@Article{IPB-2278, author = {Quint, M. and Melchinger, A. E. and Dußle, C. M. and Lübberstedt, T. and}, title = {{Breeding for virus resistance in maize}}, year = {2000}, pages = {529-545}, journal = {Genetika}, url = {http://www.dgsgenetika.org.rs/abstrakti/vol32_2000_no3_en.htm#Rad26}, volume = {32}, abstract = {Sugarcane mosaic virus (SCMV) is an important disease in maize, which is emerging in Germany since 1983. Using this pest as a model for the inheritance of oligogenic traits, we clarified the genetic ba­sis for resistance in early maturing European maize germplasm. Screening of 122 adapted European inbred lines identified three completely resistant lines, which were used for further analyses. The genetics of SCMV resis­tance was investigated by allelism tests in field experiments combined with QTL and bulked segregant analyses (BSA) on the marker level. QTL analyses revealed the presence of two major genes Scm1 and Scm2 plus three minor QTL. Involvement of Scm1 and Scm2 in the inheritance of SCMV resistance could be confirmed by BSA in a second cross. Breeders can make use of tightly linked STS markers for marker-assisted selection (MAS) as well as our SCMV resistant flint lines to improve their elite germplasm. Currently, recurrent backcrossing with phenotypic selection is the most appropriate and cost effective breeding method. With de­creasing costs of DNA chip technology, MAS can be competitive with phenotypic selection in the near future. Further objectives of our research are the isolation and cloning of Scm1 and Scm2. To achieve this goal we follow two different approaches. (1) Positional cloning based on more than 500 AFLP primer combinations resulted in Scm1/Scm2 specific markers with a resolution of approximately 0.2 cM in the respective re­gions. (2) Resistance gene analogues (RGAs), cosegregating with the tar­get genes are used to identify further candidate genes for transformation experiments.} } @Article{IPB-2271, author = {Miersch, O. and Wasternack, C. and}, title = {{Octadecanoid and Jasmonate Signaling in Tomato (Lycopersicon esculentum Mill.) Leaves: Endogenous Jasmonates Do Not Induce Jasmonate Biosynthesis}}, year = {2000}, pages = {715-722}, journal = {Biol. Chem.}, doi = {10.1515/BC.2000.092}, volume = {381}, abstract = {Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous increase of the JA level. Here we investigated the positive feedback of endogenous JA on JA formation, as well as its β-oxidation steps. JA-responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato (Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different lengths of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data revealed that β-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas evennumbered derivatives were active.After treatment of tomato leaves with (10-2H)-(–)-12-oxophytoenoic acid, (4-2H)-(–)-JA and its methyl ester were formed and could be quantified separately from the endogenously nonlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g fresh weight JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid, as well as of JA and its methyl ester, are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo.} } @Article{IPB-2270, author = {Maucher, H. and Hause, B. and Feussner, I. and Ziegler, J. and Wasternack, C. and}, title = {{Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development}}, year = {2000}, pages = {199-213}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.2000.00669.x}, volume = {21}, 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.} } @Article{IPB-2265, author = {Kramell, R. and Miersch, O. and Atzorn, R. and Parthier, B. and Wasternack, C. and}, title = {{Octadecanoid-Derived Alteration of Gene Expression and the “Oxylipin Signature” in Stressed Barley Leaves. Implications for Different Signaling Pathways}}, year = {2000}, pages = {177-188}, journal = {Plant Physiol.}, doi = {10.1104/pp.123.1.177}, volume = {123}, abstract = {Stress-induced gene expression in barley (Hordeum vulgare cv Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA), and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 h before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-JA and 9,13-didehydro-OPDA were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA-responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression, as evidenced by those genes that did not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. Application of deuterated JA showed that endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signaling pathways.