TY - JOUR ID - 2419 TI - Purification and Characterization of Allene Oxide Cyclase from Dry Corn Seeds JO - Plant Physiol. PY - 1997 SP - 565-573 AU - Ziegler, J. AU - Hamberg, M. AU - Miersch, O. AU - Parthier, B. AU - VL - 114 UR - DO - 10.1104/pp.114.2.565 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2418 TI - Concentration of Dilute Protein Solutions Prior to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis JO - Anal. Biochem. PY - 1997 SP - 257-260 AU - Ziegler, J. AU - Vogt, T. AU - Miersch, O. AU - Strack, D. AU - VL - 250 UR - DO - 10.1006/abio.1997.2248 AB - A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2409 TI - Jasmonate-signalled plant gene expression JO - Trends Plant Sci. PY - 1997 SP - 302-307 AU - Wasternack, C. AU - Parthier, B. AU - VL - 2 UR - DO - 10.1016/S1360-1385(97)89952-9 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2408 TI - Molecular modelling, synthesis and biological activity of methyl 3-methyljasmonate and related derivatives JO - Tetrahedron PY - 1997 SP - 8181-8194 AU - Ward, J. L. AU - Gaskin, P. AU - Beale, M. H. AU - Sessions, R. AU - Koda, Y. AU - Wasternack, C. AU - VL - 53 UR - DO - 10.1016/S0040-4020(97)00485-7 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2396 TI - Chiral separation of amide conjugates of jasmonic acid by liquid chromatography JO - Chromatographia PY - 1997 SP - 104-108 AU - Kramell, R. AU - Schneider, G. AU - Miersch, O. AU - VL - 45 UR - DO - 10.1007/BF02505545 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2395 TI - Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves JO - FEBS Lett. PY - 1997 SP - 197-202 AU - Kramell, R. AU - Miersch, O. AU - Hause, B. AU - Ortel, B. AU - Parthier, B. AU - Wasternack, C. AU - VL - 414 UR - DO - 10.1016/S0014-5793(97)01005-3 AB - 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. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2391 TI - Partial purification and characterization of a jasmonic acid conjugate cleaving amidohydrolase from the fungus Botryodiplodia theobromae JO - FEBS Lett. PY - 1997 SP - 105-110 AU - Hertel, S. C. AU - Knöfel, H.-D. AU - Kramell, R. AU - Miersch, O. AU - VL - 407 UR - DO - 10.1016/S0014-5793(97)00307-4 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2390 TI - Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures JO - Bot. Acta PY - 1997 SP - 452-457 AU - Hause, B. AU - Feussner, K. AU - Wasternack, C. AU - VL - 110 UR - DO - 10.1111/j.1438-8677.1997.tb00662.x AB - 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. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2389 TI - 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) JO - J. Plant Physiol. PY - 1997 SP - 127-132 AU - Hause, B. AU - Kogel, K.-H. AU - Parthier, B. AU - Wasternack, C. AU - VL - 150 UR - DO - 10.1016/S0176-1617(97)80191-5 AB - 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. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2388 TI - Overexpression of the jasmonate-inducible 23 kDa protein (JIP 23) from barley in transgenic tobacco leads to the repression of leaf proteins JO - FEBS Lett. PY - 1997 SP - 58-62 AU - Görschen, E. AU - Dunaeva, M. AU - Reeh, I. AU - Wasternack, C. AU - VL - 419 UR - DO - 10.1016/S0014-5793(97)01433-6 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2387 TI - Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation JO - Planta PY - 1997 SP - 470-478 AU - Görschen, E. AU - Dunaeva, M. AU - Hause, B. AU - Reeh, I. AU - Wasternack, C. AU - Parthier, B. AU - VL - 202 UR - DO - 10.1007/s004250050151 AB - 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. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2383 TI - Structural Elucidation of Oxygenated Storage Lipids in Cucumber Cotyledons JO - J. Biol. Chem. PY - 1997 SP - 21635-21641 AU - Feussner, I. AU - Balkenhohl, T. J. AU - Porzel, A. AU - Kühn, H. AU - Wasternack, C. AU - VL - 272 UR - DO - 10.1074/jbc.272.34.21635 AB - 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. A2 - C1 - Molecular Signal Processing; Bioorganic Chemistry ER - TY - JOUR ID - 2382 TI - Quantitative Analyse von Lipoxygenase-Metaboliten in Lipiden durch NMR-Spektroskopie JO - BIOspektrum PY - 1997 SP - 54-58 AU - Feussner, I. AU - Porzel, A. AU - Wasternack, C. AU - Kühn, H. AU - VL - 3 UR - AB - A2 - C1 - Molecular Signal Processing; Bioorganic Chemistry ER - TY - JOUR ID - 2381 TI - Isolation of a cDNA coding for an ubiquitin-conjugating enzyme UBC1 of tomato - the first stress-induced UBC of higher plants JO - FEBS Lett. PY - 1997 SP - 211-215 AU - Feussner, K. AU - Feussner, I. AU - Leopold, I. AU - Wasternack, C. AU - VL - 409 UR - DO - 10.1016/S0014-5793(97)00509-7 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2380 TI - Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid JO - Bot. Acta PY - 1997 SP - 101-108 AU - Feussner, I. AU - Fritz, I. G. AU - Hause, B. AU - Ullrich, W. R. AU - Wasternack, C. AU - VL - 110 UR - DO - 10.1111/j.1438-8677.1997.tb00616.x AB - 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. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2379 TI - Do specific linoleate 13-lipoxygenases initiate β-oxidation? JO - FEBS Lett. PY - 1997 SP - 1-5 AU - Feussner, I. AU - Kühn, H. AU - Wasternack, C. AU - VL - 406 UR - DO - 10.1016/S0014-5793(97)00218-4 AB - 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. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 185 TI - Allene Oxide Cyclase from Corn: Partial Purification and Characterization T2 - Physiology, Biochemistry and Molecular Biology of Plant Lipids PB - PY - 1997 SP - 99-101 AU - Ziegler, J. AU - Hamberg, M. AU - Miersch, O. AU - VL - UR - DO - 10.1007/978-94-017-2662-7_32 AB - 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]. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 181 TI - Do Lipoxygenases Initiate β-Oxidation? T2 - Physiology, Biochemistry and Molecular Biology of Plant Lipids PB - PY - 1997 SP - 250-252 AU - Feussner, I. AU - Kühn, H. AU - Wasternack, C. AU - VL - UR - DO - 10.1007/978-94-017-2662-7_79 AB - 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. A2 - C1 - Molecular Signal Processing ER -