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Publications - Cell and Metabolic Biology

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Wils, C. R., Brandt, W. & Manke, K. & Vogt, T. A single amino acid determines position specificity of an Arabidopsis thaliana CCoAOMT-like O-methyltransferase. FEBS Lett 587, 683-689, (2013) DOI: 10.1016/j.febslet.2013.01.040

Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT)-like proteins from plants display a conserved position specificity towards the meta-position of aromatic vicinal dihydroxy groups, consistent with the methylation pattern observed in vivo. A CCoAOMT-like enzyme identified from Arabidopsis thaliana encoded by the gene At4g26220 shows a strong preference for methylating the para position of flavanones and dihydroflavonols, whereas flavones and flavonols are methylated in the meta-position. Sequence alignments and homology modelling identified several unique amino acids compared to motifs of other CCoAOMT-like enzymes. Mutation of a single glycine, G46 towards a tyrosine was sufficient for a reversal of the unusual para- back to meta-O-methylation of flavanones and dihydroflavonols.

Books and chapters

Walter, M. H.  Role of carotenoid metabolism in the Arbuscular Mycorrhizal symbiosis.. In: Molecular Microbial Ecology of the Rhizosphere.  ( F. de Bruijn). 513-524, (2013) ISBN: 9781118297674 DOI: 10.1002/9781118297674.ch48


Nahar, K., Kyndt, T., Hause, B., Höfte, M. & Gheysen, G. Brassinosteroids suppress rice defense against root knot nematodes through antagonism with the jasmonate pathway. Mol Plant Microbe In 26, 106-115, (2013) DOI: 10.1094/MPMI-05-12-0108-FI

The importance of phytohormone balance is increasingly recognized as central to the outcome of plant?pathogen interactions. Next to their well-known developmental role, brassinosteroids (BR) were recently found to be involved in plant innate immunity. In this study, we examined the role of BR in rice (Oryza sativa) innate immunity during infection with the root-knot nematode Meloidogyne graminicola, and we studied the inter-relationship with the jasmonate (JA) pathway. Exogenous epibrassinolide (BL) supply at low concentrations induced susceptibility in the roots whereas high concentrations of BL enforced systemic defense against this nematode. Upon high exogenous BL supply on the shoot, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) confirmed a strong feedback inhibitory effect, leading to reduced BR biosynthesis in the root. Moreover, we demonstrate that the immune suppressive effect of BR is at least partly due to negative cross-talk with the JA pathway. Mutants in the BR biosynthesis or signaling pathway accumulate slightly higher levels of the immediate JA-precursor 12-oxo-phytodienoic acid, and qRT-PCR data showed that the BR and JA pathway are mutually antagonistic in rice roots. Collectively, these results suggest that the balance between the BR and JA pathway is an effective regulator of the outcome of the rice?M. graminicola interaction.

Matsuba, Y., Nguyen, T.T.H., Wiegert, K., Falara, V., Gonzales-Vigil, E., Leong, B., Schäfer, P., Kudrna, D., Wing, R.A., Bolger, A.M., Usadel, B., Tissier, A., Fernie, A.R., Barry, C.S. & Pichersky, E. Evolution of a complex locus for terpene biosynthesis in Solanum. Plant Cell 25, 2022-2036 , (2013) DOI: ​10.​1105/​tpc.​113.​111013

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate–utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.


Ischebeck, T., Werner, S., Krishnamoorthy, P., Lerche, J., Meijón, M., Stenzel, I., Löfke, C., Wiessner, T., Im, Y. J., Perera, I. Y., Iven, T., Feussner, I., Busch, W., Boss, W. F., Teichmann, T., Hause, B., Persson, S. & Ingo Heilmann, I. Phosphatidylinositol 4,5-Bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.  Plant Cell 25, 4894-4911, (2013) DOI: 10.1105/tpc.113.116582

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.
Books and chapters

Hause, B., Mielke, K. & Forner, S. Cell-specific detection of jasmonates by means of immunocytological approach.. In: Jasmonate Signaling. (Meth. Mol. Biol.; 1011) (Goossens, A.; Pauwels, L.). 135-144, (2013) ISBN: 978-1-62703-413-5 DOI: 10.1007/978-1-62703-414-2_11

To determine the location of specific molecules within tissues or cells, immunological techniques are frequently used. However, immunolocalization of small molecules, such as jasmonic acid (JA) and its bioactive amino acid conjugate, JA-isoleucine, requires proper fixation and embedding methods as well as specific antibodies. In this chapter, we present a method to prepare plant tissues for the detection of jasmonates, including the chemical fixation to immobilize JA within the tissue, the subsequent embedding in a suitable medium, and the immunolabeling procedure itself.


