hermann P23 Wicker-Thomas, Claude B3

Schmidt, Walter F. P11 Wilkinson, M.D. D5

Schnable, Patrick S. P10 Williams, M. D5

Schwender, Joerg D1 Williams,Todd D. D2

Shah, Jyoti B1 Wood, Clifford F5, P20

Shanklin, John B1, B2, B4 Wurtele, Eve Syrkin A1

Slabas, A. R. P1 Yasuno, Rie P26

Smith, Mark P8, You, Kyung Man B3

Somerville, Chris F2 Zähringer, Ulrich speaker

49

Zank, Thorsten K. C2

Zhou, Han-E D2

Zloh, M. P24

Zou, Jitao P13

zur Nieden, Uta G1