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Publikation

Floková, K.; Feussner, K.; Herrfurth, C.; Miersch, O.; Mik, V.; Tarkowská, D.; Strnad, M.; Feussner, I.; Wasternack, C.; Novák, O.; A previously undescribed jasmonate compound in flowering Arabidopsis thaliana – The identification of cis-(+)-OPDA-Ile Phytochemistry 122, 230-237, (2016) DOI: 10.1016/j.phytochem.2015.11.012

Jasmonates (JAs) are plant hormones that integrate external stress stimuli with physiological responses. (+)-7-iso-JA-L-Ile is the natural JA ligand of COI1, a component of a known JA receptor. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA) has been reported to act independently of COI1 as an essential signal in several stress-induced and developmental processes. Wound-induced increases in the endogenous levels of JA/JA-Ile are accompanied by two to tenfold increases in the concentration of OPDA, but its means of perception and metabolism are unknown. To screen for putative OPDA metabolites, vegetative tissues of flowering Arabidopsis thaliana were extracted with 25% aqueous methanol (v/v), purified by single-step reversed-phase polymer-based solid-phase extraction, and analyzed by high throughput mass spectrometry. This enabled the detection and quantitation of a low abundant OPDA analog of the biologically active (+)-7-iso-JA-L-Ile in plant tissue samples. Levels of the newly identified compound and the related phytohormones JA, JA-Ile and cis-(+)-OPDA were monitored in wounded leaves of flowering Arabidopsis lines (Col-0 and Ws) and compared to the levels observed in Arabidopsis mutants deficient in the biosynthesis of JA (dde2-2, opr3) and JA-Ile (jar1). The observed cis-(+)-OPDA-Ile levels varied widely, raising questions concerning its role in Arabidopsis stress responses.
Publikation

Floková, K.; Tarkowská, D.; Miersch, O.; Strnad, M.; Wasternack, C.; Novák, O.; UHPLC–MS/MS based target profiling of stress-induced phytohormones Phytochemistry 105, 147-157, (2014) DOI: 10.1016/j.phytochem.2014.05.015

Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20 mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA.
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

Delker, C.; Zolman, B. K.; Miersch, O.; Wasternack, C.; Jasmonate biosynthesis in Arabidopsis thaliana requires peroxisomal β-oxidation enzymes – Additional proof by properties of pex6 and aim1 Phytochemistry 68, 1642-1650, (2007) DOI: 10.1016/j.phytochem.2007.04.024

Jasmonic acid (JA) is an important regulator of plant development and stress responses. Several enzymes involved in the biosynthesis of JA from α-linolenic acid have been characterized. The final biosynthesis steps are the β-oxidation of 12-oxo-phytoenoic acid. We analyzed JA biosynthesis in the Arabidopsis mutants pex6, affected in peroxisome biogenesis, and aim1, disrupted in fatty acid β-oxidation. Upon wounding, these mutants exhibit reduced JA levels compared to wild type. pex6 accumulated the precursor OPDA. Feeding experiments with deuterated OPDA substantiate this accumulation pattern, suggesting the mutants are impaired in the β-oxidation of JA biosynthesis at different steps. Decreased expression of JA-responsive genes, such as VSP1, VSP2, AtJRG21 and LOX2, following wounding in the mutants compared to the wild type reflects the reduced JA levels of the mutants. By use of these additional mutants in combination with feeding experiments, the necessity of functional peroxisomes for JA-biosynthesis is confirmed. Furthermore an essential function of one of the two multifunctional proteins of fatty acid β-oxidation (AIM1) for wound-induced JA formation is demonstrated for the first time. These data confirm that JA biosynthesis occurs via peroxisomal fatty acid β-oxidation machinery.
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

Sharma, V. K.; Monostori, T.; Göbel, C.; Hänsch, R.; Bittner, F.; Wasternack, C.; Feussner, I.; Mendel, R. R.; Hause, B.; Schulze, J.; Transgenic barley plants overexpressing a 13-lipoxygenase to modify oxylipin signature Phytochemistry 67, 264-276, (2006) DOI: 10.1016/j.phytochem.2005.11.009

Three chimeric gene constructs were designed comprising the full length cDNA of a lipoxygenase (LOX) from barley (LOX2:Hv:1) including its chloroplast targeting sequence (cTP) under control of either (1) CaMV35S- or (2) polyubiquitin-1-promoter, whereas the third plasmid contains 35S promoter and the cDNA without cTP. Transgenic barley plants overexpressing LOX2:Hv:1 were generated by biolistics of scutella from immature embryos. Transformation frequency for 35S::LOX with or without cTP was in a range known for barley particle bombardment, whereas for Ubi::cTP-LOX no transgenic plants were detected. In general, a high number of green plantlets selected on bialaphos became yellow and finally died either in vitro or after potting. All transgenic plants obtained were phenotypically indistinguishable from wild type plants and all of them set seeds. The corresponding protein (LOX-100) in transgenic T0 and T1 plants accumulated constitutively to similar levels as in the jasmonic acid methyl ester (JAME)-treated wild type plants. Moreover, LOX-100 was clearly detectable immunocytochemically within the chloroplasts of untreated T0 plants containing the LOX-100-cDNA with the chloroplast target sequence. In contrast, an exclusive localization of LOX-100 in the cytoplasm was detectable when the target sequence was removed. In comparison to sorbitol-treated wild type leaves, analysis of oxylipin profiles in T2 progenies showed higher levels of jasmonic acid (JA) for those lines that displayed elevated levels of LOX-100 in the chloroplasts and for those lines that harboured LOX-100 in the cytoplasm, respectively. The studies demonstrate for the first time the constitutive overexpression of a cDNA coding for a 13-LOX in a monocotyledonous species and indicate a link between the occurrence of LOX-100 and senescence.
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.
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