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Publikation

Mik, V.; Pospíšil, T.; Brunoni, F.; Grúz, J.; Nožková, V.; Wasternack, C.; Miersch, O.; Strnad, M.; Floková, K.; Novák, O.; Široká, J.; Synthetic and analytical routes to the L-amino acid conjugates of cis-OPDA and their identification and quantification in plants Phytochemistry 215, 113855, (2023) DOI: 10.1016/j.phytochem.2023.113855

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.
Publikation

Wasternack, C.; Song, S.; Jasmonates: biosynthesis, metabolism, and signaling by proteins activating and repressing transciption J. Exp. Bot. 68, 1303-1321, (2017) DOI: 10.1093/jxb/erw443

The lipid-derived phytohormone jasmonate (JA) regulates plant growth, development, secondary metabolism, defense against insect attack and pathogen infection, and tolerance to abiotic stresses such as wounding, UV light, salt, and drought. JA was first identified in 1962, and since the 1980s many studies have analyzed the physiological functions, biosynthesis, distribution, metabolism, perception, signaling, and crosstalk of JA, greatly expanding our knowledge of the hormone’s action. In response to fluctuating environmental cues and transient endogenous signals, the occurrence of multilayered organization of biosynthesis and inactivation of JA, and activation and repression of the COI1–JAZ-based perception and signaling contributes to the fine-tuning of JA responses. This review describes the JA biosynthetic enzymes in terms of gene families, enzymatic activity, location and regulation, substrate specificity and products, the metabolic pathways in converting JA to activate or inactivate compounds, JA signaling in perception, and the co-existence of signaling activators and repressors.
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.; Otto, M.; Delker, C.; Kirmse, N.; Schmidt, D.; Miersch, O.; Hause, B.; Wasternack, C.; ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization J. Exp. Bot. 63, 6125-6138, (2012) DOI: 10.1093/jxb/ers261

Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.
Publikation

Wasternack, C.; Hause, B.; Emerging complexity: jasmonate-induced volatiles affect parasitoid choice J. Exp. Bot. 60, 2451-2453, (2009) DOI: 10.1093/jxb/erp197

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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

Kienow, L.; Schneider, K.; Bartsch, M.; Stuible, H.-P.; Weng, H.; Miersch, O.; Wasternack, C.; Kombrink, E.; Jasmonates meet fatty acids: functional analysis of a new acyl-coenzyme A synthetase family from Arabidopsis thaliana J. Exp. Bot. 59, 403-419, (2008) DOI: 10.1093/jxb/erm325

Arabidopsis thaliana contains a large number of genes encoding carboxylic acid-activating enzymes, including long-chain fatty acyl-CoA synthetase (LACS), 4-coumarate:CoA ligases (4CL), and proteins closely related to 4CLs with unknown activities. The function of these 4CL-like proteins was systematically explored by applying an extensive substrate screen, and it was uncovered that activation of fatty acids is the common feature of all active members of this protein family, thereby defining a new group of fatty acyl-CoA synthetase, which is distinct from the known LACS family. Significantly, four family members also displayed activity towards different biosynthetic precursors of jasmonic acid (JA), including 12-oxo-phytodienoic acid (OPDA), dinor-OPDA, 3-oxo-2(2′-[Z]-pentenyl)cyclopentane-1-octanoic acid (OPC-8), and OPC-6. Detailed analysis of in vitro properties uncovered significant differences in substrate specificity for individual enzymes, but only one protein (At1g20510) showed OPC-8:CoA ligase activity. Its in vivo function was analysed by transcript and jasmonate profiling of Arabidopsis insertion mutants for the gene. OPC-8:CoA ligase expression was activated in response to wounding or infection in the wild type but was undetectable in the mutants, which also exhibited OPC-8 accumulation and reduced levels of JA. In addition, the developmental, tissue- and cell-type specific expression pattern of the gene, and regulatory properties of its promoter were monitored by analysing promoter::GUS reporter lines. Collectively, the results demonstrate that OPC-8:CoA ligase catalyses an essential step in JA biosynthesis by initiating the β-oxidative chain shortening of the carboxylic acid side chain of its precursors, and, in accordance with this function, the protein is localized in peroxisomes.
Publikation

ten Hoopen, P.; Hunger, A.; Muller, A.; Hause, B.; Kramell, R.; Wasternack, C.; Rosahl, S.; Conrad, U.; Immunomodulation of jasmonate to manipulate the wound response J. Exp. Bot. 58, 2525-2535, (2007) DOI: 10.1093/jxb/erm122

Jasmonates are signals in plant stress responses and development. The exact mode of their action is still controversial. To modulate jasmonate levels intracellularly as well as compartment-specifically, transgenic Nicotiana tabacum plants expressing single-chain antibodies selected against the naturally occurring (3R,7R)-enantiomer of jasmonic acid (JA) were created in the cytosol and the endoplasmic reticulum. Consequently, the expression of anti-JA antibodies in planta caused JA-deficient phenotypes such as insensitivity of germinating transgenic seedlings towards methyl jasmonate and the loss of wound-induced gene expression. Results presented here suggest an essential role for cytosolic JA in the wound response of tobacco plants. The findings support the view that substrate availability takes part in regulating JA biosynthesis upon wounding. Moreover, high JA levels observed in immunomodulated plants in response to wounding suggest that tobacco plants are able to perceive a reduced level of physiologically active JA and attempt to compensate for this by increased JA accumulation.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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