@phdthesis{Zhang2014, author = {Zhang, Yi}, title = {Regulation of Agrobacterial Oncogene Expression in Host Plants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102578}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2014}, abstract = {Virulent Agrobacterium tumefaciens strains transfer and integrate a DNA region of the tumor-inducing (Ti) plasmid, the T-DNA, into the plant genome and thereby cause crown gall disease. The most essential genes required for crown gall development are the T-DNA-encoded oncogenes, IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) for auxin, and Ipt (isopentenyl transferase) for cytokinin biosynthesis. When these oncogenes are expressed in the host cell, the levels of auxin and cytokinin increase and cause cell proliferation. The aim of this study was to unravel the molecular mechanisms, which regulate expression of the agrobacterial oncogenes in plant cells. Transcripts of the three oncogenes were expressed in Arabidopsis thaliana crown galls induced by A. tumefaciens strain C58 and the intergenic regions (IGRs) between their coding sequences (CDS) were proven to have promoter activity in plant cells. These promoters possess eukaryotic sequence structures and contain cis-regulatory elements for the binding of plant transcription factors. The high-throughput protoplast transactivation (PTA) system was used and identified the Arabidopsis thaliana transcription factors WRKY18, WRKY40, WRKY60 and ARF5 to activate the Ipt oncogene promoter. No transcription factor promoted the activity of the IaaH and IaaM promoters, despite the fact that the sequences contained binding elements for type B ARR transcription factors. Likewise, the treatment of Arabidopsis mesophyll protoplasts with cytokinin (trans-zeatin) and auxin (1-NAA) exerted no positive effect on IaaH and IaaM promoter activity. In contrast, the Ipt promoter strongly responded to a treatment with auxin and only modestly to cytokinin. The three Arabidopsis WRKYs play a role in crown gall development as the wrky mutants developed smaller crown galls than wild-type plants. The WRKY40 and WRKY60 genes responded very quickly to pathogen infection, two and four hours post infection, respectively. Transcription of the WRKY18 gene was induced upon buffer infiltration, which implicates a response to wounding. The three WRKY proteins interacted with ARF5 and with each other in the plant nucleus, but only WRKY40 together with ARF5 increased activation of the Ipt promoter. Moreover, ARF5 activated the Ipt promoter in an auxin-dependent manner. The severe developmental phenotype of the arf5 mutant prevented studies on crown gall development, nevertheless, the reduced crown gall growth on the transport inhibitor response 1 (TIR1) tir1 mutant, lacking the auxin sensor, suggested that auxin signaling is required for optimal crown gall development. In conclusion, A. tumefaciens recruits the pathogen defense related WRKY40 pathway to activate Ipt expression in T-DNA-transformed plant cells. IaaH and IaaM gene expression seems not to be controlled by transcriptional activators, but the increasing auxin levels are signaled via ARF5. The auxin-depended activation of ARF5 boosts expression of the Ipt gene in combination with WRKY40 to increase cytokinin levels and induce crown gall development.}, subject = {Agrobacterium tumefaciens}, language = {en} } @article{TomeNaegeleAdamoetal.2014, author = {Tome, Filipa and N{\"a}gele, Thomas and Adamo, Mattia and Garg, Abhroop and Marco-Ilorca, Carles and Nukarinen, Ella and Pedrotti, Lorenzo and Peviani, Alessia and Simeunovic, Andrea and Tatkiewicz, Anna and Tomar, Monika and Gamm, Magdalena}, title = {The low energy signaling network}, series = {Frontiers in Plant Science}, volume = {5}, journal = {Frontiers in Plant Science}, number = {353}, issn = {1664-462X}, doi = {10.3389/fpls.2014.00353}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115813}, year = {2014}, abstract = {Stress impacts negatively on plant growth and crop productivity, causing extensive losses to agricultural production worldwide. Throughout their life, plants are often confronted with multiple types of stress that affect overall cellular energy status and activate energy-saving responses. The resulting low energy syndrome (LES) includes