372 results on '"Feussner, I."'
Search Results
352. Creating lipoxygenases with new positional specificities by site-directed mutagenesis.
- Author
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Hornung E, Rosahl S, Kühn H, and Feussner I
- Subjects
- Amino Acid Substitution, Binding Sites, Kinetics, Lipoxygenase genetics, Mutagenesis, Site-Directed, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Glycine max enzymology, Substrate Specificity, Lipoxygenase chemistry, Lipoxygenase metabolism, Plants enzymology
- Abstract
In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a gamma-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid heterogeneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngräber determinants) [3].
- Published
- 2000
353. Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh.
- Author
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Framm JJ, Peterson A, Thoeringer C, Pangert A, Hornung E, Feussner I, Luckner M, and Lindemann P
- Subjects
- Amino Acid Sequence, Blotting, Northern, Cardenolides metabolism, Cloning, Molecular, DNA, Complementary chemistry, Digitalis enzymology, Gene Expression Regulation, Enzymologic, Glucosidases metabolism, Molecular Sequence Data, RNA, Messenger genetics, RNA, Messenger metabolism, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Tissue Distribution, DNA, Complementary genetics, Digitalis genetics, Escherichia coli genetics, Glucosidases genetics, Plant Proteins, Plants, Medicinal, Plants, Toxic
- Abstract
A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.
- Published
- 2000
- Full Text
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354. Oxygenation of (3Z)-alkenals to 4-hydroxy-(2E)-alkenals in plant extracts: a nonenzymatic process.
- Author
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Noordermeer MA, Feussner I, Kolbe A, Veldink GA, and Vliegenthart JF
- Subjects
- Aldehydes chemistry, Cell Extracts, Fatty Acids, Monounsaturated metabolism, Gas Chromatography-Mass Spectrometry, Hydrogen Peroxide metabolism, Hydrogen-Ion Concentration, Linoleic Acids metabolism, Linolenic Acids metabolism, Lipid Peroxides metabolism, Lipoxygenase metabolism, Oxidation-Reduction, Plant Proteins metabolism, Recombinant Proteins metabolism, Glycine max enzymology, Aldehydes metabolism, Glycine max metabolism
- Abstract
There is large interest in 4-hydroxy-(2E)-alkenals because of their cytotoxicity in mammals. However, the biosynthetic pathway for these compounds has not been elucidated yet. In plants, 4-hydroxy-(2E)-alkenals were supposed to be derived by the subsequent actions of lipoxygenase and a peroxygenase on (3Z)-alkenals. The presence of 9-hydroxy-12-oxo-(10E)-dodecenoic acid (9-hydroxy-traumatin) in incubations of 12-oxo-(9Z)-dodecenoic acid (traumatin) in the absence of lipoxygenase or peroxygenase, has prompted us to reinvestigate its mode of formation. We show here that in vitro 9-hydroxy-traumatin, 4-hydroxy-(2E)-hexenal and 4-hydroxy-(2E)-nonenal, are formed in a nonenzymatic process. Furthermore, a novel product derived from traumatin was observed and identified as 11-hydroxy-12-oxo-(9Z)-dodecenoic acid. The results obtained here strongly suggest that the 4-hydroxy-(2E)-alkenals, observed in crude extracts of plants, are mainly due to autoxidation of (3Z)-hexenal, (3Z)-nonenal and traumatin. This may have implications for the in vivo existence and previously proposed physiological significance of these products in plants., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
355. Fatty acid 9- and 13-hydroperoxide lyases from cucumber.
- Author
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Matsui K, Ujita C, Fujimoto S, Wilkinson J, Hiatt B, Knauf V, Kajiwara T, and Feussner I
- Subjects
- Aldehyde-Lyases chemistry, Aldehyde-Lyases genetics, Aldehyde-Lyases isolation & purification, Aldehydes metabolism, Chromatography, High Pressure Liquid, Frameshift Mutation genetics, Hydro-Lyases, Hydrogen Peroxide metabolism, Hypocotyl enzymology, Intramolecular Oxidoreductases chemistry, Intramolecular Oxidoreductases metabolism, Kinetics, Molecular Sequence Data, Molecular Weight, Phylogeny, Pseudogenes genetics, RNA, Messenger analysis, RNA, Messenger genetics, RNA, Plant analysis, RNA, Plant genetics, Recombinant Proteins metabolism, Substrate Specificity, Aldehyde-Lyases metabolism, Cucumis sativus enzymology, Cytochrome P-450 Enzyme System
- Abstract
Fatty acid hydroperoxide lyase (HPL) is a novel P-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction-based cloning strategy, we isolated two HPL-related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13-hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9- and 13-hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS.
