TY - JOUR A1 - Stobiecki, Maciej A1 - Skirycz, Aleksandra A1 - Kerhoas, L. A1 - Kachlicki, P. A1 - Muth, D. A1 - Einhorn, J. A1 - Mueller-Roeber, Bernd T1 - Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS JF - Metabolomics : the official journal of the Metabolomics Society N2 - Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arahidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase H PLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated. KW - liquid chromatography-mass spectrometry KW - metabolite profiling KW - metabolomics KW - flavonoid glycosides Y1 - 2006 U6 - https://doi.org/10.1007/s11306-006-0031-5 SN - 1573-3882 VL - 2 SP - 197 EP - 219 PB - Springer CY - New York ER - TY - GEN A1 - Scarpeci, Telma E. A1 - Zanor, María I. A1 - Carrillo, Néstor A1 - Mueller-Roeber, Bernd A1 - Valle, Estela M. T1 - Generation of superoxide anion in chloroplasts of Arabidopsis thaliana during active photosynthesis BT - a focus on rapidly induced genes T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - The antioxidant defense system involves complex functional coordination of multiple components in different organelles within the plant cell. Here, we have studied the Arabidopsis thaliana early response to the generation of superoxide anion in chloroplasts during active photosynthesis. We exposed plants to methyl viologen (MV), a superoxide anion propagator in the light, and performed biochemical and expression profiling experiments using Affymetrix ATH1 GeneChip(R) microarrays under conditions in which photosynthesis and antioxidant enzymes were active. Data analysis identified superoxide-responsive genes that were compared with available microarray results. Examples include genes encoding proteins with unknown function, transcription factors and signal transduction components. A common GAAAAGTCAAAC motif containing the W-box consensus sequence of WRKY transcription factors, was found in the promoters of genes highly up-regulated by superoxide. Band shift assays showed that oxidative treatments enhanced the specific binding of leaf protein extracts to this motif. In addition, GUS reporter gene fused to WRKY30 promoter, which contains this binding motif, was induced by MV and H2O2. Overall, our study suggests that genes involved in signalling pathways and with unknown functions are rapidly activated by superoxide anion generated in photosynthetically active chloroplasts, as part of the early antioxidant response of Arabidopsis leaves. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 866 KW - antioxidant response KW - chloroplast KW - Hsp KW - oxidative stress KW - WRKY Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-434254 SN - 1866-8372 IS - 866 SP - 361 EP - 378 ER - TY - GEN A1 - Arvidsson, Samuel Janne A1 - Kwasniewski, Miroslaw A1 - Riaño- Pachón, Diego Mauricio A1 - Mueller-Roeber, Bernd T1 - QuantPrime BT - a flexible tool for reliable high-throughput primer design for quantitative PCR T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Background Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays. Results Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses. QuantPrime can be used online through the internet http://www.quantprime.de/ or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%. Conclusion QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of RNA expression assays. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 943 KW - prime pair KW - genome annotation KW - specific prime pair KW - primer pair design KW - quantification protocol Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431531 SN - 1866-8372 IS - 943 ER - TY - GEN A1 - Yang, Lei A1 - Tang, Renjie A1 - Zhu, Jinqi A1 - Liu, Hua A1 - Mueller-Roeber, Bernd A1 - Xia, Huijun A1 - Zhang, Hongxia T1 - Enhancement of stress tolerance in transgenic tobacco plants constitutively expressing AtIpk2β, an inositol polyphosphate 6-/3-kinase from Arabidopsis thaliana T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Inositol phosphates (IPs) and their turnover products have been implicated to play important roles in stress signaling in eukaryotic cells. In higher plants genes encoding inositol polyphosphate kinases have been identified previously, but their physiological functions have not been fully resolved. Here we expressed Arabidopsis inositol polyphosphate 6-/3-kinase (AtIpk2 beta) in two heterologous systems, i.e. the yeast Saccharomyces cerevisiae and in tobacco (Nicotiana tabacum), and tested the effect on abiotic stress tolerance. Expression of AtIpk2 beta rescued the salt-, osmotic- and temperature-sensitive growth defects of a yeast mutant strain (arg82 Delta) that lacks inositol polyphosphate multikinase activity encoded by the ARG82/IPK2 gene. Transgenic tobacco plants constitutively expressing AtIpk2 beta under the control of the Cauliflower Mosaic Virus 35S promoter were generated and found to exhibit improved tolerance to diverse abiotic stresses when compared to wild type plants. Expression patterns of various stress responsive genes were enhanced, and the activities of anti-oxidative enzymes were elevated in transgenic plants, suggesting a possible involvement of AtIpk2 beta in plant stress responses. