Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie

Aachen, Germany

Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie

Aachen, Germany
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Harig L.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Beinecke F.A.,University of Munster | Oltmanns J.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Muth J.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | And 7 more authors.
Plant Journal | Year: 2012

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine-binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case-by-case basis. We isolated four novel FT-like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild-type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short-day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT-like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long-day conditions, suggesting that all four tobacco FT-like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT-like clade has led to the evolution of antagonistic regulators that may help to fine-tune floral initiation in response to environmental cues. © 2012 Blackwell Publishing Ltd.

Muller B.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Noll G.A.,University of Munster | Ernst A.M.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Ruping B.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | And 5 more authors.
Applied Microbiology and Biotechnology | Year: 2010

Forisomes are mechanoproteins that undergo ATP-independent contraction-expansion cycles triggered by divalent cations, pH changes, and electrical stimuli. Although native forisomes from Medicago truncatula comprise a number of subunits encoded by separate genes, here we show that at least two of those subunits (MtSEO1 and MtSEO4) can assemble into homomeric forisome bodies that are functionally similar to their native, multimeric counterparts. We expressed these subunits in plants and yeast, resulting in the purification of large quantities of artificial forisomes with unique characteristics depending on the expression platform. These artificial forisomes were able to contract and expand in vitro like native forisomes and could respond to electrical stimulation when immobilized between interdigital transducer electrodes. These results indicate that recombinant artificial forisomes with specific characteristics can be prepared in large amounts and used as components of microscale and nanoscale devices. © Springer-Verlag 2010.

Van Deenen N.,University of Munster | Bachmann A.L.,University of Munster | Schmidt T.,University of Munster | Schaller H.,University of Strasbourg | And 3 more authors.
Molecular Biology Reports | Year: 2012

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products. © Springer Science+Business Media B.V. 2011.

Arens J.,TU Dortmund | Arens J.,Max Planck Institute For Molekulare Physiologie | Engels B.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Klopries S.,TU Dortmund | And 4 more authors.
Chemical Communications | Year: 2013

Herein preparative scale access to the shared precursor of the fusicoccane family of diterpenoids through biosynthesis in three different microbial hosts is reported. A method to purify the metabolite in high purity and on a preparative scale was explored. © 2013 The Royal Society of Chemistry.

Engels B.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Heinig U.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Grothe T.,Intermed Discovery | Stadler M.,Intermed Discovery | And 2 more authors.
Journal of Biological Chemistry | Year: 2011

Melleolides and related fungal sesquiterpenoid aryl esters are antimicrobial and cytotoxic natural products derived from cultures of the Homobasidiomycetes genus Armillaria. The initial step in the biosynthesis of all melleolides involves cyclization of the universal sesquiterpene precursor farnesyl diphosphate to produce protoilludene, a reaction catalyzed by protoilludene synthase. We achieved the partial purification of protoilludene synthase from a mycelial culture of Armillaria gallica and found that 6-protoilludene was its exclusive reaction product. Therefore, a further isomerization reaction is necessary to convert the 6-7 double bond into the 7-8 double bond found in melleolides. We expressed an A. gallica protoilludene synthase cDNA in Escherichia coli, and this also led to the exclusive production of 6-protoilludene. Sequence comparison of the isolated sesquiterpene synthase revealed a distant relationship to other fungal terpene synthases. The isolation of the genomic sequence identified the 6-protoilludene synthase to be present as a single copy gene in the genome of A. gallica, possessing an open reading frame interrupted with eight introns. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Heinig U.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Scholz S.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie | Jennewein S.,Fraunhofer Institute For Molekularbiologie Und Angewandte Okologie
Fungal Diversity | Year: 2013

Taxol (paclitaxel) is a highly-oxygenated diterpenoid natural product first isolated from the pacific yew tree (Taxus brevifolia). It is one of the most widely used anticancer drugs. Soon after the discovery of its unique mode of action and the resulting high demand, an extensive search was initiated for alternative sources to replace the slow-growing and scarce pacific yew. Thus far, however, Taxol and related compounds have only been found in the genus Taxus, which comprises a small number of slow-growing plants with a broad but generally isolated geographical distribution. In 1993, Stierle and colleagues reported the independent biosynthesis of Taxol in an endophytic fungus isolated from Taxus brevifolia, which resulted in more than 160 subsequent publications and patents addressing the biosynthesis of Taxol and related taxanes by microorganisms. The literature on fungal taxane synthesis contains numerous inconsistencies, prompting us to thoroughly re-examine Taxol biosynthesis in endophytic fungi associated with Taxus spp. Using a combination of phytochemistry, molecular biology and genome sequencing, we were unable to find any evidence for independent taxane biosynthesis in any of the endophytes, including the isolate described in the original publication (Taxomyces andreanae) and several more recent isolates from Taxus trees. Our findings therefore resolve a long-standing mystery concerning the evolution of a complex terpenoid biosynthetic pathway in two distantly-related organisms. © 2013 The Author(s).