- Published
- 2000
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356. Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies.
- Author
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Hause B, Weichert H, Höhne M, Kindl H, and Feussner I
- Subjects
- Aldehydes metabolism, Blotting, Western, Chromatography, High Pressure Liquid, Cucumis sativus genetics, Cucumis sativus growth & development, Cyclopentanes metabolism, Cytoplasmic Granules enzymology, Gas Chromatography-Mass Spectrometry, Immunohistochemistry, Linoleic Acids metabolism, Lipids, Lipoxygenase genetics, Oxylipins, Plant Leaves chemistry, Plant Leaves enzymology, Plants, Genetically Modified, Seeds enzymology, Nicotiana genetics, Cucumis sativus enzymology, Lipoxygenase metabolism, Plants, Toxic, Nicotiana enzymology
- Abstract
A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid.
- Published
- 2000
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357. Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development.
- Author
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Maucher H, Hause B, Feussner I, Ziegler J, and Wasternack C
- Subjects
- Amino Acid Sequence, Blotting, Northern, Blotting, Southern, Cloning, Molecular, DNA, Complementary genetics, Hordeum enzymology, Hordeum growth & development, Immunoblotting, In Situ Hybridization, Intramolecular Oxidoreductases metabolism, Molecular Sequence Data, Plant Development, Plant Proteins metabolism, Plants enzymology, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Gene Expression Regulation, Plant, Hordeum genetics, Intramolecular Oxidoreductases genetics, Plant Proteins genetics, Plants genetics
- Abstract
Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full-length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co-purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX-derived 9- as well as 13-hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na-salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up-regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.
- Published
- 2000
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358. Metabolic profiling of oxylipins upon salicylate treatment in barley leaves--preferential induction of the reductase pathway by salicylate(1).
- Author
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Weichert H, Stenzel I, Berndt E, Wasternack C, and Feussner I
- Subjects
- Enzyme Induction, Hordeum enzymology, Hordeum metabolism, Lipoxygenase metabolism, Peroxiredoxins, Plant Leaves drug effects, Plant Leaves enzymology, Plant Leaves metabolism, Hordeum drug effects, Linoleic Acids metabolism, Lipoxygenase biosynthesis, Peroxidases biosynthesis, Salicylates pharmacology
- Abstract
In barley leaves, 13-lipoxygenases (13-LOXs) are induced by salicylate (SA) and jasmonate. Here, we show by metabolic profiling that upon SA treatment, free linolenic acid and linoleic acid accumulate in a 10:1 ratio reflecting their relative occurrence in leaf tissues. Furthermore, 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway leading mainly to the accumulation of (13S,9Z,11E,15Z)-13-hydroxy-9, 11,15-octadecatrienoic acid (13-HOT). Under these conditions, no accumulation of other products of the LOX pathway has been found. Moreover, exogenously applied 13-HOT led to PR1b expression suggesting for the time a role of hydroxy polyenoic fatty acid derivatives in plant defense reactions.
- Published
- 1999
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359. Isolation and characterization of a calendic acid producing (8,11)-linoleoyl desaturase.
- Author
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Fritsche K, Hornung E, Peitzsch N, Renz A, and Feussner I
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Cloning, Molecular, DNA, Complementary metabolism, Fatty Acid Desaturases biosynthesis, Linoleoyl-CoA Desaturase, Molecular Sequence Data, Phylogeny, Polymerase Chain Reaction, Saccharomyces cerevisiae metabolism, Sequence Homology, Amino Acid, Calendula enzymology, Fatty Acid Desaturases genetics, Fatty Acid Desaturases isolation & purification, Fatty Acids analysis, Plants, Medicinal
- Abstract
For the biosynthesis of calendic acid a (8,11)-linoleoyl desaturase activity has been proposed. To isolate this desaturase, PCR-based cloning was used. The open reading frame of the isolated full-length cDNA is a 1131 bp sequence encoding a protein of 377 amino acids. For functional identification the cDNA was expressed in Saccharomyces cerevisiae, and formation of calendic acid was analyzed by RP-HPLC. The expression of the heterologous enzyme resulted in a significant amount of calendic acid presumably esterified within phospholipids. The results presented here identify a gene encoding a new type of (1,4)-acyl lipid desaturase.
