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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

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium, .; Corrigendum: ICTV Virus Taxonomy Profile: Ophioviridae J. Gen. Virol. 99, 949-949, (2018) DOI: 10.1099/jgv.0.001093

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Publikation

Ziegler, J.; Schmidt, S.; Strehmel, N.; Scheel, D.; Abel, S.; Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation Sci. Rep. 7, 3704, (2017) DOI: 10.1038/s41598-017-03250-6

The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.
Publikation

García, M. L.; Bó, E. D.; da Graça, J. V.; Gago-Zachert, S.; Hammond, J.; Moreno, P.; Natsuaki, T.; Pallás, V.; Navarro, J. A.; Reyes, C. A.; Luna, G. R.; Sasaya, T.; Tzanetakis, I. E.; Vaira, A. M.; Verbeek, M.; ICTV Report Consortium, .; ICTV Virus Taxonomy Profile: Ophioviridae J. Gen. Virol. 98, 1161-1162, (2017) DOI: 10.1099/jgv.0.000836

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3–12.5 kb divided into 3–4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.
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

Ziegler, J.; Schmidt, S.; Chutia, R.; Müller, J.; Böttcher, C.; Strehmel, N.; Scheel, D.; Abel, S.; Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation J. Exp. Bot. 67, 1421-1432, (2016) DOI: 10.1093/jxb/erv539

Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.
Publikation

Strehmel, N.; Mönchgesang, S.; Herklotz, S.; Krüger, S.; Ziegler, J.; Scheel, D.; Piriformospora indica Stimulates Root Metabolism of Arabidopsis thaliana Int. J. Mol. Sci. 17, 1091, (2016) DOI: 10.3390/ijms17071091

Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana’s roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes
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

Buhtz, A.; Witzel, K.; Strehmel, N.; Ziegler, J.; Abel, S.; Grosch, R.; Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae PLOS ONE 10, e0138242, (2015) DOI: 10.1371/journal.pone.0138242

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.
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