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Publikationen - Stress- und Entwicklungsbiologie

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Publikation

Furlan, G., Nakagami, H., Eschen-Lippold, L., Jiang, X., Majovsky, P., Kowarschik, K., Hoehenwarter, W., Lee, J. & Trujillo, M. Changes in PUB22 ubiquitination modes triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 dampen the immune response Plant Cell 29, 726-745, (2017) DOI: 10.1105/tpc.16.00654

Crosstalk between post-translational modifications such as ubiquitination and phosphorylation play key roles in controlling the duration and intensity of signalling events to ensure cellular homeostasis. However, the molecular mechanisms underlying the regulation of negative feedback loops remain poorly understood. Here we uncover a pathway in Arabidopsis thaliana by which a negative feedback loop involving the E3 ubiquitin ligase PUB22 that dampens the immune response is triggered by MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3), best known for its function in the activation of signalling. PUB22's stability is controlled by MPK3-mediated phosphorylation of residues localized in and adjacent to the E2 docking domain. We show that phosphorylation is critical for stabilization by inhibiting PUB22 oligomerization and thus autoubiquitination. The activity switch allows PUB22 to dampen the immune response. This regulatory mechanism also suggests that autoubiquitination, which is inherent to most single unit E3s in vitro, can function as a self-regulatory mechanism in vivo. 
Publikation

Ziegler, J., Schmidt, S., Strehmel, N., Scheel, D. & Abel, S. Arabidopsis transporter ABCG37/PDR9 contributes primarily highly oxygenated coumarins to root exudation.  Scientific 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

Strehmel, N., Hoehenwarter, W., Mönchgesang, S., Majovsky, P., Krüger, S., Scheel, D. & Lee, J. Stress-reated mitogen-activated protein kinases stimulate the accumulation of small molecules and proteins in Arabidopsis thaliana root exudates. Front Plant Sci 8 , 1292, (2017) DOI: 10.3389/fpls.2017.01292

A delicate balance in cellular signaling is required for plants to respond to microorganisms or to changes in their environment. Mitogen-activated protein kinase (MAPK) cascades are one of the signaling modules that mediate transduction of extracellular microbial signals into appropriate cellular responses. Here, we employ a transgenic system that simulates activation of two pathogen/stress-responsive MAPKs to study release of metabolites and proteins into root exudates. The premise is based on our previous proteomics study that suggests upregulation of secretory processes in this transgenic system. An advantage of this experimental set-up is the direct focus on MAPK-regulated processes without the confounding complications of other signaling pathways activated by exposure to microbes or microbial molecules. Using non-targeted metabolomics and proteomics studies, we show that MAPK activation can indeed drive the appearance of dipeptides, defense-related metabolites and proteins in root apoplastic fluid. However, the relative levels of other compounds in the exudates were decreased. This points to a bidirectional control of metabolite and protein release into the apoplast. The putative roles for some of the identified apoplastic metabolites and proteins are discussed with respect to possible antimicrobial/defense or allelopathic properties. Overall, our findings demonstrate that sustained activation of MAPKs alters the composition of apoplastic root metabolites and proteins, presumably to influence the plant-microbe interactions in the rhizosphere. The reported metabolomics and proteomics data are available via Metabolights (Identifier: MTBLS441) and ProteomeXchange (Identifier: PXD006328), respectively.
Publikationen in Druck

Palm-Forster, M. A. T., Eschen-Lippold, L., Uhrig, J., Scheel, D. & Lee, J. A novel family of proline/serine-rich proteins, which are phospho-targets of stress-related mitogen-activated protein kinases, differentially regulates growth and pathogen defense in Arabidopsis thaliana. Plant Mol Biol (2017) DOI: 10.1007/s11103-017-0641-5

The molecular actions of mitogen-activated protein kinases (MAPKs) are ultimately accomplished by the substrate proteins where phosphorylation affects their molecular properties and function(s), but knowledge regarding plant MAPK substrates is currently still fragmentary. Here, we uncovered a previously uncharacterized protein family consisting of three proline/serine-rich proteins (PRPs) that are substrates of stress-related MAPKs. We demonstrated the importance of a MAPK docking domain necessary for protein–protein interaction with MAPKs and consequently also for phosphorylation. The main phosphorylated site was mapped to a residue conserved between all three proteins, which when mutated to a non-phosphorylatable form, differentially affected their protein stability. Together with their distinct gene expression patterns, this differential accumulation of the three proteins upon phosphorylation probably contributes to their distinct function(s). Transgenic over-expression of PRP, the founding member, led to plants with enhanced resistance to Pseudomonas syringae pv. tomato DC3000. Older plants of the over-expressing lines have curly leaves and were generally smaller in stature. This growth phenotype was lost in plants expressing the phosphosite variant, suggesting a phosphorylation-dependent effect. Thus, this novel family of PRPs may be involved in MAPK regulation of plant development and / or pathogen resistance responses. As datamining associates PRP expression profiles with hypoxia or oxidative stress and PRP-overexpressing plants have elevated levels of reactive oxygen species, PRP may connect MAPK and oxidative stress signaling.
Publikation