- Published
- 1999
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360. Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis.
- Author
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Hornung E, Walther M, Kühn H, and Feussner I
- Subjects
- Amino Acid Substitution, Arabidopsis enzymology, Binding Sites, Computer Simulation, Cucumis sativus genetics, Histidine, Kinetics, Lipoxygenase genetics, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Solanum tuberosum enzymology, Substrate Specificity, Valine, Cucumis sativus enzymology, Lipoxygenase chemistry, Lipoxygenase metabolism
- Abstract
Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
- Published
- 1999
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361. Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment.
- Author
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Kohlmann M, Bachmann A, Weichert H, Kolbe A, Balkenhohl T, Wasternack C, and Feussner I
- Subjects
- Aldehydes analysis, Gas Chromatography-Mass Spectrometry, Hordeum metabolism, Lipoxygenase isolation & purification, Oxylipins, Plant Leaves drug effects, Plant Leaves metabolism, Acetates pharmacology, Aldehyde-Lyases metabolism, Aldehydes metabolism, Cyclopentanes pharmacology, Cytochrome P-450 Enzyme System, Hordeum drug effects, Lipoxygenase metabolism, Plant Growth Regulators pharmacology
- Abstract
In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment > 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)- plus (2E)-hexenal were identified as 2,4-dinitro-phenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol.g-1 fresh weight to 45 nmol.g-1 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol.g-1 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol.g-1 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment.
- Published
- 1999
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362. Cloning and expression of a new cDNA from monocotyledonous plants coding for a diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from barley (Hordeum vulgare).
- Author
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Churin J, Hause B, Feussner I, Maucher HP, Feussner K, Börner T, and Wasternack C
- Subjects
- Acid Anhydride Hydrolases metabolism, Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA, Complementary, Hordeum genetics, Humans, Molecular Sequence Data, Sequence Homology, Amino Acid, Acid Anhydride Hydrolases genetics, Hordeum enzymology
- Abstract
From a cDNA library generated from mRNA of white leaf tissues of the ribosome-deficient mutant 'albostrians' of barley (Hordeum vulgare cv. Haisa) a cDNA was isolated carrying 54.2% identity to a recently published cDNA which codes for the diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) hydrolase of Lupinus angustifolius (Maksel et al. (1998) Biochem. J. 329, 313-319), and 69% identity to four partial peptide sequences of Ap4A hydrolase of tomato. Overexpression in Escherichia coli revealed a protein of about 19 kDa, which exhibited Ap4A hydrolase activity and cross-reactivity with an antibody raised against a purified tomato Ap4A hydrolase (Feussner et al. (1996) Z. Naturforsch. 51c, 477-486). Expression studies showed an mRNA accumulation in all organs of a barley seedling. Possible functions of Ap4A hydrolase in plants will be discussed.
- Published
- 1998
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363. All three acyl moieties of trilinolein are efficiently oxygenated by recombinant His-tagged lipid body lipoxygenase in vitro.