transcriptional, translational, and metabolic reprogramming and is essential for stress adaptation. The conserved kinases sucrose-non-fermenting-1-related protein kinase-1 (SnRK1) and target of rapamycin (TOR) play central roles in the regulation of LES in response to stress conditions, affecting cellular processes and leading to growth arrest and metabolic reprogramming. We review the current understanding of how TOR and SnRK1 are involved in regulating the response of plants to low energy conditions. The central role in the regulation of cellular processes, the reprogramming of metabolism, and the phenotypic consequences of these two kinases will be discussed in light of current knowledge and potential future developments.}, language = {en} } @article{SzambowskaTessmerKursulaetal.2014, author = {Szambowska, Anna and Tessmer, Ingrid and Kursula, Petri and Usskilat, Christian and Prus, Potr and Pospiech, Helmut and Grosse, Frank}, title = {DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding}, series = {Nucleic Acids Research}, volume = {42}, journal = {Nucleic Acids Research}, number = {4}, issn = {1362-4962}, doi = {10.1093/nar/gkt1217}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117538}, pages = {2308-2319}, year = {2014}, abstract = {The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.}, language = {en} } @article{SchmittKuperEliasetal.2014, author = {Schmitt, Dominik R. and Kuper, Jochen and Elias, Agnes and Kisker, Caroline}, title = {The Structure of the TFIIH p34 Subunit Reveals a Von Willebrand Factor A Like Fold}, series = {PLoS ONE}, volume = {9}, journal = {PLoS ONE}, number = {7}, issn = {1932-6203}, doi = {10.1371/journal.pone.0102389}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119471}, pages = {e102389}, year = {2014}, abstract = {RNA polymerase II dependent transcription and nucleotide excision repair are mediated by a multifaceted interplay of subunits within the general transcription factor II H (TFIIH). A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these vital cellular processes. The importance of this complex becomes further evident in the context of severe diseases like xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy, that arise from single point mutations in TFIIH subunits. Here we describe the structure of the p34 subunit of the TFIIH complex from the eukaryotic thermophilic fungus Chaetomium thermophilum. The structure revealed that p34 contains a von Willebrand Factor A (vWA) like domain, a fold which is generally known to be involved in protein-protein interactions. Within TFIIH p34 strongly interacts with p44, a positive regulator of the helicase XPD. Putative protein-protein interfaces are analyzed and possible binding sites for the p34-p44 interaction suggested.}, language = {en} } @article{RoederGeillingerZieteketal.2014, author = {R{\"o}der, Pia V. and Geillinger, Kerstin E. and Zietek, Tamara S. and Thorens, Bernard and Koepsell, Hermann and Daniel, Hannelore}, title = {The Role of SGLT1 and GLUT2 in Intestinal Glucose Transport and Sensing}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {2}, doi = {10.1371/journal.pone.0089977}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117262}, pages = {e89977}, year = {2014}, abstract = {Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.}, language = {en} } @article{OliAbdelmohsenHentscheletal.2014, author = {Oli, Swarna and Abdelmohsen, Usama Ramadan and Hentschel, Ute and Schirmeister, Tanja}, title = {Identification of Plakortide E from the Caribbean Sponge Plakortis halichondroides as a Trypanocidal Protease Inhibitor using Bioactivity-Guided Fractionation}, series = {MARINE DRUGS}, volume = {12}, journal = {MARINE DRUGS}, number = {5}, issn = {1660-3397}, doi = {10.3390/md12052614}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116536}, pages = {2614-2622}, year = {2014}, abstract = {In this paper, we report new protease inhibitory activity of plakortide E towards cathepsins and cathepsin-like parasitic proteases. We further report on its anti-parasitic activity against Trypanosoma brucei with an IC50 value of 5 mu M and without cytotoxic effects against J774.1 macrophages at 100 mu M concentration. Plakortide E was isolated from the sponge Plakortis halichondroides using enzyme assay-guided fractionation and identified by NMR spectroscopy and mass spectrometry. Furthermore, enzyme kinetic studies confirmed plakortide E as a non-competitive, slowly-binding, reversible inhibitor of rhodesain.