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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

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.

Publikationen in Druck

Al Shweiki, M. H. D. R., Mönchgesang, S., Majovsky, P., Thieme, D., Trutschel, D. & Hoehenwarter, W. Assessment of Label-Free quantification in discovery proteomics and impact of technological factors and natural variability of protein abundance. J Proteome Res. (2017) DOI: 10.1021/acs.jproteome.6b00645

We evaluated the state of label-free discovery proteomics focusing especially on technological contributions and contributions of naturally occurring differences in protein abundance to the intersample variability in protein abundance estimates in this highly peptide-centric technology. First, the performance of popular quantitative proteomics software, Proteome Discoverer, Scaffold, MaxQuant, and Progenesis QIP, was benchmarked using their default parameters and some modified settings. Beyond this, the intersample variability in protein abundance estimates was decomposed into variability introduced by the entire technology itself and variable protein amounts inherent to individual plants of the Arabidopsis thaliana Col-0 accession. The technical component was considerably higher than the biological intersample variability, suggesting an effect on the degree and validity of reported biological changes in protein abundance. Surprisingly, the biological variability, protein abundance estimates, and protein fold changes were recorded differently by the software used to quantify the proteins, warranting caution in the comparison of discovery proteomics results. As expected, ∼99% of the proteome was invariant in the isogenic plants in the absence of environmental factors; however, few proteins showed substantial quantitative variability. This naturally occurring variation between individual organisms can have an impact on the causality of reported protein fold changes.

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. 

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