Witting, M., Ruttkies, C., Neumann, S. & Schmitt-Kopplin, P. LipidFrag: Improving reliability of in silico fragmentation of lipids and application to the Caenorhabditis elegans lipidome.  PLoS ONE 12, e0172311, (2017) DOI: 10.1371/journal.pone.0172311

Lipid identification is a major bottleneck in high-throughput lipidomics studies. However, tools for the analysis of lipid tandem MS spectra are rather limited. While the comparison against spectra in reference libraries is one of the preferred methods, these libraries are far from being complete. In order to improve identification rates, the in silico fragmentation tool MetFrag was combined with Lipid Maps and lipid-class specific classifiers which calculate probabilities for lipid class assignments. The resulting LipidFrag workflow was trained and evaluated on different commercially available lipid standard materials, measured with data dependent UPLC-Q-ToF-MS/MS acquisition. The automatic analysis was compared against manual MS/MS spectra interpretation. With the lipid class specific models, identification of the true positives was improved especially for cases where candidate lipids from different lipid classes had similar MetFrag scores by removing up to 56% of false positive results. This LipidFrag approach was then applied to MS/MS spectra of lipid extracts of the nematode Caenorhabditis elegans. Fragments explained by LipidFrag match known fragmentation pathways, e.g., neutral losses of lipid headgroups and fatty acid side chain fragments. Based on prediction models trained on standard lipid materials, high probabilities for correct annotations were achieved, which makes LipidFrag a good choice for automated lipid data analysis and reliability testing of lipid identifications.
Publikationen in Druck

Meier, R., Ruttkies, C., Treutler, H. & Neumann, S. Bioinformatics can boost metabolomics research.  J. Biotechnol. (2017) DOI: 10.1016/j.jbiotec.2017.05.018

Metabolomics is the modern term for the field of small molecule research in biology and biochemistry. Currently, metabolomics is undergoing a transition where the classic analytical chemistry is combined with modern cheminformatics and bioinformatics methods, paving the way for large-scale data analysis. We give some background on past developments, highlight current state-of-the-art approaches, and give a perspective on future requirements.
Publikation

Schymanski, E. L., Ruttkies, C., Krauss, M., Brouard, C., Kind, T., Dührkop, K., Allen, F., Vaniya, A., Verdegem, D., Böcker, S., Rousu, J., Shen, H., Tsugawa, H., Sajed, T., Fiehn, O., Ghesquière, B. & Neumann, S. Critical assessment of small molecule identification 2016: automated methods. J. Cheminformatics 9, 22, (2017) DOI: 10.1186/s13321-017-0207-1

Background
The fourth round of the Critical Assessment of Small Molecule Identification (CASMI) Contest (www.casmi-contest.org) was held in 2016, with two new categories for automated methods. This article covers the 208 challenges in Categories 2 and 3, without and with metadata, from organization, participation, results and post-contest evaluation of CASMI 2016 through to perspectives for future contests and small molecule annotation/identification.

Results
The Input Output Kernel Regression (CSI:IOKR) machine learning approach performed best in “Category 2: Best Automatic Structural Identification—In Silico Fragmentation Only”, won by Team Brouard with 41% challenge wins. The winner of “Category 3: Best Automatic Structural Identification—Full Information” was Team Kind (MS-FINDER), with 76% challenge wins. The best methods were able to achieve over 30% Top 1 ranks in Category 2, with all methods ranking the correct candidate in the Top 10 in around 50% of challenges. This success rate rose to 70% Top 1 ranks in Category 3, with candidates in the Top 10 in over 80% of the challenges. The machine learning and chemistry-based approaches are shown to perform in complementary ways.

Conclusions
The improvement in (semi-)automated fragmentation methods for small molecule identification has been substantial. The achieved high rates of correct candidates in the Top 1 and Top 10, despite large candidate numbers, open up great possibilities for high-throughput annotation of untargeted analysis for “known unknowns”. As more high quality training data becomes available, the improvements in machine learning methods will likely continue, but the alternative approaches still provide valuable complementary information. Improved integration of experimental context will also improve identification success further for “real life” annotations. The true “unknown unknowns” remain to be evaluated in future CASMI contests.
Publikationen in Druck

Herz, K., Dietz, S., Haider, S., Jandt, U., Scheel, D. & Bruelheide, H.  Drivers of intraspecific trait variation of grass and forb species in German meadows and pastures J. Vegetation Sci (2017) DOI: 10.1111/jvs.12534

Questions
To what extent is trait variation in grasses and forbs driven by land-use intensity, climate, soil conditions and plant diversity of the local neighbourhood? Do grass and forb species differ in the degree of intraspecific trait variation?

