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Publikationen - Molekulare Signalverarbeitung

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Publikation

Miersch, O.; Neumerkel, J.; Dippe, M.; Stenzel, I.; Wasternack, C.; Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling New Phytol. 177, 114-127, (2008) DOI: 10.1111/j.1469-8137.2007.02252.x

In potato 12‐hydroxyjasmonic acid (12‐OH‐JA) is a tuber‐inducing compound. Here, it is demonstrated that 12‐OH‐JA, as well as its sulfated and glucosylated derivatives, are constituents of various organs of many plant species. All accumulate differentially and usually to much higher concentrations than jasmonic acid (JA).In wounded tomato leaves, 12‐OH‐JA and its sulfated, as well as glucosylated, derivative accumulate after JA, and their diminished accumulation in wounded leaves of the JA‐deficient mutants spr2 and acx1 and also a JA‐deficient 35S::AOCantisense line suggest their JA‐dependent formation.To elucidate how signaling properties of JA/JAME (jasmonic acid methyl ester) are affected by hydroxylation and sulfation, germination and root growth were recorded in the presence of the different jasmonates, indicating that 12‐OH‐JA and 12‐hydroxyjasmonic acid sulfate (12‐HSO4‐JA) were not bioactive. Expression analyses for 29 genes showed that expression of wound‐inducible genes such as those coding for PROTEINASE INHIBITOR2, POLYPHENOL OXIDASE, THREONINE DEAMINASE or ARGINASE was induced by JAME and less induced or even down‐regulated by 12‐OH‐JA and 12‐HSO4‐JA. Almost all genes coding for enzymes in JA biosynthesis were up‐regulated by JAME but down‐regulated by 12‐OH‐JA and 12‐HSO4‐JA.The data suggest that wound‐induced metabolic conversion of JA/JAME into 12‐OH‐JA alters expression pattern of genes including a switch off in JA signaling for a subset of genes.
Publikation

Kienow, L.; Schneider, K.; Bartsch, M.; Stuible, H.-P.; Weng, H.; Miersch, O.; Wasternack, C.; Kombrink, E.; Jasmonates meet fatty acids: functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana J. Exp. Bot. 59, 403-419, (2008) DOI: 10.1093/jxb/erm325

Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systematically explored by applying an extensive substrate screen, and it was uncovered that activation of fatty acids is the common feature of all active members of this protein family, thereby defining a new group of fatty acyl-CoA synthetase, which is distinct from the known LACS family. Significantly, four family members also displayed activity towards different biosynthetic precursors of jasmonic acid (JA), including 12-oxo-phytodienoic acid (OPDA), dinor-OPDA, 3-oxo-2(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8), and OPC-6. Detailed analysis of in vitro properties uncovered significant differences in substrate specificity for individual enzymes, but only one protein (At1g20510) showed OPC-8:CoA ligase activity. Its in vivo function was analysed by transcript and jasmonate profiling of Arabidopsis insertion mutants for the gene. OPC-8:CoA ligase expression was activated in response to wounding or infection in the wild type but was undetectable in the mutants, which also exhibited OPC-8 accumulation and reduced levels of JA. In addition, the developmental, tissue- and cell-type specific expression pattern of the gene, and regulatory properties of its promoter were monitored by analysing promoter::GUS reporter lines. Collectively, the results demonstrate that OPC-8:CoA ligase catalyses an essential step in JA biosynthesis by initiating the β-oxidative chain shortening of the carboxylic acid side chain of its precursors, and, in accordance with this function, the protein is localized in peroxisomes.
Publikation

Brüx, A.; Liu, T.-Y.; Krebs, M.; Stierhof, Y.-D.; Lohmann, J. U.; Miersch, O.; Wasternack, C.; Schumacher, K.; Reduced V-ATPase Activity in the trans-Golgi Network Causes Oxylipin-Dependent Hypocotyl Growth Inhibition in Arabidopsis Plant Cell 20, 1088-1100, (2008) DOI: 10.1105/tpc.108.058362

