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Publikationen - Molekulare Signalverarbeitung

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Publikation

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.
Publikation

Fonseca, S.; Chini, A.; Hamberg, M.; Adie, B.; Porzel, A.; Kramell, R.; Miersch, O.; Wasternack, C.; Solano, R.; (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate Nat. Chem. Biol. 5, 344-350, (2009) DOI: 10.1038/nchembio.161

Hormone-triggered activation of the jasmonate signaling pathway in Arabidopsis thaliana requires SCFCOI1-mediated proteasome degradation of JAZ repressors. (−)-JA-L-Ile is the proposed bioactive hormone, and SCFCOI1 is its likely receptor. We found that the biological activity of (−)-JA-L-Ile is unexpectedly low compared to coronatine and the synthetic isomer (+)-JA-L-Ile, which suggests that the stereochemical orientation of the cyclopentanone-ring side chains greatly affects receptor binding. Detailed GC-MS and HPLC analyses showed that the (−)-JA-L-Ile preparations currently used in ligand binding studies contain small amounts of the C7 epimer (+)-7-iso-JA-L-Ile. Purification of each of these molecules demonstrated that pure (−)-JA-L-Ile is inactive and that the active hormone is (+)-7-iso-JA-L-Ile, which is also structurally more similar to coronatine. In addition, we show that pH changes promote conversion of (+)-7-iso-JA-L-Ile to the inactive (−)-JA-L-Ile form, thus providing a simple mechanism that can regulate hormone activity through epimerization.
Publikation

Gao, X.; Stumpe, M.; Feussner, I.; Kolomiets, M.; A novel plastidial lipoxygenase of maize (Zea mays) ZmLOX6 encodes for a fatty acid hydroperoxide lyase and is uniquely regulated by phytohormones and pathogen infection Planta 227, 491-503, (2008) DOI: 10.1007/s00425-007-0634-8

Lipoxygenases (LOXs) are members of a large enzyme family that catalyze oxygenation of free polyunsaturated fatty acids into diverse hydroperoxide compounds, collectively called oxylipins. Although LOXs have been well studied in dicot species, reports of the genes encoding these enzymes are scarce for monocots, especially maize. Herein, we reported the cloning, characterization and molecular functional analysis of a novel maize LOX gene, ZmLOX6. The ZmLOX6 nucleotide sequence encodes a deduced translation product of 892 amino acids. Phylogenetic analysis showed that ZmLOX6 is distantly related to previously reported 9- or 13-LOXs from maize and other plant species, including rice and Arabidopsis. Although sequence prediction suggested cytoplasmic localization of this protein, ZmLOX6 protein has been reportedly isolated from mesophyll cell chloroplasts, emphasizing the unique features of this protein. Plastidial localization was confirmed by chloroplast uptake experiments with the in vitro translated protein. Analysis of recombinant protein revealed that ZmLOX6 has lost fatty acid hydroperoxide forming activity but 13-LOX-derived fatty acid hydroperoxides were cleaved into odd-chain ω-oxo fatty acids and as yet not identified C5-compound. In line with its reported abundance in mesophyll cells, ZmLOX6 was predominantly expressed in leaf tissue. Northern blot analysis demonstrated that ZmLOX6 was induced by jasmonic acid, but repressed by abscisic acid, salicylic acid and ethylene and was not responsive to wounding or insects. Further, this gene was strongly induced by the fungal pathogen Cochliobolus carbonum during compatible interactions, suggesting that ZmLOX6 may contribute to susceptibility to this pathogen. The potential involvement of ZmLOX6 in maize interactions with pathogens is discussed.
Publikation

Delker, C.; Raschke, A.; Quint, M.; Auxin dynamics: the dazzling complexity of a small molecule’s message Planta 227, 929-941, (2008) DOI: 10.1007/s00425-008-0710-8

The phytohormone auxin is a potent regulator of plant development. Since its discovery in the beginning of the twentieth century many aspects of auxin biology have been extensively studied, ranging from biosynthesis and metabolism to the elucidation of molecular components of downstream signaling. With the identification of the F-box protein TIR1 as an auxin receptor a major breakthrough in understanding auxin signaling has been achieved and recent modeling approaches have shed light on the putative mechanisms underlying the establishment of auxin gradients and maxima essential for many auxin-regulated processes. Here, we review these and other recent advances in unraveling the entanglement of biosynthesis, polar transport and cellular signaling events that allow small auxinic molecules to facilitate their complex regulatory action.
Publikation

Meixner, C.; Ludwig-Müller, J.; Miersch, O.; Gresshoff, P.; Staehelin, C.; Vierheilig, H.; Lack of mycorrhizal autoregulation and phytohormonal changes in the supernodulating soybean mutant nts1007 Planta 222, 709-715, (2005) DOI: 10.1007/s00425-005-0003-4

Autoregulatory mechanisms have been reported in the rhizobial and the mycorrhizal symbiosis. Autoregulation means that already existing nodules or an existing root colonization by an arbuscular mycorrhizal fungus systemically suppress subsequent nodule formation/root colonization in other parts of the root system. Mutants of some legumes lost their ability to autoregulate the nodule number and thus display a supernodulating phenotype. On studying the effect of pre-inoculation of one side of a split-root system with an arbuscular mycorrhizal fungus on subsequent mycorrhization in the second side of the split-root system of a wild-type soybean (Glycine max L.) cv. Bragg and its supernodulating mutant nts1007, we observed a clear suppressional effect in the wild-type, whereas further root colonization in the split-root system of the mutant nts1007 was not suppressed. These data strongly indicate that the mechanisms involved in supernodulation also affect mycorrhization and support the hypothesis that the autoregulation in the rhizobial and the mycorrhizal symbiosis is controlled in a similar manner. The accumulation patterns of the plant hormones IAA, ABA and Jasmonic acid (JA) in non-inoculated control plants and split-root systems of inoculated plants with one mycorrhizal side of the split-root system and one non-mycorrhizal side, indicate an involvement of IAA in the autoregulation of mycorrhization. Mycorrhizal colonization of soybeans also resulted in a strong induction of ABA and JA levels, but on the basis of our data the role of these two phytohormones in mycorrhizal autoregulation is questionable.
Publikation

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.
Publikation

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.
Publikation

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.
Publikation

Feussner, I.; Hause, B.; Nellen, A.; Wasternack, C.; Kindl, H.; Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms Planta 198, 288-293, (1996) DOI: 10.1007/BF00206255

Lipid bodies are degraded during germination. Whereas some proteins, e.g. oleosins, are synthesized during the formation of lipid bodies of maturating seeds, a new set of proteins, including a specific form of lipoxygenase (LOX; EC 1.13.11.12), is detectable in lipid bodies during the stage of fat degradation in seed germination. In cotyledons of cucumber (Cucumis sativus L.) seedlings at day 4 of germination, the most conspicuous staining with anti-LOX antibodies was observed in the cytosol. At very early stages of germination, however, the LOX form present in large amounts and synthesized preferentially was the lipid-body LOX. This was demonstrated by immunocytochemical staining of cotyledons from 1-h and 24-h-old seedlings: the immunodecoration of sections of 24-h-old seedlings with anti-LOX antiserum showed label exclusively correlated with lipid bodies of around 3 μm in diameter. In accordance, the profile of LOX protein isolated from lipid bodies during various stages of germination showed a maximum at day 1. By measuring biosynthesis of the protein in vivo we demonstrated that the highest rates of synthesis of lipid-body LOX occurred at day 1 of germination. The early and selective appearance of a LOX form associated with lipid bodies at this stage of development is discussed.
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