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Preprints

Mik, V.; Poslíšil, 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 ChemRxiv (2023) DOI: 10.26434/chemrxiv-2023-qlzj4

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, similar to other phytohormones. 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 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 reached a maximum of pmol/g. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Current synthetic and analytical methodologies support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.
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

Serra, P.; Carbonell, A.; Navarro, B.; Gago-Zachert, S.; Li, S.; Di Serio, F.; Flores, R.; Symptomatic plant viroid infections in phytopathogenic fungi: A request for a critical reassessment Proc. Natl. Acad. Sci. U.S.A. 117, 10126-10128, (2020) DOI: 10.1073/pnas.1922249117

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Bücher und Buchkapitel

Flores, R.; Gago-Zachert, S.; Serra, P.; De la Peña, M.; Navarro, B.; Chrysanthemum Chlorotic Mottle Viroid (Hadidi, A., et al., eds.). 331-338, (2017) DOI: 10.1016/B978-0-12-801498-1.00031-0

Chrysanthemum chlorotic mottle viroid (CChMVd) (398–401 nt) belongs to genus Pelamoviroid, family Avsunviroidae and, like other members of this family, replicates in plastids through a rolling-circle mechanism involving hammerhead ribozymes. CChMVd RNA adopts a branched conformation stabilized by a kissing-loop interaction, resembling peach latent mosaic viroid in this respect. Chrysanthemum is the only natural and experimental host for CChMVd, which in the most sensitive varieties induces leaf mottling and chlorosis, delay in flowering, and dwarfing. The viroid has been found in major chrysanthemum growing areas including Europe and Asia. There are natural variants in which the change (UUUC→GAAA) mapping at a tetraloop in the CChMVd branched conformation is sufficient to change the symptomatic phenotype into a nonsymptomatic one without altering the viroid titer. Preinfection with nonsymptomatic variants prevents challenge inoculation with symptomatic ones. Moreover, experimental coinoculation with symptomatic and nonsymptomatic CChMVd variants results in symptomless phenotypes only when the latter is in vast excess, thus indicating its lower fitness.
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

Arnold, M. D.; Gruber, C.; Floková, K.; Miersch, O.; Strnad, M.; Novák, O.; Wasternack, C.; Hause, B.; The Recently Identified Isoleucine Conjugate of cis-12-Oxo-Phytodienoic Acid Is Partially Active in cis-12-Oxo-Phytodienoic Acid-Specific Gene Expression of Arabidopsis thaliana PLOS ONE 11, e0162829, (2016) DOI: 10.1371/journal.pone.0162829

Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.
Publikation

Flores, R.; Gago-Zachert, S.; Serra, P.; Sanjuán, R.; Elena, S. F.; Viroids: Survivors from the RNA World? Annu. Rev. Microbiol. 68, 395-414, (2014) DOI: 10.1146/annurev-micro-091313-103416

Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario—the so-called RNA world—existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G + C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.
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

Reginato, M.; Abdala, G.; Miersch, O.; Ruiz, O.; Moschetti, E.; Luna, V.; Changes in the levels of jasmonates and free polyamines induced by Na2SO4 and NaCl in roots and leaves of the halophyte Prosopis strombulifera Biologia 67, 689-697, (2012) DOI: 10.2478/s11756-012-0052-7

Prosopis strombulifera, a common legume in high-salinity soils of Argentina, is a useful model for elucidation of salt tolerance mechanisms and specific biochemical pathways in halophytes, since its NaCl tolerance exceeds the limit described for most halophytic plants. We analyzed the effects of the increasing concentration of two main soil salts, Na2SO4 and NaCl, on growth parameters of P. strombulifera, chlorophyll levels, and content of jasmonates (JAs) and polyamines (PAs), which are key molecules involved in stress responses. P. strombulifera showed a halophytic response (growth promotion) to NaCl, but strong growth inhibition by iso-osmotic solutions of Na2SO4. Chlorophyll levels, number of leaves and leaf area were also differentially affected. An important finding was the partial alleviation of SO42− toxicity by treatment with two-salt mixture. JAs are not directly involved in salt tolerance in this species since its levels decrease under all salt treatments. Beneficial effects of Putrescine (Put) accumulation in NaCl treated plants maybe inferred probably associated with the antioxidative defense system. Another novel finding is the accumulation of the uncommon PA cadaverine in roots under high Na2SO4, which may be related to SO42− toxicity.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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