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Leon-Reyes, A.; Van der Does, D.; De Lange, E. S.; Delker, C.; Wasternack, C.; Van Wees, S. C. M.; Ritsema, T.; Pieterse, C. M. J.; Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway Planta 232, 1423-1432, (2010) DOI: 10.1007/s00425-010-1265-z

Jasmonates (JAs) and salicylic acid (SA) are plant hormones that play pivotal roles in the regulation of induced defenses against microbial pathogens and insect herbivores. Their signaling pathways cross-communicate providing the plant with a regulatory potential to finely tune its defense response to the attacker(s) encountered. In Arabidopsis thaliana, SA strongly antagonizes the jasmonic acid (JA) signaling pathway, resulting in the downregulation of a large set of JA-responsive genes, including the marker genes PDF1.2 and VSP2. Induction of JA-responsive marker gene expression by different JA derivatives was equally sensitive to SA-mediated suppression. Activation of genes encoding key enzymes in the JA biosynthesis pathway, such as LOX2, AOS, AOC2, and OPR3 was also repressed by SA, suggesting that the JA biosynthesis pathway may be a target for SA-mediated antagonism. To test this, we made use of the mutant aos/dde2, which is completely blocked in its ability to produce JAs because of a mutation in the ALLENE OXIDE SYNTHASE gene. Mutant aos/dde2 plants did not express the JA-responsive marker genes PDF1.2 or VSP2 in response to infection with the necrotrophic fungus Alternaria brassicicola or the herbivorous insect Pieris rapae. Bypassing JA biosynthesis by exogenous application of methyl jasmonate (MeJA) rescued this JA-responsive phenotype in aos/dde2. Application of SA suppressed MeJA-induced PDF1.2 expression to the same level in the aos/dde2 mutant as in wild-type Col-0 plants, indicating that SA-mediated suppression of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway.

Rudus, I.; Kepczynska, E.; Kepczynski, J.; Wasternack, C.; Miersch, O.; Changes in jasmonates and 12-oxophytodienoic acid contents of Medicago sativa L. during somatic embryogenesis Acta Physiol. Plant. 27, 497-504, (2005) DOI: 10.1007/s11738-005-0055-x

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.

Gerhardt, B.; Fischer, K.; Balkenhohl, T. J.; Pohnert, G.; Kühn, H.; Wasternack, C.; Feussner, I.; Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and β-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes Planta 220, 919-930, (2005) DOI: 10.1007/s00425-004-1408-1

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via β-oxidation. However, β-oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain β-oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as β-oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain β-oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous β-oxidation by glyoxysomes.

Weichert, H.; Kolbe, A.; Kraus, A.; Wasternack, C.; Feussner, I.; Metabolic profiling of oxylipins in germinating cucumber seedlings - lipoxygenase-dependent degradation of triacylglycerols and biosynthesis of volatile aldehydes Planta 215, 612-619, (2002) DOI: 10.1007/s00425-002-0779-4

A particular isoform of lipoxygenase (LOX) localized on lipid bodies was shown by earlier investigations to play a role in initiating the mobilization of triacylglycerols during seed germination. Here, further physiological functions of LOXs within whole cotyledons of cucumber (Cucumis sativus L.) were analyzed by measuring the endogenous amounts of LOX-derived products. The lipid-body LOX-derived esterified (13S)-hydroperoxy linoleic acid was the dominant metabolite of the LOX pathway in this tissue. It accumulated to about 14 µmol/g fresh weight, which represented about 6% of the total amount of linoleic acid in cotyledons. This LOX product was not only reduced to its hydroxy derivative, leading to degradation by β-oxidation, but alternatively it was metabolized by fatty acid hydroperoxide lyase leading to formation of hexanal as well. Furthermore, the activities of LOX forms metabolizing linolenic acid were detected by measuring the accumulation of volatile aldehydes and the allene oxide synthase-derived metabolite jasmonic acid. The first evidence is presented for an involvement of a lipid-body LOX form in the production of volatile aldehydes.

Nibbe, M.; Hilpert, B.; Wasternack, C.; Miersch, O.; Apel, K.; Cell death and salicylate- and jasmonate-dependent stress responses in Arabidopsis are controlled by single cet genes Planta 216, 120-128, (2002) DOI: 10.1007/s00425-002-0907-1

The jasmonic acid (JA)-dependent regulation of the Thi2.1 gene had previously been exploited for setting up a genetic screen for the isolation of signal transduction mutants of Arabidopsis thaliana (L.) Heynh. that constitutively express the thionin gene. Several cet mutants had been isolated which showed a constitutive expression of the thionin gene. These cet mutants, except for one, also showed spontaneous leaf cell necrosis and were up-regulated in the expression of the PR1 gene, reactions often associated with the systemic acquired resistance (SAR) pathway. Four of these cet mutants, cet1, cet2, cet3 and cet4.1 were crossed with the fad triple and coi1 mutants that are blocked at two steps within the JA-dependent signaling pathway, and with transgenic NahG plants that are deficient in salicylic acid (SA) and are unable to activate SAR. Analysis of the various double-mutant lines revealed that the four cet genes act within a signaling cascade at or prior to branch points from which not only JA-dependent signals but also SA-dependent signaling and cell death pathways diverge.

Weichert, H.; Kohlmann, M.; Wasternack, C.; Feussner, I.; Metabolic profiling of oxylipins upon sorbitol treatment in barley leaves Biochem. Soc. Trans. 28, 861-862, (2001) DOI: 10.1042/bst0280861

In barley leaves 13-lipoxygenases (LOXs) are induced by salicylate and jasmonate. Here, we analyse by metabolic profiling the accumulation of oxylipins upon sorbitol treatment. Although 13-LOX-derived products are formed and specifically directed into the reductase branch of the LOX pathway, accumulation is much later than in the cases of salicylate and jasmonate treatment. In addition, under these conditions only the accumulation of jasmonates as additional products of the LOX pathway has been found.

Weichert, H.; Kolbe, A.; Wasternack, C.; Feussner, I.; Formation of 4-hydroxy-2-alkenals in barley leaves Biochem. Soc. Trans. 28, 850-851, (2000) DOI: 10.1042/bst0280850

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.

Hause, B.; Feussner, K.; Wasternack, C.; Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures Bot. Acta 110, 452-457, (1997) DOI: 10.1111/j.1438-8677.1997.tb00662.x

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.

Görschen, E.; Dunaeva, M.; Hause, B.; Reeh, I.; Wasternack, C.; Parthier, B.; Expression of the ribosome-inactivating protein JIP60 from barley in transgenic tobacco leads to an abnormal phenotype and alterations on the level of translation Planta 202, 470-478, (1997) DOI: 10.1007/s004250050151

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.

Feussner, I.; Fritz, I. G.; Hause, B.; Ullrich, W. R.; Wasternack, C.; Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid Bot. Acta 110, 101-108, (1997) DOI: 10.1111/j.1438-8677.1997.tb00616.x

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.
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