Location
Managed grasslands in three regions of Germany.

Methods
Using a phytometer approach, we raised 20 common European grassland species (ten forbs and ten grasses) and planted them into 54 plots of different land-use types (pasture, meadow, mown pasture). After 1 yr in the field, we measured above- and below-ground plant functional traits. Linear mixed effects models (LMEM) were used to identify the most powerful predictors for every trait. Variation partitioning was applied to assess the amount of inter- and intraspecific trait variation in grasses and forbs explained by environmental conditions (land-use intensity, climate and soil conditions) and plant species diversity of the local neighbourhood.

Results
For 12 out of the 14 traits studied, either land-use intensity or local neighbourhood diversity were predictors in the best LMEM. Land-use intensity had considerably stronger effects than neighbourhood diversity. Root dry matter content and root phosphorus concentration of forbs were more affected by land-use intensity than those of grasses. For almost all traits, intraspecific trait variation of grasses was much higher than that of forbs, while traits of forbs varied more among species. Overall, inter- and intraspecific variation was of the same magnitude.

Conclusion
The similar magnitude of intra- and interspecific trait variation suggests that both sources should be considered in grassland studies at a scale similar to that of our study. The high amount of intraspecific trait variation that was explained by environmental factors and local neighbourhood diversity clearly demonstrates the high potential of species to adjust to local conditions, which would be ignored when only considering species mean trait values.

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Publikation

Nakano, R. T., Piślewska-Bednarek, M., Yamada, K., Edger, P. P., Miyahara, M., Kondo, M., Böttcher, C., Mori, M., Nishimura, M., Schulze-Lefert, P., Hara-Nishimura, I. & Bednarek, P. PYK10 myrosinase reveals a functional coordination between endoplasmic reticulum bodies and glucosinolates in Arabidopsis thaliana Plant J. 89, 204-220, (2017) DOI: 10.1111/tpj.13377

The endoplasmic reticulum body (ER body) is an organelle derived from the ER that occurs in only three families of the order Brassicales and is suggested to be involved in plant defense. ER bodies in Arabidopsis thaliana contain large amounts of β-glucosidases, but the physiological functions of ER bodies and these enzymes remain largely unclear. Here we show that PYK10, the most abundant β-glucosidase in A. thaliana root ER bodies, hydrolyzes indole glucosinolates (IGs) in addition to the previously reported in vitro substrate scopolin. We found a striking co-expression between ER body-related genes (including PYK10), glucosinolate biosynthetic genes and the genes for so-called specifier proteins affecting the terminal products of myrosinase-mediated glucosinolate metabolism, indicating that these systems have been integrated into a common transcriptional network. Consistent with this, comparative metabolite profiling utilizing a number of A. thaliana relatives within Brassicaceae identified a clear phylogenetic co-occurrence between ER bodies and IGs, but not between ER bodies and scopolin. Collectively, our findings suggest a functional link between ER bodies and glucosinolate metabolism in planta. In addition, in silico three-dimensional modeling, combined with phylogenomic analysis, suggests that PYK10 represents a clade of 16 myrosinases that arose independently from the other well-documented class of six thioglucoside glucohydrolases. These findings provide deeper insights into how glucosinolates are metabolized in cruciferous plants and reveal variation of the myrosinase–glucosinolate system within individual plants.
Publikation

Küster, N., Rosahl, S. & Dräger, B. Potato plants with genetically engineered tropane alkaloid precursors Planta 245 , 355-365, (2017) DOI: 10.1007/s00425-016-2610-7

Solanum tuberosumtropinone reductase I reduced tropinone in vivo. Suppression of tropinone reductase II strongly reduced calystegines in sprouts. Overexpression of putrescineN-methyltransferase did not alter calystegine accumulation.

Calystegines are hydroxylated alkaloids formed by the tropane alkaloid pathway. They accumulate in potato (Solanum tuberosum L., Solanaceae) roots and sprouting tubers. Calystegines inhibit various glycosidases in vitro due to their sugar-mimic structure, but functions of calystegines in plants are not understood. Enzymes participating in or competing with calystegine biosynthesis, including putrescine N-methyltransferase (PMT) and tropinone reductases (TRI and TRII), were altered in their activity in potato plants by RNA interference (RNAi) and by overexpression. The genetically altered potato plants were investigated for the accumulation of calystegines and for intermediates of their biosynthesis. An increase in N-methylputrescine provided by DsPMT expression was not sufficient to increase calystegine accumulation. Overexpression and gene knockdown of StTRI proved that S. tuberosum TRI is a functional tropinone reductase in vivo, but no influence on calystegine accumulation was observed. When StTRII expression was suppressed by RNAi, calystegine formation was severely compromised in the transformed plants. Under phytochamber and green house conditions, the StTRII RNAi plants did not show phenotypic alterations. Further investigation of calystegines function in potato plants under natural conditions is enabled by the calystegine deprived StTRII RNAi plants.

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