Frolov, A., Henning, A., Böttcher, C., Tissier, A. & Strack, D. An UPLC-MS/MS method for the simultaneous identification and quantitation of cell wall phenolics in Brassica napus seeds J Agricult Food Chem 61 (6), 1219-1227, (2013) DOI: 10.1021/jf3042648

The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development.
Books and chapters

Engler, C. & Marillonnet, S Combinatorial DNA assembly using Golden Gate cloning.. In: Synthetic biology.(Meth. Mol. Biol.; 1073) (Polizzi, K. M.). 1073, 141-156, (2013) ISBN: 978-1-62703-624-5 DOI: 10.1007/978-1-62703-625-2_12

A basic requirement for synthetic biology is the availability of efficient DNA assembly methods. We have previously reported the development of Golden Gate cloning, a method that allows parallel assembly of multiple DNA fragments in a one-tube reaction. Golden Gate cloning can be used for different levels of construct assembly: from gene fragments to complete gene coding sequences, from basic genetic elements to full transcription units, and finally from transcription units to multigene constructs. We provide here a protocol for DNA assembly using Golden Gate cloning, taking as an example the level of assembly of gene fragments to complete coding sequences, a level of cloning that can be used to perform DNA shuffling. Such protocol requires the following steps: (1) selecting fusion sites within parental sequences (sites at which parental sequences will be recombined), (2) amplifying all DNA fragments by PCR to add flanking restriction sites, (3) cloning the amplified fragments in intermediate constructs, and (4) assembling all or selected sets of intermediate constructs in a compatible recipient vector using a one-pot restriction-ligation.

Brückner, K. & Tissier, A. High-level diterpene production by transient expression in Nicotiana benthamiana Plant Meth 9:46, (2013) DOI: 10.1186/1746-4811-9-46

Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.

Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.

With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.


Kathagen, A., Schulte, A., Balcke, G., Phillips, H. S., Martens, T., Matschke, J., Günther, H. S., Soriano, R., Modrusan, Z., Sandmann, T., Kuhl, C., Tissier, A., Holz, M., Krawinkel, L. A., Glatzel, M., Westphal, M. & Lamszus, K. Hypoxia and oxygenation induce a metabolic switch between pentose phosphate pathway and glycolysis in glioma stem like cells Acta Neuropathol 126, 763-780, (2013) DOI: 10.1007/s00401-013-1173-y

Fluctuations in oxygen tension during tissue remodeling impose a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastoma, the most common malignant brain tumor, possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. We identified a reciprocal metabolic switch between the pentose phosphate pathway (PPP) and glycolysis in glioblastoma stem-like (GS) cells. Expression of PPP enzymes is upregulated by acute oxygenation but downregulated by hypoxia, whereas glycolysis enzymes, particularly those of the preparatory phase, are regulated inversely. Glucose flux through the PPP is reduced under hypoxia in favor of flux through glycolysis. PPP enzyme expression is elevated in human glioblastomas compared to normal brain, especially in highly proliferative tumor regions, whereas expression of parallel preparatory phase glycolysis enzymes is reduced in glioblastomas, except for strong upregulation in severely hypoxic regions. Hypoxia stimulates GS cell migration but reduces proliferation, whereas oxygenation has opposite effects, linking the metabolic switch to the “go or grow” potential of the cells. Our findings extend Warburg’s observation that tumor cells predominantly utilize glycolysis for energy production, by suggesting that PPP activity is elevated in rapidly proliferating tumor cells but suppressed by acute severe hypoxic stress, favoring glycolysis and migration to protect cells against hypoxic cell damage.

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