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Publikationen - Stoffwechsel- und Zellbiologie

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Publikation

Mielke, K.; Forner, S.; Kramell, R.; Conrad, U.; Hause, B.; Cell-specific visualization of jasmonates in wounded tomato and Arabidopsis leaves using jasmonate-specific antibodies New Phytol. 190, 1069-1080, (2011) DOI: 10.1111/j.1469-8137.2010.03638.x

Jasmonates are well‐characterized signals in the development of plants and their response to abiotic and biotic stresses, such as touch and wounding by herbivores. A gap in our knowledge on jasmonate‐induced processes, however, is the cellular localization of jasmonates.Here, a novel antibody‐based approach was developed to visualize jasmonates in cross‐sections of plant material. Antibodies raised in rabbits against BSA‐coupled jasmonic acid (JA) are specific for JA, its methyl ester and isoleucine conjugate. They do not bind to 12‐oxophytodienoic acid, 12‐hydoxy‐JA or coronatine. These antibodies were used in combination with newly established fixation and embedding methods.Jasmonates were rapidly and uniformly distributed within all cells near the site of damage of a mechanically wounded tomato (Solanum lycopersicum) leaf. Leaf tissue distally located to the wound site exhibited identical distribution, but had a lower signal intensity. The occurrence of jasmonates in all cell types of a wounded leaf was accompanied by transcript accumulation of early JA‐induced genes visualized by in situ hybridization.With these new antibodies, a powerful tool is available to detect cell‐specifically the occurrence of jasmonates in any jasmonate‐dependent stress response or developmental process of plants.
Publikation

Helber, N.; Wippel, K.; Sauer, N.; Schaarschmidt, S.; Hause, B.; Requena, N.; A Versatile Monosaccharide Transporter That Operates in the Arbuscular Mycorrhizal Fungus Glomus sp Is Crucial for the Symbiotic Relationship with Plants Plant Cell 23, 3812-3823, (2011) DOI: 10.1105/tpc.111.089813

For more than 400 million years, plants have maintained a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This evolutionary success can be traced to the role of these fungi in providing plants with mineral nutrients, particularly phosphate. In return, photosynthates are given to the fungus, which support its obligate biotrophic lifestyle. Although the mechanisms involved in phosphate transfer have been extensively studied, less is known about the reciprocal transfer of carbon. Here, we present the high-affinity Monosaccharide Transporter2 (MST2) from Glomus sp with a broad substrate spectrum that functions at several symbiotic root locations. Plant cell wall sugars can efficiently outcompete the Glc uptake capacity of MST2, suggesting they can serve as alternative carbon sources. MST2 expression closely correlates with that of the mycorrhiza-specific PhosphateTransporter4 (PT4). Furthermore, reduction of MST2 expression using host-induced gene silencing resulted in impaired mycorrhiza formation, malformed arbuscules, and reduced PT4 expression. These findings highlight the symbiotic role of MST2 and support the hypothesis that the exchange of carbon for phosphate is tightly linked. Unexpectedly, we found that the external mycelium of AM fungi is able to take up sugars in a proton-dependent manner. These results imply that the sugar uptake system operating in this symbiosis is more complex than previously anticipated.
Publikation

Escalante-Pérez, M.; Krol, E.; Stange, A.; Geiger, D.; Al-Rasheid, K. A. S.; Hause, B.; Neher, E.; Hedrich, R.; A special pair of phytohormones controls excitability, slow closure, and external stomach formation in the Venus flytrap Proc. Natl. Acad. Sci. U.S.A. 108, 15492-15497, (2011) DOI: 10.1073/pnas.1112535108

Venus flytrap's leaves can catch an insect in a fraction of a second. Since the time of Charles Darwin, scientists have struggled to understand the sensory biology and biomechanics of this plant, Dionaea muscipula. Here we show that insect-capture of Dionaea traps is modulated by the phytohormone abscisic acid (ABA) and jasmonates. Water-stressed Dionaea, as well as those exposed to the drought-stress hormone ABA, are less sensitive to mechanical stimulation. In contrast, application of 12-oxo-phytodienoic acid (OPDA), a precursor of the phytohormone jasmonic acid (JA), the methyl ester of JA (Me-JA), and coronatine (COR), the molecular mimic of the isoleucine conjugate of JA (JA-Ile), triggers secretion of digestive enzymes without any preceding mechanical stimulus. Such secretion is accompanied by slow trap closure. Under physiological conditions, insect-capture is associated with Ca2+ signaling and a rise in OPDA, Apparently, jasmonates bypass hapto-electric processes associated with trap closure. However, ABA does not affect OPDA-dependent gland activity. Therefore, signals for trap movement and secretion seem to involve separate pathways. Jasmonates are systemically active because application to a single trap induces secretion and slow closure not only in the given trap but also in all others. Furthermore, formerly touch-insensitive trap sectors are converted into mechanosensitive ones. These findings demonstrate that prey-catching Dionaea combines plant-specific signaling pathways, involving OPDA and ABA with a rapidly acting trigger, which uses ion channels, action potentials, and Ca2+ signals.
Publikation

