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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

Groß, N.; Wasternack, C.; Köck, M.; Wound-induced RNaseLE expression is jasmonate and systemin independent and occurs only locally in tomato (Lycopersicon esculentum cv. Lukullus) Phytochemistry 65, 1343-1350, (2004) DOI: 10.1016/j.phytochem.2004.04.036

Tomato RNaseLE is induced by phosphate deficiency and wounding and may play a role in macromolecular recycling as well as wound healing. Here, we analyzed the role of jasmonate and systemin in the wound-induced RNaseLE activation. The rapid expression of RNaseLE upon wounding of leaves leading to maximal RNase activity within 10 h, appeared only locally. Jasmonic acid (JA) or its molecular mimic ethyl indanoyl isoleucine conjugate did not induce RNaseLE expression. Correspondingly, RNaseLE was expressed upon wounding of 35S::allene oxide cyclase antisense plants known to be JA deficient. RNaseLE was not expressed upon systemin treatment, but was locally expressed in the spr1 mutant which is affected in systemin perception. In tomato plants carrying a PromLE::uidA construct, GUS activity could be detected upon wounding, but not following treatment with JA or systemin. The data indicate a locally acting wound-inducible systemin- and JA-independent signaling pathway for RNaseLE expression.RNaseLE expression was analyzed by pharmacological studies of different tomato lines and upon wounding of leaves. The gene is only locally activated via a new type of wound-induced signaling pathway in a jasmonate/systemin-independent manner.
Publikation

Wasternack, C.; Introductory Remarks on Biosynthesis and Diversity in Actions J. Plant Growth Regul. 23, 167-169, (2004) DOI: 10.1007/s00344-004-0051-1

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Bücher und Buchkapitel

Wasternack, C.; Jasmonates—Biosynthesis and Role in Stress Responses and Developmental Processes 143-155, (2004) DOI: 10.1016/B978-012520915-1/50012-6

This chapter presents jasmonates and their related compounds and discusses jasmonate-induced alteration of gene expression. Jasmonates exerts two different changes in gene expression— decrease in the expression of nuclear- and chloroplast-encoded genes and increase in the expression of specific genes. Jasmonates are shown to alter sink-source relationships such as JA promotes formation of the N-rich vegetative storage proteins—VSPα and VSPβ—of soybean, including reallocation in pod filling. In addition to such nutrient reallocation to other parts of the plant, jasmonates cause decreases in photosynthesis and chlorophyll content, the most significant manifestations of senescence in leaves. The rise of endogenous jasmonates upon stress or exogenous treatment with jasmonates correlates in time with the expression of various genes. The promotion of senescence by jasmonates is counteracted by cytokinins. The capacity of jasmonates to down regulate photosynthetic genes may also be one determinant in the onset of senescence.
Publikation

Stenzel, I.; Hause, B.; Miersch, O.; Kurz, T.; Maucher, H.; Weichert, H.; Ziegler, J.; Feussner, I.; Wasternack, C.; Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana Plant Mol. Biol. 51, 895-911, (2003) DOI: 10.1023/A:1023049319723

In biosynthesis of octadecanoids and jasmonate (JA), the naturally occurring enantiomer is established in a step catalysed by the gene cloned recently from tomato as a single-copy gene (Ziegler et al., 2000). Based on sequence homology, four full-length cDNAs were isolated from Arabidopsis thaliana ecotype Columbia coding for proteins with AOC activity. The expression of AOCgenes was transiently and differentially up-regulated upon wounding both locally and systemically and was induced by JA treatment. In contrast, AOC protein appeared at constitutively high basal levels and was slightly increased by the treatments. Immunohistochemical analyses revealed abundant occurrence of AOC protein as well as of the preceding enzymes in octadecanoid biosynthesis, lipoxygenase (LOX) and allene oxide synthase (AOS), in fully developed tissues, but much less so in 7-day old leaf tissues. Metabolic profiling data of free and esterified polyunsaturated fatty acids and lipid peroxidation products including JA and octadecanoids in wild-type leaves and the jasmonate-deficient mutant OPDA reductase 3 (opr3) revealed preferential activity of the AOS branch within the LOX pathway. 13-LOX products occurred predominantly as esterified derivatives, and all 13-hydroperoxy derivatives were below the detection limits. There was a constitutive high level of free 12-oxo-phytodienoic acid (OPDA) in untreated wild-type and opr3 leaves, but an undetectable expression of AOC. Upon wounding opr3 leaves exhibited only low expression of AOC, wounded wild-type leaves, however, accumulated JA and AOC mRNA. These and further data suggest regulation of JA biosynthesis by OPDA compartmentalization and a positive feedback by JA during leaf development.
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

Schilling, S.; Niestroj, A. J.; Rahfeld, J.-U.; Hoffmann, T.; Wermann, M.; Zunkel, K.; Wasternack, C.; Demuth, H.-U.; Identification of Human Glutaminyl Cyclase as a Metalloenzyme J. Biol. Chem. 278, 49773-49779, (2003) DOI: 10.1074/jbc.M309077200

Human glutaminyl cyclase (QC) was identified as a metalloenzyme as suggested by the time-dependent inhibition by the heterocyclic chelators 1,10-phenanthroline and dipicolinic acid. The effect of EDTA on QC catalysis was negligible. Inactivated enzyme could be fully restored by the addition of Zn2+ in the presence of equimolar concentrations of EDTA. Little reactivation was observed with Co2+ and Mn2+. Other metal ions such as K+, Ca2+, and Ni2+ were inactive under the same conditions. Additionally, imidazole and imidazole derivatives were identified as competitive inhibitors of QC. An initial structure activity-based inhibitor screening of imidazole-derived compounds revealed potent inhibition of QC by imidazole N-1 derivatives. Subsequent data base screening led to the identification of two highly potent inhibitors, 3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one and 1,4-bis-(imidazol-1-yl)-methyl-2,5-dimethylbenzene, which exhibited respective Ki values of 818 ± 1 and 295 ± 5 nm. The binding properties of the imidazole derivatives were further analyzed by the pH dependence of QC inhibition. The kinetically obtained pKa values of 6.94 ± 0.02, 6.93 ± 0.03, and 5.60 ± 0.05 for imidazole, methylimidazole, and benzimidazole, respectively, match the values obtained by titrimetric pKa determination, indicating the requirement for an unprotonated nitrogen for binding to QC. Similarly, the pH dependence of the kinetic parameter Km for the QC-catalyzed conversion of H-Gln-7-ami-no-4-methylcoumarin also implies that only N-terminally unprotonated substrate molecules are bound to the active site of the enzyme, whereas turnover is not affected. The results reveal human QC as a metal-dependent transferase, suggesting that the active site-bound metal is a potential site for interaction with novel, highly potent competitive inhibitors.
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