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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

Floß, D. S.; Hause, B.; Lange, P. R.; Küster, H.; Strack, D.; Walter, M. H.; Knock-down of the MEP pathway isogene 1-deoxy-d-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes Plant J. 56, 86-100, (2008) DOI: 10.1111/j.1365-313X.2008.03575.x

The first step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by two isoforms of 1‐deoxy‐d‐ xylulose 5‐phosphate synthase (DXS1 and DXS2). In Medicago truncatula , MtDXS1 and MtDXS2 genes exhibit completely different expression patterns. Most prominently, colonization by arbuscular mycorrhizal (AM) fungi induces the accumulation of certain apocarotenoids (cyclohexenone and mycorradicin derivatives) correlated with the expression of MtDXS2 but not of MtDXS1. To prove a distinct function of DXS2, a selective RNAi approach on MtDXS2 expression was performed in transgenic hairy roots of M. truncatula. Repression of MtDXS2 consistently led to reduced transcript levels in mycorrhizal roots, and to a concomitant reduction of AM‐induced apocarotenoid accumulation. The transcript levels of MtDXS1 remained unaltered in RNAi plants, and no phenotypical changes in non‐AM plants were observed. Late stages of the AM symbiosis were adversely affected, but only upon strong repression with residual MtDXS2‐1 transcript levels remaining below approximately 10%. This condition resulted in a strong decrease in the transcript levels of MtPT4 , an AM‐specific plant phosphate transporter gene, and in a multitude of other AM‐induced plant marker genes, as shown by transcriptome analysis. This was accompanied by an increased proportion of degenerating and dead arbuscules at the expense of mature ones. The data reveal a requirement for DXS2‐dependent MEP pathway‐based isoprenoid products to sustain mycorrhizal functionality at later stages of the symbiosis. They further validate the concept of a distinct role for DXS2 in secondary metabolism, and offer a novel tool to selectively manipulate the levels of secondary isoprenoids by targeting their precursor supply.
Publikation

Fellenberg, C.; Milkowski, C.; Hause, B.; Lange, P.-R.; Böttcher, C.; Schmidt, J.; Vogt, T.; Tapetum-specific location of a cation-dependent O-methyltransferase in Arabidopsis thaliana Plant J. 56, 132-145, (2008) DOI: 10.1111/j.1365-313X.2008.03576.x

Cation‐ and S ‐adenosyl‐l ‐methionine (AdoMet)‐dependent plant natural product methyltransferases are referred to as CCoAOMTs because of their preferred substrate, caffeoyl coenzyme A (CCoA). The enzymes are encoded by a small family of genes, some of which with a proven role in lignin monomer biosynthesis. In Arabidopsis thaliana individual members of this gene family are temporally and spatially regulated. The gene At1g67990 is specifically expressed in flower buds, and is not detected in any other organ, such as roots, leaves or stems. Several lines of evidence indicate that the At1g67990 transcript is located in the flower buds, whereas the corresponding CCoAOMT‐like protein, termed AtTSM1, is located exclusively in the tapetum of developing stamen. Flowers of At1g67990 RNAi‐suppressed plants are characterized by a distinct flower chemotype with severely reduced levels of the N  ′,N  ′′‐ bis‐(5‐hydroxyferuloyl)‐N  ′′′‐sinapoylspermidine compensated for by N1 ,N5 ,N10 ‐tris‐(5‐hydroxyferuloyl)spermidine derivative, which is characterized by the lack of a single methyl group in the sinapoyl moiety. This severe change is consistent with the observed product profile of AtTSM1 for aromatic phenylpropanoids. Heterologous expression of the recombinant protein shows the highest activity towards a series of caffeic acid esters, but 5‐hydroxyferuloyl spermidine conjugates are also accepted substrates. The in vitro substrate specificity and the in vivo RNAi‐mediated suppression data of the corresponding gene suggest a role of this cation‐dependent CCoAOMT‐like protein in the stamen/pollen development of A. thaliana .
Publikation

Schilling, S.; Stenzel, I.; von Bohlen, A.; Wermann, M.; Schulz, K.; Demuth, H.-U.; Wasternack, C.; Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions Biol. Chem. 388, 145-153, (2007) DOI: 10.1515/BC.2007.016

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 105- to 106-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.
Publikation

Delker, C.; Stenzel, I.; Hause, B.; Miersch, O.; Feussner, I.; Wasternack, C.; Jasmonate Biosynthesis in Arabidopsis thaliana - Enzymes, Products, Regulation Plant Biol. 8, 297-306, (2006) DOI: 10.1055/s-2006-923935

Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.
Publikation

Stenzel, I.; Hause, B.; Maucher, H.; Pitzschke, A.; Miersch, O.; Ziegler, J.; Ryan, C. A.; Wasternack, C.; Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling Plant J. 33, 577-589, (2003) DOI: 10.1046/j.1365-313X.2003.01647.x

The allene oxide cyclase (AOC)‐catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue‐specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i) activation of systemin‐dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii) the tissue‐specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii) the tissue‐specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.
Publikation

Schilling, S.; Manhart, S.; Hoffmann, T.; Ludwig, H.-H.; Wasternack, C.; Demuth, H.-U.; Substrate Specificity of Glutaminyl Cyclases from Plants and Animals Biol. Chem. 384, 1583-1592, (2003) DOI: 10.1515/BC.2003.175

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-?homoglutaminyl residue in the peptide H-?homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo( N?-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates gastrin, neurotensin and [GlN1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.
Publikation

Bachmann, A.; Hause, B.; Maucher, H.; Garbe, E.; Vörös, K.; Weichert, H.; Wasternack, C.; Feussner, I.; Jasmonate-Induced Lipid Peroxidation in Barley Leaves Initiated by Distinct 13-LOX Forms of Chloroplasts Biol. Chem. 383, 1645-1657, (2002) DOI: 10.1515/BC.2002.185

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [Vörös et al., Eur. J. Biochem. 251 (1998), 36 44], two fulllength cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonatetreated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenasederived products in the stroma and in the envelope. These data revealed jasmonateinduced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.
Publikation

Miersch, O.; Wasternack, C.; Octadecanoid and Jasmonate Signaling in Tomato (Lycopersicon esculentum Mill.) Leaves: Endogenous Jasmonates Do Not Induce Jasmonate Biosynthesis Biol. Chem. 381, 715-722, (2000) DOI: 10.1515/BC.2000.092

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.
Publikation

Maucher, H.; Hause, B.; Feussner, I.; Ziegler, J.; Wasternack, C.; Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development Plant J. 21, 199-213, (2000) DOI: 10.1046/j.1365-313x.2000.00669.x

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.
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