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 954 KW - arabidopsis thaliana KW - AtIpk2 beta KW - inositol phosphate KW - IP3 KW - stress tolerance KW - transgenic tobacco Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431225 SN - 1866-8372 IS - 954 ER - TY - JOUR A1 - Messerschmidt, Katrin A1 - Hochrein, Lena A1 - Dehm, Daniel A1 - Schulz, Karina A1 - Mueller-Roeber, Bernd T1 - Characterizing seamless ligation cloning extract for synthetic biological applications JF - Analytical biochemistry : methods in the biological sciences N2 - Synthetic biology aims at designing and engineering organisms. The engineering process typically requires the establishment of suitable DNA constructs generated through fusion of multiple protein coding and regulatory sequences. Conventional cloning techniques, including those involving restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments must be joined or scar-free fusions are mandatory. Overlap-based-cloning methods have the potential to overcome such limitations. One such method uses seamless ligation cloning extract (SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone (up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology regions are easily tolerated and the deletion of unwanted vector sequences concomitant with targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion method suitable for cloning projects in virtually all molecular. and synthetic biology projects. (C) 2016 Elsevier Inc. All rights reserved. KW - SLiCE KW - Seamless ligation cloning KW - Homologous recombination KW - Synthetic biology Y1 - 2016 U6 - https://doi.org/10.1016/j.ab.2016.05.029 SN - 0003-2697 SN - 1096-0309 VL - 509 SP - 24 EP - 32 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Hilker, Monika A1 - Schwachtje, Jens A1 - Baier, Margarete A1 - Balazadeh, Salma A1 - Bäurle, Isabel A1 - Geiselhardt, Sven A1 - Hincha, Dirk K. A1 - Kunze, Reinhard A1 - Mueller-Roeber, Bernd A1 - Rillig, Matthias G. A1 - Rolff, Jens A1 - Schmülling, Thomas A1 - Steppuhn, Anke A1 - van Dongen, Joost A1 - Whitcomb, Sarah J. A1 - Wurst, Susanne A1 - Zuther, Ellen A1 - Kopka, Joachim T1 - Priming and memory of stress responses in organisms lacking a nervous system JF - Biological reviews KW - priming KW - stress signalling KW - epigenetics KW - memory KW - fitness KW - stress tolerance KW - defence KW - bet hedging Y1 - 2016 U6 - https://doi.org/10.1111/brv.12215 SN - 1464-7931 SN - 1469-185X VL - 91 SP - 1118 EP - 1133 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Hochrein, Lena A1 - Machens, Fabian A1 - Gremmels, Juergen A1 - Schulz, Karina A1 - Messerschmidt, Katrin A1 - Mueller-Roeber, Bernd T1 - AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies JF - Nucleic acids research N2 - The assembly of large DNA constructs coding for entire pathways poses a major challenge in the field of synthetic biology. Here, we present AssemblX, a novel, user-friendly and highly efficient multi-gene assembly strategy. The software-assisted AssemblX process allows even unexperienced users to rapidly design, build and test DNA constructs with currently up to 25 functional units, from 75 or more subunits. At the gene level, AssemblX uses scar-free, overlap-based and sequence-independent methods, allowing the unrestricted design of transcriptional units without laborious parts domestication. The assembly into multi-gene modules is enabled via a standardized, highly efficient, polymerase chain reaction-free and virtually sequence-independent scheme, which relies on rare cutting restriction enzymes and optimized adapter sequences. Selection and marker switching strategies render the whole process reliable, rapid and very effective. The assembly product can be easily transferred to any desired expression host, making AssemblX useful for researchers from various fields. Y1 - 2017 U6 - https://doi.org/10.1093/nar/gkx034 SN - 0305-1048 SN - 1362-4962 VL - 45 PB - Oxford Univ. Press CY - Oxford ER - TY - JOUR A1 - Shahnejat-Bushehri, Sara A1 - Allu, Annapurna Devi A1 - Mehterov, Nikolay A1 - Thirumalaikumar, Venkatesh P. A1 - Alseekh, Saleh A1 - Fernie, Alisdair R. A1 - Mueller-Roeber, Bernd A1 - Balazadeh, Salma T1 - Arabidopsis