- Author
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Feussner I, Bachmann A, Höhne M, and Kindl H
- Subjects
- Chromatography, Affinity, DNA, Complementary, Hydrogen-Ion Concentration, Lipoxygenase chemistry, Lipoxygenase genetics, Recombinant Proteins metabolism, Glycine max enzymology, Substrate Specificity, Histidine chemistry, Lipoxygenase metabolism, Oxygen metabolism, Triglycerides metabolism
- Abstract
Recently, we found a 13-lipoxygenase in germinating cucumber cotyledons, which was located at the lipid body membrane. Based on its products formed mobilization of storage lipids seems to be initiated by this 13-lipoxygenase. For biochemical characterization its cDNA was expressed as His-tagged protein. Active recombinant enzyme was obtained from low temperature cultivation of E. coli after affinity purification. It (i) exhibited an unchanged region specificity, and (ii) showed a pH optimum of 7.2 against trilinolein as substrate. We compared its ability to oxygenate trilinolein with the one of another 13-lipoxygenase, soybean lipoxygenase-1. At the pH optimum of soybean lipoxygenase-1 (9.0), trilinolein was oxygenated only to 28% of the amount converted by the lipid body lipoxygenase. Moreover, trilinolein oxygenation by soybean lipoxygenase-1 leads mainly to monohydroperoxy derivatives, whereas oxygenation by lipid body LOX leads to a trihydroperoxy derivative.
- Published
- 1998
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364. Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves.
- Author
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Vörös K, Feussner I, Kühn H, Lee J, Graner A, Löbler M, Parthier B, and Wasternack C
- Subjects
- Amino Acid Sequence, Base Sequence, Chromosome Mapping, DNA, Complementary isolation & purification, Gene Expression Regulation, Plant drug effects, Lipoxygenase drug effects, Lipoxygenase metabolism, Molecular Sequence Data, Oxylipins, Plant Growth Regulators pharmacology, Plant Leaves enzymology, Sequence Analysis, DNA, Sorbitol pharmacology, Acetates pharmacology, Cyclopentanes pharmacology, Hordeum enzymology, Lipoxygenase genetics
- Abstract
We found three methyl jasmonate-induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX-92, -98 and -100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7, 949-957]. At least two of them (LOX-92 and LOX-100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX-100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2-type lipoxygenase and was therefore designated LOX2:Hv:1. The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13-LOX and arachidonate 15-LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2:Hv:1, suggesting a separate signalling pathway compared with other jasmonate-induced proteins of barley. The properties of LOX2:Hv:1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.
- Published
- 1998
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365. Lipoxygenase-2 oxygenates storage lipids in embryos of germinating barley.
- Author
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Holtman WL, Vredenbregt-Heistek JC, Schmitt NF, and Feussner I
- Subjects
- Chromatography, High Pressure Liquid, Isoenzymes isolation & purification, Isoenzymes metabolism, Kinetics, Lipids chemistry, Lipoxygenase isolation & purification, Substrate Specificity, Germination physiology, Hordeum physiology, Lipid Metabolism, Lipoxygenase metabolism
- Abstract
Besides the pre-existing lipoxygenase (LOX-1) present in quiescent grains, a new lipoxygenase (LOX-2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. & Douma, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 form different products after incubation with linoleic acid, the (9S)- and (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1991) FEBS Lett. 280, 159-162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. & Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-1 oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. Km values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2-8-fold for LOX-2). Interestingly, LOX-1 showed significantly higher Km values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di- and tri-linoleoylglycerol; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-1, with a corresponding larger extent of oxygenation. In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for beta-oxidation and therefore as a carbon source for the growing barley embryo.
- Published
- 1997
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366. Structural elucidation of oxygenated storage lipids in cucumber cotyledons. Implication of lipid body lipoxygenase in lipid mobilization during germination.
- Author
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Feussner I, Balkenhohl TJ, Porzel A, Kühn H, and Wasternack C
- Subjects
- Chromatography, High Pressure Liquid, Cucumis sativus enzymology, Cucumis sativus growth & development, Helianthus metabolism, Linoleic Acid, Linoleic Acids metabolism, Lipid Mobilization, Lipid Peroxides metabolism, Seeds metabolism, Triglycerides metabolism, Cucumis sativus metabolism, Lipid Metabolism, Lipoxygenase metabolism
- Abstract
At early stages of germination, a special lipoxygenase is expressed in cotyledons of cucumber and several other plants. This enzyme is localized at the lipid storage organelles and oxygenates their storage triacylglycerols. We have isolated this lipid body lipoxygenase from cucumber seedlings and found that it is capable of oxygenating in vitro di- and trilinolein to the corresponding mono-, di-, and trihydroperoxy derivatives. To investigate the in vivo activity of this enzyme during germination, lipid bodies were isolated from cucumber seedlings at different stages of germination, and the triacylglycerols were analyzed for oxygenated derivatives by a combination of high pressure liquid chromatography, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy. We identified as major oxygenation products triacylglycerols that contained one, two, or three 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid residues. During germination, the amount of oxygenated lipids increased strongly, reaching a maximum after 72 h and declining afterward. The highly specific pattern of hydroperoxy lipids formed suggested the involvement of the lipid body lipoxygenase in their biosynthesis. These data suggest that this lipoxygenase may play an important role during the germination process of cucumber and other plants and support our previous hypothesis that the specific oxygenation of the storage lipids may initiate their mobilization as a carbon and energy source for the growing seedling.
