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Publikation

Brückner, K.; Schäfer, P.; Weber, E.; Grützner, R.; Marillonnet, S.; Tissier, A.; A library of synthetic transcription activator-like effector-activated promoters for coordinated orthogonal gene expression in plants Plant J. 82, 707-716, (2015) DOI: 10.1111/tpj.12843

A library of synthetic promoters containing the binding site of a single designer transcription activator‐like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18‐base long dTALE‐binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty‐three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.
Publikation

Božić, D.; Papaefthimiou, D.; Brückner, K.; de Vos, R. C. H.; Tsoleridis, C. A.; Katsarou, D.; Papanikolaou, A.; Pateraki, I.; Chatzopoulou, F. M.; Dimitriadou, E.; Kostas, S.; Manzano, D.; Scheler, U.; Ferrer, A.; Tissier, A.; Makris, A. M.; Kampranis, S. C.; Kanellis, A. K.; Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis PLOS ONE 10, e0124106, (2015) DOI: 10.1371/journal.pone.0124106

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
Publikation

Bergau, N.; Bennewitz, S.; Syrowatka, F.; Hause, G.; Tissier, A.; The development of type VI glandular trichomes in the cultivated tomato Solanum lycopersicum and a related wild species S. habrochaites BMC Plant Biol. 15, 289, (2015) DOI: 10.1186/s12870-015-0678-z

BackgroundType VI glandular trichomes represent the most abundant trichome type on leaves and stems of tomato plants and significantly contribute to herbivore resistance, particularly in the wild species. Despite this, their development has been poorly studied so far. The goal of this study is to fill this gap. Using a variety of cell imaging techniques, a detailed record of the anatomy and developmental stages of type VI trichomes in the cultivated tomato (Solanum lycopersicum) and in a related wild species (S. habrochaites) is provided.ResultsIn both species, the development of these structures follows a highly reproducible cell division pattern. The two species differ in the shape of the trichome head which is round in S. habrochaites and like a four-leaf clover in S. lycopersicum, correlating with the presence of a large intercellular cavity in S. habrochaites where the produced metabolites accumulate. In both species, the junction between the intermediate cell and the four glandular cells constitute a breaking point facilitating the decapitation of the trichome and thereby the quick release of the metabolites. A strongly auto-fluorescent compound transiently accumulates in the early stages of development suggesting a potential role in the differentiation process. Finally, immuno-labelling with antibodies recognizing specific cell wall components indicate a key role of pectin and arabinogalactan components in the differentiation of type VI trichomes.ConclusionsOur observations explain the adaptive morphologies of type VI trichomes for metabolite storage and release and provide a framework for further studies of these important metabolic cellular factories. This is required to better exploit their potential, in particular for the breeding of pest resistance in tomato.
Publikation

Patron, N. J.; Orzaez, D.; Marillonnet, S.; Warzecha, H.; Matthewman, C.; Youles, M.; Raitskin, O.; Leveau, A.; Farré, G.; Rogers, C.; Smith, A.; Hibberd, J.; Webb, A. A. R.; Locke, J.; Schornack, S.; Ajioka, J.; Baulcombe, D. C.; Zipfel, C.; Kamoun, S.; Jones, J. D. G.; Kuhn, H.; Robatzek, S.; Van Esse, H. P.; Sanders, D.; Oldroyd, G.; Martin, C.; Field, R.; O'Connor, S.; Fox, S.; Wulff, B.; Miller, B.; Breakspear, A.; Radhakrishnan, G.; Delaux, P.-M.; Loqué, D.; Granell, A.; Tissier, A.; Shih, P.; Brutnell, T. P.; Quick, W. P.; Rischer, H.; Fraser, P. D.; Aharoni, A.; Raines, C.; South, P. F.; Ané, J.-M.; Hamberger, B. R.; Langdale, J.; Stougaard, J.; Bouwmeester, H.; Udvardi, M.; Murray, J. A. H.; Ntoukakis, V.; Schäfer, P.; Denby, K.; Edwards, K. J.; Osbourn, A.; Haseloff, J.; Standards for plant synthetic biology: a common syntax for exchange of DNA parts New Phytol. 208, 13-19, (2015) DOI: 10.1111/nph.13532