NAC Transcription Factor JUNGBRUNNEN1 Exerts Conserved Control Over Gibberellin and Brassinosteroid Metabolism and Signaling Genes in Tomato JF - Frontiers in plant science N2 - The Arabidopsis thaliana NAC transcription factor JUNGBRUNNEN1 (AtJUB1) regulates growth by directly repressing GA3ox1 and DWF4, two key genes involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis, respectively, leading to GA and BR deficiency phenotypes. AtJUB1 also reduces the expression of PIF4, a bHLH transcription factor that positively controls cell elongation, while it stimulates the expression of DELLA genes, which are important repressors of growth. Here, we extend our previous findings by demonstrating that AtJUB1 induces similar GA and BR deficiency phenotypes and changes in gene expression when overexpressed in tomato (Solanum lycopersicum). Importantly, and in accordance with the growth phenotypes observed, AtJUB1 inhibits the expression of growth-supporting genes, namely the tomato orthologs of GA3ox1, DWF4 and PIF4, but activates the expression of DELLA orthologs, by directly binding to their promoters. Overexpression of AtJUB1 in tomato delays fruit ripening, which is accompanied by reduced expression of several ripeningrelated genes, and leads to an increase in the levels of various amino acids (mostly proline, beta-alanine, and phenylalanine), gamma-aminobutyric acid (GABA), and major organic acids including glutamic acid and aspartic acid. The fact that AtJUB1 exerts an inhibitory effect on the GA/BR biosynthesis and PIF4 genes but acts as a direct activator of DELLA genes in both, Arabidopsis and tomato, strongly supports the model that the molecular constituents of the JUNGBRUNNEN1 growth control module are considerably conserved across species. KW - Arabidopsis KW - tomato KW - fruit KW - growth KW - transcription factor KW - gibberellic acid KW - brassinosteroid KW - DELLA proteins Y1 - 2017 U6 - https://doi.org/10.3389/fpls.2017.00214 SN - 1664-462X VL - 8 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Ebrahimian-Motlagh, Saghar A1 - Ribone, Pamela A. A1 - Thirumalaikumar, Venkatesh P. A1 - Allu, Annapurna Devi A1 - Chan, Raquel L. A1 - Mueller-Roeber, Bernd A1 - Balazadeh, Salma T1 - JUNGBRUNNEN1 Confers Drought Tolerance Downstream of the HD-Zip I Transcription Factor AtHB13 JF - Frontiers in plant science N2 - Low water availability is the major environmental factor limiting growth and productivity of plants and crops and is therefore considered of high importance for agriculture affected by climate change. Identifying regulatory components controlling the response and tolerance to drought stress is thus of major importance. The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) from Arabidopsis thaliana extends leaf longevity under non-stress growth conditions, lowers cellular hydrogen peroxide (H2O2) level, and enhances tolerance against heat stress and salinity. Here, we additionally find that JUB1 strongly increases tolerance to drought stress in Arabidopsis when expressed from both, a constitutive (CaMV 35S) and an abiotic stress-induced (RD29A) promoter. Employing a yeast one-hybrid screen we identified HD-Zip class I TF AtHB13 as an upstream regulator of JUB1. AtHB13 has previously been reported to act as a positive regulator of drought tolerance. AtHB13 and JUB1 thereby establish a joint drought stress control module. KW - Arabidopsis KW - transcription factor KW - drought KW - JUB1 KW - HB13 Y1 - 2017 U6 - https://doi.org/10.3389/fpls.2017.02118 SN - 1664-462X VL - 8 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Omidbakhshfard, Mohammad Amin A1 - Fujikura, Ushio A1 - Olas, Justyna Jadwiga A1 - Xue, Gang-Ping A1 - Balazadeh, Salma A1 - Mueller-Roeber, Bernd T1 - GROWTH-REGULATING FACTOR 9 negatively regulates arabidopsis leaf growth by controlling ORG3 and restricting cell proliferation in leaf primordia JF - PLoS Genetics : a peer-reviewed, open-access journal N2 - Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the Arabidopsis thaliana TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF OBP3-RESPONSIVE GENE 3 (ORG3). While grf9 knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing GRF9 (GRF9ox). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that GRF9 restricts cell proliferation in the early developing leaf. Performing in vitro binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified ORG3 as a direct downstream, and positively regulated target of GRF9. Genetic analysis of grf9 org3 and GRF9ox org3 double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf. Y1 - 2018 U6 - https://doi.org/10.1371/journal.pgen.1007484 SN - 1553-7404 VL - 14 IS - 7 PB - PLoS CY - San Fransisco ER -