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Publikationen - Molekulare Signalverarbeitung

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Publikation

Bochnia, M., Scheidemann, W., Ziegler, J., Sander, J., Vollstedt, S., Glatter, M., Janzen, N., Terhardt, M. & Zeyner, A. Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Veterinary Education 30, 24-28, (2018) DOI: 10.1111/eve.12596

Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]-conjugates) increased in body fluids of the AM horse (MCPA-carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA-carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA-conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA-conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA-conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.

Publikation

Ibañez, C., Delker, C., Martinez, C., Bürstenbinder, K., Janitza, P., Lippmann, R., Ludwig, W., Sun, H., James, G. V., Klecker, M., Grossjohann, A., Schneeberger, K., Prat, S. & Quint, M. Brassinosteroids dominate hormonal regulation of plant thermomorphogenesis via BZR1 Curr Biol 28, 303-310.e3, (2018) DOI: 10.1016/j.cub.2017.11.077

Thermomorphogenesis is defined as the suite of morphological changes that together are likely to contribute to adaptive growth acclimation to usually elevated ambient temperature [ 1, 2 ]. While many details of warmth-induced signal transduction are still elusive, parallels to light signaling recently became obvious (reviewed in [ 3 ]). It involves photoreceptors that can also sense changes in ambient temperature [ 3–5 ] and act, for example, by repressing protein activity of the central integrator of temperature information PHYTOCHROME-INTERACTING FACTOR 4 (PIF4 [ 6 ]). In addition, PIF4 transcript accumulation is tightly controlled by the evening complex member EARLY FLOWERING 3 [ 7, 8 ]. According to the current understanding, PIF4 activates growth-promoting genes directly but also via inducing auxin biosynthesis and signaling, resulting in cell elongation. Based on a mutagenesis screen in the model plant Arabidopsis thaliana for mutants with defects in temperature-induced hypocotyl elongation, we show here that both PIF4 and auxin function depend on brassinosteroids. Genetic and pharmacological analyses place brassinosteroids downstream of PIF4 and auxin. We found that brassinosteroids act via the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), which accumulates in the nucleus at high temperature, where it induces expression of growth-promoting genes. Furthermore, we show that at elevated temperature BZR1 binds to the promoter of PIF4, inducing its expression. These findings suggest that BZR1 functions in an amplifying feedforward loop involved in PIF4 activation. Although numerous negative regulators of PIF4 have been described, we identify BZR1 here as a true temperature-dependent positive regulator of PIF4, acting as a major growth coordinator.
Publikation

Gantner, J., Ordon, J., Ilse, T., Kretschmer, C., Gruetzner, R., Löfke, C., Dagdas, Y., Bürstenbinder, K., Marillonnet, S. & Stuttmann, J. Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system PLoS ONE 13, e0197185, (2018) DOI: 10.1371/journal.pone.0197185

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
Publikation

Bagchi, R., Melnyk, C. W., Christ, G., Winkler, M., Kirchsteiner, K., Salehin, M., Mergner, J., Niemeyer, M., Schwechheimer, C., Calderón Villalobos, L. I. A. & Estelle, M. The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases. EMBO J 37, 255-268, (2018) DOI: 10.15252/embj.201797159

The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.
Publikation

Wasternack, C. & Feussner, I. The Oxylipin Pathways: Biochemistry and Function Annu Rev Plant Biol 69, 363-386, (2018) DOI: 10.1146/annurev-arplant-042817-040440

Plant oxylipins form a constantly growing group of signaling molecules that comprise oxygenated fatty acids and metabolites derived therefrom. In the last decade, the understanding of biosynthesis, metabolism, and action of oxylipins, especially jasmonates, has dramatically improved. Additional mechanistic insights into the action of enzymes and insights into signaling pathways have been deepened for jasmonates. For other oxylipins, such as the hydroxy fatty acids, individual signaling properties and cross talk between different oxylipins or even with additional phytohormones have recently been described. This review summarizes recent understanding of the biosynthesis, regulation, and function of oxylipins.
Publikation

Wasternack, C. & Hause, B. A Bypass in Jasmonate Biosynthesis – the OPR3-independent Formation Trends Plant Sci 23, 276-279, (2018) DOI: 10.1016/j.tplants.2018.02.011

For the first time in 25 years, a new pathway for biosynthesis of jasmonic acid (JA) has been identified. JA production takes place via 12-oxo-phytodienoic acid (OPDA) including reduction by OPDA reductases (OPRs). A loss-of-function allele, opr3-3, revealed an OPR3-independent pathway converting OPDA to JA.
Publikation

Krägeloh, T., Cavalleri, J. M. V., Ziegler, J., Sander, J., Terhardt, M., Breves, G. & Cehak, A. Identification of hypoglycin a binding adsorbents as potential preventive measures in co-grazers of atypical myopathy affected horses. Equine Vet J. 50, 220-227, (2018) DOI: 10.1111/evj.12723

Summary

Background

Intestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches.

