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Publikationen - Molekulare Signalverarbeitung

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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

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

Kogel, K.-H.; Ortel, B.; Jarosch, B.; Atzorn, R.; Schiffer, R.; Wasternack, C.; Resistance in barley against the powdery mildew fungus (Erysiphe graminis f.sp.hordei) is not associated with enhanced levels of endogenous jasmonates Eur. J. Plant Pathol. 101, 319-332, (1995) DOI: 10.1007/BF01874788

Onset of acquired resistance of barley (Hordeum vulgare) chemically induced by 2,6-dichloroisonicotinic acid (DCINA) correlated with the accumulation of mRNA homologous to cDNA pHvJ256 which codes for a soluble leaf-thionin with a Mr. of 6 kDa [Wasternacket al., 1994a]. In the present work, we extend this finding by showing that the thionin transcript also accumulated following treatment of barley with the resistance-inducing compounds 3,5-dichlorosalicylic acid (DCSA), salicylic acid (SA), and an extract fromBacillus subtilis. The polypeptide showed antifungal activity against the biotrophic cereal pathogensErysiphe graminis f.sp.hordei andPuccinia graminis f.sp.tritici which may indicate a possible role in the mechanism of acquired resistance in barley. A thionin transcript hybridizing to pHvJ256 accumulated also in response to application of jasmonates, or treatments that elevated endogenous amounts of the plant growth substance, pointing to the possibility that signaling mediating defense responses in barley involves jasmonates. However, a topical spray application of jasmonic acid (JA) or jasmonate methyl ester (JM) did not protect barley leaves against infection byE. graminis. Performing a kinetic analysis by an enzyme immunoassay specific for (−)-JA, (−)-JM, and its amino acid conjugates, accumulation of jasmonates was detected in osmotically stressed barley but not at the onset of chemically induced or genetically based resistance governed by the powdery mildew resistance genesMlg, Mla 12, ormlo 5. Furthermore, the jasmonate-inducible proteins JIP-23 and JIP-60 were strongly induced following JM- but not DCINA-treatment or inoculation withE. graminis. Hence, in barley, no indications were found in favour for the previously proposed model of a lipid-based signaling pathway via jasmonates mediating expression of resistance in plants against pathogens.
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