} } @Article{IPB-2257, author = {Hause, B. and Stenzel, I. and Miersch, O. and Maucher, H. and Kramell, R. and Ziegler, J. and Wasternack, C. and}, title = {{Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles}}, year = {2000}, pages = {113-126}, journal = {Plant J.}, doi = {10.1046/j.1365-313x.2000.00861.x}, volume = {24}, abstract = {A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis (\+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato.} } @Article{IPB-2255, author = {Gross, H. B. and Dalebout, T. and Grubb, C. D. and Abel, S. and}, title = {{Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana}}, year = {2000}, pages = {265-272}, journal = {Plant Sci.}, doi = {10.1016/S0168-9452(00)00354-X}, volume = {159}, abstract = {Natural isothiocyanates, derived from glucosinolates by myrosinase-catalyzed hydrolysis, are potent chemopreventive agents that favorably modify carcinogen metabolism in mammals by inhibiting metabolic activation of carcinogens and/or by inducing carcinogen-detoxifying enzymes. Methylsulfinylalkyl isothiocyanates are potent selective inducers of mammalian Phase 2 detoxification enzymes such as quinone reductase [NADP(H):quinone-acceptor oxidoreductase, EC 1.6.99.2]. Members of the Cruciferae family, including the model plant species Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates. We have adapted a colorimetric bioassay for quinone reductase activity in Hepa 1c1c7 murine hepatoma cells as a versatile tool to rapidly monitor methylsulfinylalkyl glucosinolate content in A. thaliana leaf extracts. Using wild type plants and mutant plants defective in the synthesis of 4-methylsulfinylbutyl glucosinolate (glucoraphanin), we have demonstrated that A. thaliana (ecotype Columbia) is a rich source of Phase 2 enzyme inducers and that methylsulfinylalkyl glucosinolates, predominantly glucoraphanin, account for about 80% of the quinone reductase inducer potency of Columbia leaf extracts. We have optimized leaf extraction conditions and the quinone reductase bioassay to allow for screening of large numbers of plant extracts in a molecular genetic approach to dissecting glucosinolate biosynthesis in A. thaliana.} } @Article{IPB-2253, author = {Colón-Carmona, A. and Chen, D. L. and Yeh, K.-C. and Abel, S. and}, title = {{Aux/IAA Proteins Are Phosphorylated by Phytochrome in Vitro}}, year = {2000}, pages = {1728-1738}, journal = {Plant Physiol.}, doi = {10.1104/pp.124.4.1728}, volume = {124}, abstract = {Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes,SHY2/IAA3, AXR3/IAA17, andAXR2/IAA7 cause pleiotropic phenotypes consistent with enhanced auxin responses, possibly by increasing Aux/IAA protein stability. Semidominant mutations shy2-1D,shy2-2, axr3-1, and axr2-1induce ectopic light responses in dark-grown seedlings. Because genetic studies suggest that the shy2-1D andshy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/IAA proteins interact directly with phytochrome and whether they are substrates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with recombinant phytochrome A from oat (Avena sativa). We further show that recombinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated by recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indicates that phytochrome A phosphorylation occurs on the N-terminal half of the protein. Metabolic labeling and immunoprecipitation studies with affinity-purified antibodies to IAA3 demonstrate increased in vivo steady-state levels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 protein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is proposed to provide one molecular mechanism for integrating auxin and light signaling in plant development.} } @Article{IPB-2252, author = {Chen, D. L. and Delatorre, C. A. and Bakker, A. and Abel, S. and}, title = {{Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana}}, year = {2000}, pages = {13-22}, journal = {Planta}, doi = {10.1007/s004250000271}, volume = {211}, abstract = {Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants.} } @Article{IPB-2244, author = {Abel, S. and Nürnberger, T. and Ahnert, V. and Krauss, G.-J. and Glund, K. and}, title = {{Induction of an Extracellular Cyclic Nucleotide Phosphodiesterase as an Accessory Ribonucleolytic Activity during Phosphate Starvation of Cultured Tomato Cells}}, year = {2000}, pages = {543-552}, journal = {Plant Physiol.}, doi = {10.1104/pp.122.2.543}, volume = {122}, abstract = {During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2′:3′-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970–976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2′:3′-cyclic NMP to 3′-NMP and the 3′:5′-cyclic isomers to a mixture of 3′-NMP and 5′-NMP. Specific activities of the enzyme are 2-fold higher for 2′:3′-cyclic NMP than for 3′:5′-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared −Pi culture medium as a source of 3′-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.