- Published
- 1997
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367. Isolation of a cDNA coding for an ubiquitin-conjugating enzyme UBC1 of tomato--the first stress-induced UBC of higher plants.
- Author
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Feussner K, Feussner I, Leopold I, and Wasternack C
- Subjects
- Amino Acid Sequence, Base Sequence, DNA, Complementary chemistry, Enzyme Induction drug effects, Genes, Plant, Ligases biosynthesis, Ligases chemistry, Solanum lycopersicum chemistry, Molecular Sequence Data, Multigene Family, Plant Proteins biosynthesis, Plant Proteins chemistry, RNA, Messenger drug effects, RNA, Messenger metabolism, Sequence Homology, Amino Acid, Cadmium pharmacology, DNA, Complementary isolation & purification, Hot Temperature, Ligases genetics, Solanum lycopersicum enzymology, Solanum lycopersicum genetics, Plant Proteins genetics, Ubiquitin-Conjugating Enzymes
- Abstract
A clone of an ubiquitin-conjugating enzyme (UBC) was isolated from a lambda-ZAP-cDNA library, generated from mRNA of tomato (Lycopersicon esculentum) cells grown in suspension for 3 days. The open reading frame called LeUBC1, encodes for a polypeptide with a predicted molecular mass of 21.37 kDa, which was confirmed by bacterial overexpression and SDS-PAGE. Database searches with LeUBC1 showed highest sequence similarities to UBC1 of bovine and yeast. By Southern blot analysis LeUBC1 was identified as a member of a small E2 subfamily of tomato, presumably consisting of at least two members. As revealed by Northern blot analysis LeUBC1 is constitutively expressed in an exponentially growing tomato cell culture. In response to heat shock an increase in LeUBC1-mRNA was detectable. A strong accumulation of the LeUBC1-transcript was observed by exposure to heavy metal stress which was performed by treatment with cadmium chloride (CdCl2). The cellular uptake of cadmium was controlled via ICP-MS measurements. The data suggest that like in yeast, in plants a certain subfamily of UBC is specifically involved in the proteolytic degradation of abnormal proteins as result of stress.
- Published
- 1997
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368. Differential Induction of Lipoxygenase Isoforms in Wheat upon Treatment with Rust Fungus Elicitor, Chitin Oligosaccharides, Chitosan, and Methyl Jasmonate.
- Author
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Bohland C, Balkenhohl T, Loers G, Feussner I, and Grambow HJ
- Abstract
A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.C. 1.13.11.12) in wheat (Triticum aestivum) leaves. Immunoblot analysis using anti-LOX antibodies revealed the induction of 92- and 103-kD LOX species after Pgt elicitor treatment. In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants. The effects of chitin oligomers and chitosan resembled those caused by MJ. In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action. LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions. It is most active at pH 5.5 to 6.0, and product formation from linoleic and [alpha]-linolenic acid is clearly in favor of the 9-LOOHs. It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves.
- Published
- 1997
- Full Text
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369. Do specific linoleate 13-lipoxygenases initiate beta-oxidation?