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease‐mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.
Publikation

Nagel, R.; Bernholz, C.; Vranová, E.; Košuth, J.; Bergau, N.; Ludwig, S.; Wessjohann, L.; Gershenzon, J.; Tissier, A.; Schmidt, A.; Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate Plant J. 84, 847-859, (2015) DOI: 10.1111/tpj.13064

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C10), farnesyl diphosphate (FDP, C15) or geranylgeranyl diphosphate (GGDP, C20). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C25) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C25 versus C20 product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C20) as a product and a larger R group (Met) resulting in GFDP (C25).
Publikation

Walter, M. H.; Stauder, R.; Tissier, A.; Evolution of root-specific carotenoid precursor pathways for apocarotenoid signal biogenesis Plant Sci. 233, 1-10, (2015) DOI: 10.1016/j.plantsci.2014.12.017

Various cleavage products of C40 carotenoid substrates are formed preferentially or exclusively in roots. Such apocarotenoid signaling or regulatory compounds differentially induced in roots during environmental stress responses including root colonization by arbuscular mycorrhizal fungi include ABA, strigolactones and C13 α-ionol/C14 mycorradicin derivatives. The low carotenoid levels in roots raise the question of whether there is a regulated precursor supply channeled into apocarotenoid formation distinct from default carotenoid pathways. This review describes root-specific isogene components of carotenoid pathways toward apocarotenoid formation, highlighting a new PSY3 class of phytoene synthase genes in dicots. It is clearly distinct from the monocot PSY3 class co-regulated with ABA formation. At least two members of the exclusive dicot PSY3s are regulated by nutrient stress and mycorrhization. This newly recognized dicot PSY3 (dPSY3 vs. mPSY3 from monocots) class probably represents an ancestral branch in the evolution of the plant phytoene synthase family. The evolutionary history of PSY genes is compared with the evolution of MEP pathway isogenes encoding 1-deoxy-d-xylulose 5-phosphate synthases (DXS), particularly DXS2, which is co-regulated with dPSY3s in mycorrhizal roots. Such stress-inducible isoforms for rate-limiting steps in root carotenogenesis might be components of multi-enzyme complexes committed to apocarotenoid rather than to carotenoid formation.
Bücher und Buchkapitel

Tissier, A.; Ziegler, J.; Vogt, T.; Specialized Plant Metabolites: Diversity and Biosynthesis (Krauss, G.-J. & Nies, D. H., eds.). 14-37, (2015) ISBN: 9783527686063 DOI: 10.1002/9783527686063.ch2

Plant secondary metabolites, also termed specialized plant metabolites, currently comprise more than 200 000 natural products that are all based on a few biosynthetic pathways and key primary metabolites. Some pathways like flavonoid and terpenoid biosynthesis are universally distributed in the plant kingdom, whereas others like alkaloid or cyanogenic glycoside biosynthesis are restricted to a limited set of taxa. Diversification is achieved by an array of mechanisms at the genetic and enzymatic level including gene duplications, substrate promiscuity of enzymes, cell‐specific regulatory systems, together with modularity and combinatorial aspects. Specialized metabolites reflect adaptations to a specific environment. The observed diversity illustrates the heterogeneity and multitude of ecological habitats and niches that plants have colonized so far and constitutes a reservoir of potential new metabolites that may provide adaptive advantage in the face of environmental changes. The code that connects the observed chemical diversity to this ecological diversity is largely unknown. One way to apprehend this diversity is to realize its tremendous plasticity and evolutionary potential. This chapter presents an overview of the most widespread and popular secondary metabolites, which provide a definite advantage to adapt to or to colonize a particular environment, making the boundary between the “primary” and the “secondary” old fashioned and blurry.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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