} } @Article{IPB-2292, author = {Ziegler, J. and Stenzel, I. and Hause, B. and Maucher, H. and Hamberg, M. and Grimm, R. and Ganal, M. and Wasternack, C. and}, title = {{Molecular Cloning of Allene Oxide Cyclase}}, year = {2000}, pages = {19132-19138}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.M002133200}, volume = {275}, abstract = {Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (\>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.} } @Article{IPB-2289, author = {Weichert, H. and Kolbe, A. and Wasternack, C. and Feussner, I. and}, title = {{Formation of 4-hydroxy-2-alkenals in barley leaves}}, year = {2000}, pages = {850-851}, journal = {Biochem. Soc. Trans.}, doi = {10.1042/bst0280850}, volume = {28}, abstract = {In barley leaves 13-lipoxygenases are induced by jasmonates. This leads to induction of lipid peroxidation. Here we show by in vitro studies that these processes may further lead to autoxidative formation of (2E)-4-hydroxy-2-hexenal from (3Z)-hexenal.} } @Article{IPB-2288, author = {Wasternack, C. and Hause, B. and}, title = {{Stressabwehr und Entwicklung: Jasmonate — chemische Signale in Pflanzen}}, year = {2000}, pages = {312-320}, journal = {Biologie in unserer Zeit}, doi = {10.1002/1521-415X(200011)30:6<312::AID-BIUZ312>3.0.CO;2-8}, volume = {30}, abstract = {Chemische Signale wurden bereits im 19.Jahrhundert als Regulatoren von Wachstum und Entwicklung der Pflanzen postuliert.In den letzten 70 Jahren wurde die Wirkungsweise der klassischen Pflanzenhormone wie der Auxine, Gibberelline, Cytokinine, Ethylen und Abscisinsäure aufgeklärt. Doch erst im letzten Jahrzehnt entdeckte man die Bedeutung der Brassinosteroide, der Peptidhormone und der Jasmonate.} } @Article{IPB-2415, author = {Wasternack, C. and Parthier, B. and}, title = {{Jasmonate-signalled plant gene expression}}, year = {1997}, pages = {302-307}, journal = {Trends Plant Sci.}, doi = {10.1016/S1360-1385(97)89952-9}, volume = {2}, abstract = {Jasmonic acid is distributed throughout higher plants, synthesized from linolenic acid via the octadecanoic pathway. An important and probably essential role seems to be its operation as a ‘master switch’, responsible for the activation of signal transduction pathways in response to predation and pathogen attack. Proteins encoded by jasmonate-induced genes include enzymes of alkaloid and phytoalexin synthesis, storage proteins, cell wall constituents and stress protectants. The wound-induced formation of proteinase inhibitors is a well-studied example, in which jasmonic acid combines with abscisic acid and ethylene to protect the plant from predation.} } @Article{IPB-2414, author = {Ward, J. L. and Gaskin, P. and Beale, M. H. and Sessions, R. and Koda, Y. and Wasternack, C. and}, title = {{Molecular modelling, synthesis and biological activity of methyl 3-methyljasmonate and related derivatives}}, year = {1997}, pages = {8181-8194}, journal = {Tetrahedron}, doi = {10.1016/S0040-4020(97)00485-7}, volume = {53}, abstract = {Methyl 3-methyljasmonate was synthesised from methyl jasmonate via methyl 3,7-dehydrojasmonate. Molecular modelling predicted an increase in the proportion of cis-orientated side-chains for equilibrated 3-methyl-substituted jasmonate. The synthetic 3-methyljasmonate was shown by gc-ms analysis to equilibrate to a 2:1 ratio of isomers, which appeared from the NMR spectra to comprise mainly the cis-isomer. Surprisingly, both 3,7-dehydro- and 3-methyl-derivatives were inactive in four well established jasmonate bioassays. Methyl-2-methyljasmonate was synthesised and also found to be inactive. Methyl 4,5-dehydrojasmonate was prepared, via the 5-diazo derivative. Both of these compounds have low activity. Our results are discussed with reference to previous knowledge of jasmonate structure-activity relationships and indicate that there are stringent steric demands in jasmonate-receptor interactions.} } @Article{IPB-2402, author = {Kramell, R. and Schneider, G. and Miersch, O. and}, title = {{Chiral separation of amide conjugates of jasmonic acid by liquid chromatography}}, year = {1997}, pages = {104-108}, journal = {Chromatographia}, doi = {10.1007/BF02505545}, volume = {45}, abstract = {Synthetic amide conjugates of (−)-jasmonic acid and its (\+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (\+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism.} } @Article{IPB-2401, author = {Kramell, R. and Miersch, O. and Hause, B. and Ortel, B. and Parthier, B. and Wasternack, C. and}, title = {{Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves}}, year = {1997}, pages = {197-202}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)01005-3}, volume = {414}, abstract = {Leaves of barley (Hordeum vulgare L. cv. Salome ) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up‐regulation of specific genes and down‐regulation of house‐keeping genes. This transcriptional regulation exhibits several specificities. (i) The (−)‐enantiomers are more active, and conjugates are mainly active if they carry an l ‐amino acid moiety. (ii) The various JA‐responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.} } @Article{IPB-2397, author = {Hertel, S. C. and Knöfel, H.-D. and Kramell, R. and Miersch, O. and}, title = {{Partial purification and characterization of a jasmonic acid conjugate cleaving amidohydrolase from the fungus Botryodiplodia theobromae}}, year = {1997}, pages = {105-110}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)00307-4}, volume = {407}, abstract = {A protein preparation from the mycelium of the tropical pathogenic fungus Botryodiplodia theobromae revealed a novel peptidase activity. This enzyme was capable of cleaving conjugates of jasmonic acid with α-amino acids. The protein was enriched 108-fold by gel filtration, ion exchange and hydrophobic interaction chromatography. The enzyme was found to be a glycoprotein with a molecular mass of about 107 kDa. The amidohydrolase seems to be very specific with regard to (−)-jasmonic acid and α-amino acids with (S)-configuration.} } @Article{IPB-2396, author = {Hause, B. and Feussner, K. and Wasternack, C. and}, title = {{Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures}}, year = {1997}, pages = {452-457}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1997.tb00662.x}, volume = {110}, abstract = {Diadenosine 5′,5′”‐P1,P4‐tetraphosphate (Ap4A) cleaving enzymes are assumed to regulate intracellular levels of Ap4A, a compound known to affect cell proliferation and stress responses. From plants an Ap4A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap4A hydrolase in 4‐day‐old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein‐5‐isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap4A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6‐diamidino‐2‐phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap4A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap4A in triggering DNA synthesis and cell proliferation.} } @Article{IPB-2395, author = {Hause, B. and Kogel, K.-H. and Parthier, B. and Wasternack, C. and}, title = {{In barley leaf cells, jasmonates do not act as a signal during compatible or incompatible interactions with the powdery mildew fungus (Erysiphe graminis f. sp. hordei)}}, year = {1997}, pages = {127-132}, journal = {J. Plant Physiol.}, doi = {10.1016/S0176-1617(97)80191-5}, volume = {150}, abstract = {We have studied a possible function of jasmonates as mediators in the host-pathogen interaction of barley (Hordeum vulgare L.) with the powdery mildew fungus Egh (Erysiphe graminis f. sp. hordei). Previous findings from whole-leaf extracts demonstrated that (i) extracts from infected barley leaves did not contain enhanced levels of jasmonates, (ii) transcripts of jasmonate-inducible genes were not expressed upon infection, and (iii) exogenous application of jasmonates did not induce resistance to Egh (Kogel et al., 1995). Nevertheless, the question arises whether or not jasmonates are involved in the interaction of barley with the powdery mildew fungus at the local site of infection. Using an immunocytological approach the analysis of leaf cross-sections from a susceptible barley cultivar and its near-isogenic mlo5-resistant line revealed no accumulation of JIP-23, the most abundant jasmonate inducible protein, neither in epidermal cells attacked by the pathogen nor in adjacent mesophyll cells. As a positive control, cross-sections from methyl jasmonate-treated leaf segments showed a strong signal for JIP-23 accumulation. Because the presence of the jasmonate-inducible protein is highly indicative for an already low threshold level of endogenous jasmonate (Lehmann et al., 1995), the lack of JIP-23 accumulation at the sites of attempted fungal infection clearly demonstrates the absence of enhanced levels of jasmonates. This excludes even a local rise of jasmonate confined to those single cells penetrated (Mlo genotype) or attacked (mlo5 genotype) by the fungus.