Objectives

To identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin.

Study design

Experimental in vitro study.

Methods

Substances commonly used in equine practice (activated charcoal composition, di-tri-octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)-conjugates was investigated by analysing their concentrations in samples from the Ussing chambers.

Results

Activated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism.

Main limitations

Binding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration.

Conclusions

For the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication.
Publikation

Nishiyama, T., Sakayama, H., de Vries, J., Buschmann, H., Saint-Marcoux, D., Ullrich, K. K., Haas, F. B., Vanderstraeten, L., Becker, D., Lang, D., Vosolsobě, S., Rombauts, S., Wilhelmsson, P. K. I., Janitza, P., Kern, R., Heyl, A., Rümpler, F, Calderón Villalobos, L. I. A., Clay, J. M., Skokan, R., Toyoda, A., Suzuki, Y., Kagoshima, H., Schijlen, E., Tajeshwar, N., Catarino, B., Hetherington, A. J., Saltykova, A., Bonnot, C., Breuninger, H., Symeonidi, A., Radhakrishnan, G. V., Van Nieuwerburgh, F., Deforce, D., Chang, C., Karol, K. G., Hedrich, R., Ulvskov, P., Glöckner, G., Delwiche, C. F., Petrášek, J., Van de Peer, Y., Friml, J., Beilby, M., Dolan, L., Kohara, Y., Sugano, S., Fujiyama, A., Delaux, P.-M., Quint, M., Theißen, G., Hagemann, M., Harholt, J., Dunand, C., Zachgo, S., Langdale, J., Maumus, F., Van Der Straeten, D., Gould, S. B. & Rensing, S. A. The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization Cell 174, 448-464.e24, (2018) DOI: 10.1016/j.cell.2018.06.033

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.
Publikation

White, M. D., Klecker, M., Hopkinson, R. J., Weits, D. A., Mueller, C., Naumann, C., O’Neill, R., Wickens, J., Yang, J., Brooks-Bartlett, J. C., Garman, E. F., Grossmann, T. N., Dissmeyer, N. & Flashman, E. Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets Nature Commun 8, 14690, (2017) DOI: 10.1038/ncomms14690

Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.
Publikation

Möller, B., Pöschl, Y., Plötner, R. & Bürstenbinder, K. PaCeQuant: A tool for high-throughput quantification of pavement cell shape characteristics. Plant Physiol. 175, 988-1017, (2017) DOI: 10.1104/pp.17.00961

Pavement cells (PCs) are the most frequently occurring cell type in the leaf epidermis and play important roles in leaf growth and function. In many plant species, PCs form highly complex jigsaw puzzle shaped cells with interlocking lobes. Understanding of their development is of high interest for plant science research because of their importance for leaf growth and hence for plant fitness and crop yield. Studies of PC development, however, are limited because robust methods are lacking that enable automatic segmentation and quantification of PC shape parameters suitable to reflect their cellular complexity. Here, we present our new ImageJ-based tool, PaCeQuant, which provides a fully automatic image analysis workflow for PC shape quantification. PaCeQuant automatically detects cell boundaries of PCs from confocal input images, and enables manual correction of automatic segmentation results or direct import of manually segmented cells. PaCeQuant simultaneously extracts 27 shape features that include global, contour-based, skeleton-based and PC-specific object descriptors. In addition, we included a method for classification and analysis of lobes at two-cell-junctions and three-cell-junctions, respectively. We provide an R script for graphical visualization and statistical analysis. We validated PaCeQuant by extensive comparative analysis to manual segmentation and existing quantification tools, and demonstrated its usability to analyze PC shape characteristics during development and between different genotypes. PaCeQuant thus provides a platform for robust, efficient and reproducible quantitative analysis of PC shape characteristics that can easily be applied to study PC development in large data sets.
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