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Publikation

Wasternack, C.; How Jasmonates Earned their Laurels: Past and Present J. Plant Growth Regul. 34, 761-794, (2015) DOI: 10.1007/s00344-015-9526-5

The histories of research regarding all plant hormones are similar. Identification and structural elucidation have been followed by analyses of their biosynthesis, distributions, signaling cascades, roles in developmental or stress response programs, and crosstalk. Jasmonic acid (JA) and its derivatives comprise a group of plant hormones that were discovered recently, compared to auxin, abscisic acid, cytokinins, gibberellic acid, and ethylene. Nevertheless, there have been tremendous advances in JA research, following the general progression outlined above and parallel efforts focused on several other “new” plant hormones (brassinosteroids, salicylate, and strigolactones). This review focuses on historical aspects of the identification of jasmonates, and characterization of their biosynthesis, distribution, perception, signaling pathways, crosstalk with other hormones and roles in plant stress responses and development. The aim is to illustrate how our present knowledge on jasmonates was generated and how that influences current efforts to extend our knowledge.
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

Fortes, A. M.; Miersch, O.; Lange, P. R.; Malhó, R.; Testillano, P. S.; Risueño, M. d. C.; Wasternack, C.; Pais, M. S.; Expression of Allene Oxide Cyclase and Accumulation of Jasmonates during Organogenic Nodule Formation from Hop (Humulus lupulus var. Nugget) Internodes Plant Cell Physiol. 46, 1713-1723, (2005) DOI: 10.1093/pcp/pci187

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its stereoisomeric precursor, cis-(+)-12-oxophytodienoic acid (OPDA), which is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). A cDNA of AOC was isolated from Humulus lupulus var. Nugget. The ORF of 765 bp encodes a 255 amino acid protein, which carries a putative chloroplast targeting sequence. The recombinant protein without its putative chloroplast target sequence showed significant AOC activity. Previously we demonstrated that wounding induces organogenic nodule formation in hop. Here we show that the AOC transcript level increases in response to wounding of internodes, peaking between 2 and 4 h after wounding. In addition, Western blot analysis showed elevated levels of AOC peaking 24 h after internode inoculation. The AOC increase was accompanied by increased JA levels 24 h after wounding, whereas OPDA had already reached its highest level after 12 h. AOC is mostly present in the vascular bundles of inoculated internodes. During prenodule and nodule formation, AOC levels were still high. JA and OPDA levels decreased down to 10 and 118 pmol (g FW)–1, respectively, during nodule formation, but increased during plantlet regeneration. Double immunolocalization analysis of AOC and Rubisco in connection with lugol staining showed that AOC is present in amyloplasts of prenodular cells and in the chloroplasts of vacuolated nodular cells, whereas meristematic cells accumulated little AOC. These data suggest a role of AOC and jasmonates in organogenic nodule formation and plantlet regeneration from these nodules.
Publikation

Sharma, V. K.; Monostori, T.; Hause, B.; Maucher, H.; Göbel, C.; Hornung, E.; Hänsch, R.; Bittner, F.; Wasternack, C.; Feussner, I.; Mendel, R. R.; Schulze, J.; Genetic transformation of barley to modify expression of a 13-lipoxygenase Acta Biol. Szeged. 49, 33-34, (2005)

Immature scutella of barley were transformed with cDNA coding for a 13-lipoxygenase of barley (LOX-100) via particle bombardment. Regenerated plants were tested by PAT-assay, Western-analysis and PCR-screening. Immunocytochemical assay of T0 plants showed expression of the LOX cDNA both in the chloroplasts and in the cytosol, depending on the presence of the chloroplast signal peptide sequences in the cDNA. A few transgenic plants containing higher amounts of LOX-derived products have been found. These are the candidates for further analysis concerning pathogen resistance.
Publikation

Schneider, K.; Kienow, L.; Schmelzer, E.; Colby, T.; Bartsch, M.; Miersch, O.; Wasternack, C.; Kombrink, E.; Stuible, H.-P.; A New Type of Peroxisomal Acyl-Coenzyme A Synthetase from Arabidopsis thaliana Has the Catalytic Capacity to Activate Biosynthetic Precursors of Jasmonic Acid J. Biol. Chem. 280, 13962-13972, (2005) DOI: 10.1074/jbc.M413578200

Arabidopsis thaliana contains a large number of genes that encode carboxylic acid-activating enzymes, including nine long-chain fatty acyl-CoA synthetases, four 4-coumarate:CoA ligases (4CL), and 25 4CL-like proteins of unknown biochemical function. Because of their high structural and sequence similarity with bona fide 4CLs and their highly hydrophobic putative substrate-binding pockets, the 4CL-like proteins At4g05160 and At5g63380 were selected for detailed analysis. Following heterologous expression, the purified proteins were subjected to a large scale screen to identify their preferred in vitro substrates. This study uncovered a significant activity of At4g05160 with medium-chain fatty acids, medium-chain fatty acids carrying a phenyl substitution, long-chain fatty acids, as well as the jasmonic acid precursors 12-oxo-phytodienoic acid and 3-oxo-2-(2′-pentenyl)-cyclopentane-1-hexanoic acid. The closest homolog of At4g05160, namely At5g63380, showed high activity with long-chain fatty acids and 12-oxo-phytodienoic acid, the latter representing the most efficiently converted substrate. By using fluorescent-tagged variants, we demonstrated that both 4CL-like proteins are targeted to leaf peroxisomes. Collectively, these data demonstrate that At4g05160 and At5g63380 have the capacity to contribute to jasmonic acid biosynthesis by initiating the β-oxidative chain shortening of its precursors.
Publikation

Rudus, I.; Kepczynska, E.; Kepczynski, J.; Wasternack, C.; Miersch, O.; Changes in jasmonates and 12-oxophytodienoic acid contents of Medicago sativa L. during somatic embryogenesis Acta Physiol. Plant. 27, 497-504, (2005) DOI: 10.1007/s11738-005-0055-x

Jasmonic acid (JA), its methyl ester (MeJA) and the biosynthetic precursor 12-oxophytodienoic acid (OPDA) were detected quantitatively during somatic embryogenesis of Medicago sativa L. Using GC-MS analysis, these compounds were found in initial explants, in calli and in somatic embryos in the nanogram range per gram of fresh weight. In distinct stages of somatic embryogenesis, JA and 12-OPDA accumulated preferentially in cotyledonary embryos. Initial explants exhibited about five-fold higher JA content than OPDA content, whereas in other stages OPDA accumulated predominantly. These data suggest that also in embryogenic tissues OPDA and JA may have individual signalling properties.
Publikation

Ludwig, A. A.; Saitoh, H.; Felix, G.; Freymark, G.; Miersch, O.; Wasternack, C.; Boller, T.; Jones, J. D. G.; Romeis, T.; Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants Proc. Natl. Acad. Sci. U.S.A. 102, 10736-10741, (2005) DOI: 10.1073/pnas.0502954102

Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.
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.
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