Zdyb, A.; Demchenko, K.; Heumann, J.; Mrosk, C.; Grzeganek, P.; Göbel, C.; Feussner, I.; Pawlowski, K.; Hause, B.; Jasmonate biosynthesis in legume and actinorhizal nodules New Phytol. 189, 568-579, (2011) DOI: 10.1111/j.1469-8137.2010.03504.x

Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells.The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild‐type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance.To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1‐sense or MtAOC1‐RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation.These data suggest that jasmonates are not involved in the development and function of root nodules.
Publikation

Wirsing, L.; Naumann, K.; Vogt, T.; Arabidopsis methyltransferase fingerprints by affinity-based protein profiling Anal. Biochem. 408, 220-225, (2011) DOI: 10.1016/j.ab.2010.09.029

Precise annotation of time and spatial distribution of enzymes involved in plant secondary metabolism by gel electrophoresis are usually difficult due to their low abundance. Therefore, effective methods to enrich these enzymes are required to correlate available transcript and metabolite data with the actual presence of active enzymes in wild-type and mutant plants or to monitor variations of these enzymes under various types of biotic and abiotic stress conditions. S-Adenosyl-L-methionine-dependent O-methyltransferases play important roles in the modification of natural products such as phenylpropanoids or alkaloids. In plants they occur as small superfamilies with defined roles for each of its members in different organs and tissues. We explored the use of S-adenosyl-L-homocysteine as a selectivity function in affinity-based protein profiling supported by capture compound mass spectrometry. Due to their high affinity to this ligand it was possible to identify developmental changes of flower-specific patterns of plant natural product O-methyltransferases and corroborate the absence of individual O-methyltransferases in the corresponding Arabidopsis knockout lines. Developmental changes in the OMT pattern were correlated with transcript data obtained by qPCR.
Publikation

Weber, E.; Gruetzner, R.; Werner, S.; Engler, C.; Marillonnet, S.; Assembly of Designer TAL Effectors by Golden Gate Cloning PLOS ONE 6, e19722, (2011) DOI: 10.1371/journal.pone.0019722

Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
Publikation

Weber, E.; Engler, C.; Gruetzner, R.; Werner, S.; Marillonnet, S.; A Modular Cloning System for Standardized Assembly of Multigene Constructs PLOS ONE 6, e16765, (2011) DOI: 10.1371/journal.pone.0016765

The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.
Publikation

Walter, M. H.; Strack, D.; Carotenoids and their cleavage products: Biosynthesis and functions Nat. Prod. Rep. 28, 663-692, (2011) DOI: 10.1039/C0NP00036A

Covering: up to mid-2010This review focuses on plant carotenoids, but it also includes progress made on microbial and animal carotenoid metabolism to better understand the functions and the evolution of these structurally diverse compounds with a common backbone. Plants have evolved isogenes for specific key steps of carotenoid biosynthesis with differential expression profiles, whose characteristic features will be compared. Perhaps the most exciting progress has been made in studies of carotenoid cleavage products (apocarotenoids) with an ever-expanding variety of novel functions being discovered. This review therefore covers structural, molecular genetic and functional aspects of carotenoids and apocarotenoids alike. Apocarotenoids are specifically tailored from carotenoids by a family of oxidative cleavage enzymes, but whether there are contributions to their generation from chemical oxidation, photooxidation or other mechanisms is largely unknown. Control of carotenoid homeostasis is discussed in the context of biosynthetic and degradative reactions but also in the context of subcellular environments for deposition and sequestration within and outside of plastids. Other aspects of carotenoid research, including metabolic engineering and synthetic biology approaches, will only be covered briefly.
Publikation

Thieme, F.; Engler, C.; Kandzia, R.; Marillonnet, S.; Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes PLOS ONE 6, e20556, (2011) DOI: 10.1371/journal.pone.0020556

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.
Bücher und Buchkapitel

Engler, C.; Marillonnet, S.; Generation of Families of Construct Variants Using Golden Gate Shuffling (Lu, C. et al., eds.). Methods Mol. Biol. 729, 167-181, (2011) ISBN: 978-1-61779-065-2 DOI: 10.1007/978-1-61779-065-2_11

Current standard cloning methods based on the use of restriction enzymes and ligase are very versatile, but are not well suited for high-throughput cloning projects or for assembly of many DNA fragments from several parental plasmids in a single step. We have previously reported the development of an efficient cloning method based on the use of type IIs restriction enzymes and restriction–ligation. Such method allows seamless assembly of multiple fragments from several parental plasmids with high efficiency, and also allows performing DNA shuffling if fragments prepared from several homologous genes are assembled together in a single restriction–ligation. Such protocol, called Golden Gate shuffling, requires performing the following steps: (1) sequences from several homologous genes are aligned, and recombination sites defined on conserved sequences; (2) modules defined by the position of these recombination sites are amplified by PCR with primers designed to equip them with flanking BsaI sites; (3) the amplified fragments are cloned as intermediate constructs and sequenced; and (4) finally, the intermediate modules are assembled together in a compatible recipient vector in a one-pot restriction–ligation. Depending on the needs of the user, and because of the high cloning efficiency, the resulting constructs can either be screened and analyzed individually, or, if required in larger numbers, directly used in functional screens to detect improved protein variants.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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