}, language = {en} } @article{MacintyreZhangViegelmannetal.2014, author = {Macintyre, Lynsey and Zhang, Tong and Viegelmann, Christina and Martinez, Ignacio Juarez and Cheng, Cheng and Dowdells, Catherine and Abdelmohsen, Usama Ramadan and Gernert, Christine and Hentschel, Ute and Edrada-Ebel, RuAngelie}, title = {Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts}, series = {Marine Drugs}, volume = {12}, journal = {Marine Drugs}, number = {6}, issn = {1660-3397}, doi = {10.3390/md12063416}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116097}, pages = {3416-3448}, year = {2014}, abstract = {Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR H-1 and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.}, language = {en} } @article{KreuzwieserScheererKruseetal.2014, author = {Kreuzwieser, J{\"u}rgen and Scheerer, Ursel and Kruse, J{\"o}rg and Burzlaff, Tim and Honsel, Anne and Alfarraj, Saleh and Georgiev, Palmen and Schnitzler, J{\"o}rg-Peter and Ghirardo, Andrea and Kreuzer, Ines and Hedrich, Rainer and Rennenberg, Heinz}, title = {The Venus flytrap attracts insects by the release of volatile organic compounds}, series = {Journal of Experimental Botany}, volume = {65}, journal = {Journal of Experimental Botany}, number = {2}, doi = {10.1093/jxb/ert455}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121161}, pages = {755-66}, year = {2014}, abstract = {Does Dionaea muscipula, the Venus flytrap, use a particular mechanism to attract animal prey? This question was raised by Charles Darwin 140 years ago, but it remains unanswered. This study tested the hypothesis that Dionaea releases volatile organic compounds (VOCs) to allure prey insects. For this purpose, olfactory choice bioassays were performed to elucidate if Dionaea attracts Drosophila melanogaster. The VOCs emitted by the plant were further analysed by GC-MS and proton transfer reaction-mass spectrometry (PTR-MS). The bioassays documented that Drosophila was strongly attracted by the carnivorous plant. Over 60 VOCs, including terpenes, benzenoids, and aliphatics, were emitted by Dionaea, predominantly in the light. This work further tested whether attraction of animal prey is affected by the nutritional status of the plant. For this purpose, Dionaea plants were fed with insect biomass to improve plant N status. However, although such feeding altered the VOC emission pattern by reducing terpene release, the attraction of Drosophila was not affected. From these results it is concluded that Dionaea attracts insects on the basis of food smell mimicry because the scent released has strong similarity to the bouquet of fruits and plant flowers. Such a volatile blend is emitted to attract insects searching for food to visit the deadly capture organ of the Venus flytrap.}, language = {en} } @article{JahnSchmidtMock2014, author = {Jahn, Martin T. and Schmidt, Katrin and Mock, Thomas}, title = {A novel cost effective and high-throughput isolation and identification method for marine microalgae}, series = {Plant Methods}, volume = {10}, journal = {Plant Methods}, number = {26}, doi = {10.1186/1746-4811-10-26}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121255}, year = {2014}, abstract = {BACKROUND: Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures. RESULTS: In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms. CONCLUSIONS: We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures."}, language = {en} } @article{HyunvanderGraaffAlbaceteetal.2014, author = {Hyun, Tae Kyung and van der Graaff, Eric and Albacete, Alfonso and Eom, Seung Hee and Grosskinsky, Dominik K. and B{\"o}hm, Hannah and Janschek, Ursula and Rim, Yeonggil and Ali, Walid Wahid and Kim, Soo Young and Roitsch, Thomas}, title = {The Arabidopsis PLAT Domain Protein1 is Critically Involved in Abiotic Stress Tolerance}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {11}, doi = {10.1371/journal.pone.0112946}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114648}, pages = {e112946}, year = {2014}, abstract = {Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.}, language = {en} }