} } @Article{IPB-2394, author = {Görschen, E. and Dunaeva, M. and Reeh, I. and Wasternack, C. and}, title = {{Overexpression of the jasmonate-inducible 23 kDa protein (JIP 23) from barley in transgenic tobacco leads to the repression of leaf proteins}}, year = {1997}, pages = {58-62}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)01433-6}, volume = {419}, abstract = {We investigated transgenic tobacco lines which express different amounts of the barley JIP 23. In these plants the amount of several proteins decreased proportionally to increasing amounts of JIP 23 whereas the transcript levels were constant as determined for the small and the large subunit of RuBPCase. However, the translation initiation of the rbcS transcript was found to be less efficient than in the wild type. In contrast, the jip 23 transcript was efficiently initiated, indicating that no unspecific impairment of initiation occurred. The data suggest that the barley JIP 23 leads to discrimination among certain tobacco transcripts during translation initiation.} } @Article{IPB-2393, author = {Görschen, E. and Dunaeva, M. and Hause, B. and Reeh, I. and Wasternack, C. and Parthier, B. and}, title = {{Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation}}, year = {1997}, pages = {470-478}, journal = {Planta}, doi = {10.1007/s004250050151}, volume = {202}, abstract = {In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm.} } @Article{IPB-2389, author = {Feussner, I. and Balkenhohl, T. J. and Porzel, A. and Kühn, H. and Wasternack, C. and}, title = {{Structural Elucidation of Oxygenated Storage Lipids in Cucumber Cotyledons}}, year = {1997}, pages = {21635-21641}, journal = {J. Biol. Chem.}, doi = {10.1074/jbc.272.34.21635}, volume = {272}, 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.} } @Article{IPB-2388, author = {Feussner, I. and Porzel, A. and Wasternack, C. and Kühn, H. and}, title = {{Quantitative Analyse von Lipoxygenase-Metaboliten in Lipiden durch NMR-Spektroskopie}}, year = {1997}, pages = {54-58}, journal = {BIOspektrum}, volume = {3}, } @Article{IPB-2387, author = {Feussner, K. and Feussner, I. and Leopold, I. and Wasternack, C. and}, title = {{Isolation of a cDNA coding for an ubiquitin-conjugating enzyme UBC1 of tomato - the first stress-induced UBC of higher plants}}, year = {1997}, pages = {211-215}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)00509-7}, volume = {409}, abstract = {A clone of an ubiquitin‐conjugating enzyme (UBC) was isolated from a λ‐ZAP‐cDNA library, generated from mRNA of tomato (Lycopersicon esculentum) cells grown in suspension for 3 days. The open reading frame called Le UBC1, 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 Le UBC1 showed highest sequence similarities to UBC1 of bovine and yeast. By Southern blot analysis Le UBC1 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 Le UBC1 is constitutively expressed in an exponentially growing tomato cell culture. In response to heat shock an increase in Le UBC1‐mRNA was detectable. A strong accumulation of the Le UBC1‐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.} } @Article{IPB-2386, author = {Feussner, I. and Fritz, I. G. and Hause, B. and Ullrich, W. R. and Wasternack, C. and}, title = {{Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid}}, year = {1997}, pages = {101-108}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1997.tb00616.x}, volume = {110}, abstract = {Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6‐dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX‐95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX‐97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.} } @Article{IPB-2385, author = {Feussner, I. and Kühn, H. and Wasternack, C. and}, title = {{Do specific linoleate 13-lipoxygenases initiate β-oxidation?}}, year = {1997}, pages = {1-5}, journal = {FEBS Lett.}, doi = {10.1016/S0014-5793(97)00218-4}, volume = {406}, 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 β-oxidation during lipid mobilization of oilseed plants.} } @Article{IPB-2425, author = {Ziegler, J. and Hamberg, M. and Miersch, O. and Parthier, B. and}, title = {{Purification and Characterization of Allene Oxide Cyclase from Dry Corn Seeds}}, year = {1997}, pages = {565-573}, journal = {Plant Physiol.