Schmidt T.,University of Munster | Hillebrand A.,University of Munster | Wurbs D.,University of Munster | Wahler D.,University of Munster | And 4 more authors.
Plant Molecular Biology Reporter | Year: 2010

Rubber biosynthesis requires the action of specific enzymes known as cis-prenyltransferases (CPTs). These enzymes are responsible for the sequential addition of isopentenyl pyrophosphate units to the growing polyisoprene chain, a biochemical reaction thought to be stimulated by the presence of small rubber particle proteins (SRPPs). We have cloned, characterized, and analyzed the expression of three CPT genes (TkCPT1-3) and five SRPP genes (TkSRPP1-5) from the rubber-producing plant Taraxacum koksaghyz. The deduced TkCPT amino acid sequences showed significant levels of sequence identity with Hevea brasiliensis CPTs. We also found no obvious differences between SRPPs from T. koksaghyz, another rubber producer, and a non-rubber plant. The roles of the individual TkCPTs and TkSRRPs in rubber biosynthesis are discussed. © Springer-Verlag 2009.

Masani M.Y.A.,Malaysian Palm Oil Board | Noll G.,University of Munster | Parveez G.K.A.,Malaysian Palm Oil Board | Sambanthamurthi R.,Malaysian Palm Oil Board | And 2 more authors.
Plant Science | Year: 2013

Oil palm protoplasts are suitable as a starting material for the production of oil palm plants with new traits using approaches such as somatic hybridization, but attempts to regenerate viable plants from protoplasts have failed thus far. Here we demonstrate, for the first time, the regeneration of viable plants from protoplasts isolated from cell suspension cultures. We achieved a protoplast yield of 1.14×106 per gram fresh weight with a viability of 82% by incubating the callus in a digestion solution comprising 2% cellulase, 1% pectinase, 0.5% cellulase onuzuka R10, 0.1% pectolyase Y23, 3% KCl, 0.5% CaCl2 and 3.6% mannitol. The regeneration of protoplasts into viable plants required media optimization, the inclusion of plant growth regulators and the correct culture technique. Microcalli derived from protoplasts were obtained by establishing agarose bead cultures using Y3A medium supplemented with 10μM naphthalene acetic acid, 2μM 2,4-dichlorophenoxyacetic acid, 2μM indole-3-butyric acid, 2μM gibberellic acid and 2μM 2-γ-dimethylallylaminopurine. Small plantlets were regenerated from microcalli by somatic embryogenesis after successive subculturing steps in medium with limiting amounts of growth regulators supplemented with 200mg/l ascorbic acid. © 2013 Elsevier Ireland Ltd.

Munt O.,University of Munster | Arias M.,NEIKER | Hernandez M.,NEIKER | Ritter E.,NEIKER | And 3 more authors.
Industrial Crops and Products | Year: 2012

Taraxacum brevicorniculatum produces high-quality natural rubber in its roots and could be developed as an alternative commercial source of this valuable raw material. However, current wild type accessions have a low biomass and branched roots that make them difficult to harvest. We set out to determine the optimum fertilizer and spacing requirements for T. brevicorniculatum plants in greenhouse and field trials, aiming to increase root biomass and reduce branching thus maximizing rubber yield and reducing losses during harvesting. Our preliminary data from greenhouse trials show that fertilizers containing calcium and magnesium in addition to NPK (nitrogen, phosphorus and potassium) increase root biomass by 25% compared to standard NPK fertilizer plus boron, and by 15% compared to NPK plus calcium. Fertilizers with a low N:P ratio increased the root biomass in greenhouse and field trials. Dense sowing (5-10. cm between plants) inhibits root branching and leads to the formation of single taproots that can be harvested easily. We therefore provisionally recommend the application of low N:P ratio NPK fertilizer supplemented with calcium and magnesium to closely-spaced plants in order to achieve the greatest increase in plant biomass per hectare. © 2011 Elsevier B.V.

Masani M.Y.A.,Malaysian Palm Oil Board | Noll G.A.,University of Munster | Parveez G.K.A.,Malaysian Palm Oil Board | Sambanthamurthi R.,Malaysian Palm Oil Board | And 2 more authors.
PLoS ONE | Year: 2014

Background: Genetic engineering remains a major challenge in oil palm (Elaeis guineensis) because particle bombardment and Agrobacterium-mediated transformation are laborious and/or inefficient in this species, often producing chimeric plants and escapes. Protoplasts are beneficial as a starting material for genetic engineering because they are totipotent, and chimeras are avoided by regenerating transgenic plants from single cells. Novel approaches for the transformation of oil palm protoplasts could therefore offer a new and efficient strategy for the development of transgenic oil palm plants. Methodology/Principal Findings: We recently achieved the regeneration of healthy and fertile oil palms from protoplasts. Therefore, we focused on the development of a reliable PEG-mediated transformation protocol for oil palm protoplasts by establishing and validating optimal heat shock conditions, concentrations of DNA, PEG and magnesium chloride, and the transfection procedure. We also investigated the transformation of oil palm protoplasts by DNA microinjection and successfully regenerated transgenic microcalli expressing green fluorescent protein as a visible marker to determine the efficiency of transformation. Conclusions/Significance: We have established the first successful protocols for the transformation of oil palm protoplasts by PEG-mediated transfection and DNA microinjection. These novel protocols allow the rapid and efficient generation of non-chimeric transgenic callus and represent a significant milestone in the use of protoplasts as a starting material for the development of genetically-engineered oil palm plants. © 2014 Masani et al.

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