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Publikationen - Molekulare Signalverarbeitung

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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

Meena, S.; Wagner, C.; Caggegi, L.; Baumann-Kaschig, K.; Ried, M. K.; A user-friendly protocol for the cultivation and successful crossing of Lotus japonicus Bio Protoc. (2021) DOI: 10.21769/p1464

This is a detailed and user-friendly protocol for the cultivation and successful crossing of Lotus japonicus (L. japonicus) e.g. for the generation of higher order mutants, based on methods previously reported (Grant et al., 1962; Handberg and Stougaards, 1992; Jiang and Gresshoff, 1997; Pajuelo and Stougaard, 2005).
Publikationen in Druck

Ried, M. K.; Wild, R.; Zhu, J.; Broger, L.; Harmel, R. K.; Hothorn, L. A.; Fiedler, D.; Hothorn, M.; Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis bioRxiv (2019) DOI: 10.1101/2019.12.13.875393

Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signalling cascades, enabling them to maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signalling molecules (PP-InsPs), which are sensed by SPX-domain containing proteins. In plants, PP-InsP bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8 – SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors.
Publikation

Ried, M. K.; Banhara, A.; Hwu, F.-Y.; Binder, A.; Gust, A. A.; Höfle, C.; Hückelhoven, R.; Nürnberger, T.; Parniske, M.; A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis PLOS Pathog. 15, e1007747, (2019) DOI: 10.1371/journal.ppat.1007747

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved.
Publikation

Girardin, A.; Wang, T.; Ding, Y.; Keller, J.; Buendia, L.; Gaston, M.; Ribeyre, C.; Gasciolli, V.; Auriac, M.-C.; Vernié, T.; Bendahmane, A.; Ried, M. K.; Parniske, M.; Morel, P.; Vandenbussche, M.; Schorderet, M.; Reinhardt, D.; Delaux, P.-M.; Bono, J.-J.; Lefebvre, B.; LCO Receptors Involved in Arbuscular Mycorrhiza Are Functional for Rhizobia Perception in Legumes Curr. Biol. 29, 4249-4259.e5, (2019) DOI: 10.1016/j.cub.2019.11.038

Bacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes.
Publikation

Jung, J.-Y.; Ried, M. K.; Hothorn, M.; Poirier, Y.; Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain Curr. Opin. Biotech. 49, 156-162, (2018) DOI: 10.1016/j.copbio.2017.08.012

Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input.
Publikation

López-Carrasco, A.; Ballesteros, C.; Sentandreu, V.; Delgado, S.; Gago-Zachert, S.; Flores, R.; Sanjuán, R.; Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing PLOS Pathog. 13, e1006547, (2017) DOI: 10.1371/journal.ppat.1006547

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.
Publikation

López-Carrasco, A.; Gago-Zachert, S.; Mileti, G.; Minoia, S.; Flores, R.; Delgado, S.; The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations RNA Biol. 13, 83-97, (2016) DOI: 10.1080/15476286.2015.1119365

Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (−) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.
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.
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