}, doi = {10.1104/pp.114.2.565}, volume = {114}, abstract = {Allene oxide cyclase (AOC; EC 5.3.99.6) catalyzes the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid to 12-oxo- 10,15(Z)-phytodienoic acid, the precursor of jasmonic acid (JA). This soluble enzyme was purified 2000-fold from dry corn (Zea mays L.) kernels to apparent homogeneity. The dimeric protein has a molecular mass of 47 kD. Allene oxide cyclase activity was not affected by divalent ions and was not feedback-regulated by its product, 12-oxo-l0,15(Z)-phytodienoic acid, or by JA. ([plus or minus])-cis- 12,13-Epoxy-9(Z)-octadecenoic acid, a substrate analog, strongly inhibited the enzyme, with 50% inhibition at 20 [mu]M. Modification of the inhibitor, such as methylation of the carboxyl group or a shift in the position of the epoxy group, abolished the inhibitory effect, indicating that both structural elements and their position are essential for binding to AOC. Nonsteroidal anti-inflammatory drugs, which are often used to interfere with JA biosynthesis, did not influence AOC activity. The purified enzyme catalyzed the cyclization of 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid derived from linolenic acid, but not that of 12,13(S)-epoxy-9(Z),11- octadecadienoic acid derived from linoleic acid.} } @Article{IPB-2424, author = {Ziegler, J. and Vogt, T. and Miersch, O. and Strack, D. and}, title = {{Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis}}, year = {1997}, pages = {257-260}, journal = {Anal. Biochem.}, doi = {10.1006/abio.1997.2248}, volume = {250}, } @INBOOK{IPB-185, author = {Ziegler, J. and Hamberg, M. and Miersch, O. and}, title = {{Physiology, Biochemistry and Molecular Biology of Plant Lipids}}, year = {1997}, pages = {99-101}, chapter = {{Allene Oxide Cyclase from Corn: Partial Purification and Characterization}}, doi = {10.1007/978-94-017-2662-7_32}, abstract = {In plants, the oxylipin pathway gives rise to several oxygenated fatty acid derivatives such as hydroxy- and keto fatty acids as well as volatile aldehydes and cyclic compounds, which are, in part, physiologically active [1]. Among these, jasmonic acid is discussed as signalling molecule during several stress responses, wounding, senescense and plant pathogen interactions [2].} } @INBOOK{IPB-181, author = {Feussner, I. and Kühn, H. and Wasternack, C. and}, title = {{Physiology, Biochemistry and Molecular Biology of Plant Lipids}}, year = {1997}, pages = {250-252}, chapter = {{Do Lipoxygenases Initiate β-Oxidation?}}, doi = {10.1007/978-94-017-2662-7_79}, 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 seedlings growth. During this stage the lipid storing organelles, the lipid bodies, are degraded and a new set of proteins, including a specific form of lipoxygenase (LOX), is detectable at their membranes in different plants [1,2]. LOXs are widely distributed in plants and animals and catalyze the regio- and stereo-specific oxygenation of polyunsaturated fatty acids [3]. The enzymatic transformations of the resulting fatty acid hydroperoxides have been extensively studied [4]. Three well characterized enzymes, a lyase, an allene oxide synthase, and a peroxygenase, were shown to degrade hydroperoxides into compounds of physiological importance, such as odors, oxylipins, and jasmonates. We have recently reported a new LOX reaction in plants where a specific LOX, the lipid body LOX, metabolizes esterified fatty acids. This reaction resulted in the formation of 13(S)-hydroxy-linoleic acid (13-HODE) and lead us to propose an additional branch of the LOX pathway: the reductase pathway. Besides a specific LOX form we suggest two additional enzyme activities, a lipid hydroperoxide reductase and a lipid hydroxide-specific lipase which lead to the formation of 13-HODE. 13-HODE might be the endogenous substrate for β-oxidation in the glyoxysomes during germination of oilseeds containing high amounts of polyunsaturated fatty acids.} } @Article{IPB-2473, author = {Hause, B. and zur Nieden, U. and Lehmann, J. and Wasternack, C. and Parthier, B. and}, title = {{Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves}}, year = {1994}, pages = {333-341}, journal = {Bot. Acta}, doi = {10.1111/j.1438-8677.1994.tb00804.x}, volume = {107}, abstract = {The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA.} } @Article{IPB-2469, author = {Abel, S. and Theologis, A. and}, title = {{Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression}}, year = {1994}, pages = {421-427}, journal = {Plant J.}, doi = {10.1111/j.1365-313X.1994.00421.x}, volume = {5}, abstract = {An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β‐glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)‐mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin‐mediated induction of chloramphenicol acetyl‐transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin‐inducible PS‐IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.} } @Article{IPB-2468, author = {Abel, S. and Oeller, P. W. and Theologis, A. and}, title = {{Early auxin-induced genes encode short-lived nuclear proteins.}}, year = {1994}, pages = {326-330}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, doi = {10.1073/pnas.91.1.326}, volume = {91}, abstract = {The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses.} }