- Author
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Feussner I, Kühn H, and Wasternack C
- Subjects
- Cucumis sativus enzymology, Cucumis sativus metabolism, Lipid Bilayers, Oxidation-Reduction, Lipoxygenase metabolism
- Abstract
The germination process of oilseed plants is characterized by a mobilization of the storage lipids which constitute the major carbon source for the growing seedling. Despite the physiological importance of the lipid mobilization, the mechanism of this process is not well understood. Recently, it was found that a specific linoleate 13-lipoxygenase is induced during the stage of lipid mobilization in various oilseed plants and that this enzyme is translocated to the membranes of the lipid storage organelles, the so called lipid bodies. Lipoxygenase expression was paralleled by the occurrence of enantiospecific hydro(pero)xy polyenoic fatty acid derivatives in the storage lipids suggesting the in vivo action of the enzyme. Furthermore, it was reported that oxygenated polyenoic fatty acids, in particular as 13(S)-hydro(pero)xy-9(Z),11(E)-octadecanoic acid [13(S)-H(P)ODE], are cleaved preferentially from the storage lipids when compared with their non-oxygenated linoleate residues. These findings may suggest that 13(S)-H(P)ODE may constitute the endogenous substrate for beta-oxidation during lipid mobilization of oilseed plants.
- Published
- 1997
- Full Text
- View/download PDF
370. Lipoxygenase-catalyzed oxygenation of storage lipids is implicated in lipid mobilization during germination.
- Author
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Feussner I, Wasternack C, Kindl H, and Kühn H
- Abstract
The etiolated germination process of oilseed plants is characterized by the mobilization of storage lipids, which serve as a major carbon source for the seedling. We found that during early stages of germination in cucumber, a lipoxygenase (linoleate: oxygen oxidoreductase, EC 1.13.11.12) form is induced that is capable of oxygenating the esterified fatty acids located in the lipid-storage organelles, the so-called lipid bodies. Large amounts of esterified (13S)-hydroxy-(9Z,11E)-octadecadienoic acid were detected in the lipid bodies, whereas only traces of other oxygenated fatty acid isomers were found. This specific product pattern confirms the in vivo action of this lipoxygenase form during germination. Lipid fractionation studies of lipid bodies indicated the presence of lipoxygenase products both in the storage triacylglycerols and, to a higher extent, in the phospholipids surrounding the lipid stores as a monolayer. The degree of oxygenation of the storage lipids increased drastically during the time course of germination. We show that oxygenated fatty acids are preferentially cleaved from the lipid bodies and are subsequently released into the cytoplasm. We suggest that they may serve as substrate for beta-oxidation. These data suggest that during the etiolated germination, a lipoxygenase initiates the mobilization of storage lipids. The possible mechanisms of this implication are discussed.
- Published
- 1995
- Full Text
- View/download PDF
371. The lipid body lipoxygenase from cucumber seedlings exhibits unusual reaction specificity.
- Author
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Feussner I and Kühn H
- Subjects
- Binding Sites, Lipid Metabolism, Lipids chemistry, Lipoxygenase chemistry, Substrate Specificity, Cucumis sativus enzymology, Lipoxygenase metabolism
- Abstract
The lipid body lipoxygenase of cucumber seedlings is at a high level expressed during the germinating process which is the stage of triglyceride mobilisation. This enzyme exhibits an unusual positional specificity which has not been described so far for any plant and animal lipoxygenase. The purified enzyme converts arachidonic acid to 15-S-hydroperoxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid (15S-HPETE), 12-S-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid (12S-HPETE), and 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) in a ratio of 76:4:20 with the corresponding R-isomers being only minor contaminants. Binding to the lipid bodies enhances the arachidonic acid dioxygenase activity more than 4-times and alters positional specificity of the enzyme in favour of 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) formation.
- Published
- 1995
- Full Text
- View/download PDF
372. A lipoxygenase is the main lipid body protein in cucumber and soybean cotyledons during the stage of triglyceride mobilization.
- Author
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Feussner I and Kindl H
- Subjects
- Amino Acid Sequence, Lipoxygenase chemistry, Molecular Sequence Data, Plants embryology, Sequence Alignment, Glycine max embryology, Lipoxygenase metabolism, Plants enzymology, Glycine max enzymology, Triglycerides metabolism
- Abstract
The 90-kDa lipid body protein characterized earlier by its high expression during the stage of fat degradation was identified as a form of lipoxygenase. This organelle form was compared with lipoxygenase species purified from the cytosol. It is further shown that the antibodies raised against the lipid body membrane lipoxygenase from cucumber cotyledons cross-react with both cytosolic and lipid body lipoxygenase from soybean.
- Published
- 1992
- Full Text
- View/download PDF
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