Regulated cell expansion allows plants to adapt their morphogenesis to prevailing environmental conditions. Cell expansion is driven by turgor pressure created by osmotic water uptake and is restricted by the extensibility of the cell wall, which in turn is regulated by the synthesis, incorporation, and cross-linking of new cell wall components. The vacuolar H+-ATPase (V-ATPase) could provide a way to coordinately regulate turgor pressure and cell wall synthesis, as it energizes the secondary active transport of solutes across the tonoplast and also has an important function in the trans-Golgi network (TGN), which affects synthesis and trafficking of cell wall components. We have previously shown that det3, a mutant with reduced V-ATPase activity, has a severe defect in cell expansion. However, it was not clear if this is caused by a defect in turgor pressure or in cell wall synthesis. Here, we show that inhibition of the tonoplast-localized V-ATPase subunit isoform VHA-a3 does not impair cell expansion. By contrast, inhibition of the TGN-localized isoform VHA-a1 is sufficient to restrict cell expansion. Furthermore, we provide evidence that the reduced hypocotyl cell expansion in det3 is conditional and due to active, hormone-mediated growth inhibition caused by a cell wall defect.
Publikation

Stenzel, I.; Hause, B.; Proels, R.; Miersch, O.; Oka, M.; Roitsch, T.; Wasternack, C.; The AOC promoter of tomato is regulated by developmental and environmental stimuli Phytochemistry 69, 1859-1869, (2008) DOI: 10.1016/j.phytochem.2008.03.007

The allene oxide cyclase (AOC) catalyzes the formation of cis-(+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the β-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.
Publikation

Schilling, S.; Wasternack, C.; Demuth, H.-U.; Glutaminyl cyclases from animals and plants: a case of functionally convergent protein evolution Biol. Chem. 389, (2008) DOI: 10.1515/BC.2008.111

Several mammalian peptide hormones and proteins from plant and animal origin contain an N-terminal pyroglutamic acid (pGlu) residue. Frequently, the moiety is important in exerting biological function in either mediating interaction with receptors or stabilizing against N-terminal degradation. Glutaminyl cyclases (QCs) were isolated from different plants and animals catalyzing pGlu formation. The recent resolution of the 3D structures of Carica papaya and human QCs clearly supports different evolutionary origins of the proteins, which is also reflected by different enzymatic mechanisms. The broad substrate specificity is revealed by the heterogeneity of physiological substrates of plant and animal QCs, including cytokines, matrix proteins and pathogenesis-related proteins. Moreover, recent evidence also suggests human QC as a catalyst of pGlu formation at the N-terminus of amyloid peptides, which contribute to Alzheimer's disease. Obviously, owing to its biophysical properties, the function of pGlu in plant and animal proteins is very similar in terms of stabilizing or mediating protein and peptide structure. It is possible that the requirement for catalysis of pGlu formation under physiological conditions may have triggered separate evolution of QCs in plants and animals.
Publikation

Wasternack, C.; Feussner, I.; Multifunctional Enzymes in Oxylipin Metabolism ChemBioChem 9, 2373-2375, (2008) DOI: 10.1002/cbic.200800582

For the first time a member of the CYP74 enzyme subfamily (9‐AOS) from tomato has been shown by chemical and analytical approaches to catalyze multiple reactions. These multifunctional properties of 9‐AOS from the oxylipin‐forming lipoxygenase (LOX) pathway raise several new questions on lipid‐derived signaling.
Publikation

Schüler, G.; Mithöfer, A.; Baldwin, I. T.; BERGER, S.; Ebel, J.; Santos, J. G.; Herrmann, G.; Hölscher, D.; Kramell, R.; Kutchan, T. M.; Maucher, H.; Schneider, B.; Stenzel, I.; Wasternack, C.; Boland, W.; Coronalon: a powerful tool in plant stress physiology FEBS Lett. 563, 17-22, (2004) DOI: 10.1016/S0014-5793(04)00239-X

Coronalon, a synthetic 6‐ethyl indanoyl isoleucine conjugate, has been designed as a highly active mimic of octadecanoid phytohormones that are involved in insect and disease resistance. The spectrum of biological activities that is affected by coronalon was investigated in nine different plant systems specifically responding to jasmonates and/or 12‐oxo‐phytodienoic acid. In all bioassays analyzed, coronalon demonstrated a general strong activity at low micromolar concentrations. The results obtained showed the induction of (i) defense‐related secondary metabolite accumulation in both cell cultures and plant tissues, (ii) specific abiotic and biotic stress‐related gene expression, and (iii) root growth retardation. The general activity of coronalon in the induction of plant stress responses together with its simple and efficient synthesis suggests that this compound might serve as a valuable tool in the examination of various aspects in plant stress physiology. Moreover, coronalon might become employed in agriculture to elicit plant resistance against various aggressors.
Publikation

Miersch, O.; Weichert, H.; Stenzel, I.; Hause, B.; Maucher, H.; Feussner, I.; Wasternack, C.; Constitutive overexpression of allene oxide cyclase in tomato (Lycopersicon esculentum cv. Lukullus) elevates levels of some jasmonates and octadecanoids in flower organs but not in leaves Phytochemistry 65, 847-856, (2004) DOI: 10.1016/j.phytochem.2004.01.016

The allene oxide cyclase (AOC), an enzyme in jasmonate biosynthesis, occurs in vascular bundles and ovules of tomato flowers which exhibit a tissue-specific oxylipin signature (Plant J. 24, 113-126, 2000). Constitutive overexpression of the AOC did not led to altered levels of jasmonates in leaves, but these levels increased upon wounding or other stresses suggesting regulation of jasmonate biosynthesis by substrate availability (Plant J. 33, 577-589, 2003). Here, we show dramatic changes in levels of jasmonic acid (JA), of 12-oxo-phytodienoic acid (OPDA), their methyl esters (JAME, OPDAME), and of dinor-OPDA in most flower organs upon constitutive overexpression of AOC. Beside a dominant occurrence of OPDAME and JA in most flower organs, the ratio among the various compounds was altered differentially in the organs of transgenic flowers, e.g. OPDAME increased up to 53-fold in stamen, and JA increased about 51-fold in buds and 7.5-fold in sepals. The increase in jasmonates and octadecanoids was accompanied by decreased levels of free lipid hydro(per)oxy compounds. Except for 16:2, the AOC overexpression led to a significant increase in free but not esterified polyunsaturated fatty acids in all flower organs. The data suggest different regulation of JA biosynthesis in leaves and flowers of tomato.Constitutive overexpression of the AOC increases in all flower organs levels of some jasmonates and octadecanoids, alters the ratios among the compounds, decreases levels of free lipid hydro(per)oxy compounds and increases levels of free but not of esterified polyunsaturated fatty acids.
Publikation

Maucher, H.; Stenzel, I.; Miersch, O.; Stein, N.; Prasad, M.; Zierold, U.; Schweizer, P.; Dorer, C.; Hause, B.; Wasternack, C.; The allene oxide cyclase of barley (Hordeum vulgare L.)—cloning and organ-specific expression Phytochemistry 65, 801-811, (2004) DOI: 10.1016/j.phytochem.2004.01.009

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199–213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.Barley plants contain one allene oxide cyclase and three allene oxide synthases which are up-regulated during seedling development accompanied by elevated levels of jasmonate.
Publikation

Grubb, C. D.; Zipp, B. J.; Ludwig-Müller, J.; Masuno, M. N.; Molinski, T. F.; Abel, S.; Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis Plant J. 40, 893-908, (2004) DOI: 10.1111/j.1365-313X.2004.02261.x

Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis.
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