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Publikationen - Stoffwechsel- und Zellbiologie

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Balcke, G., Bennewitz, S., Bergau, N., Athmer, B., Henning, A., Majovsky, P., Jiménez-Gómez, J. M., Hoehenwarter, W. & Tissier, A. F Multi-Omics of tomato glandular trichomes reveals distinct features of central carbon metabolism supporting high productivity of specialized metabolites Plant Cell 29 , 960-983, (2017) DOI: 10.1105/tpc.17.00060

Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics and 13C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2. Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity. 
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Bilova, T., Paudel, G., Shilyaev, N., Schmidt, R., Brauch, D., Tarakhovskaya, E., Milrud, S., Smolikova, G., Tissier, A., Vogt, T., Sinz, A., Brandt, W., Birkemeyer, C., Wessjohann, L. A. & Frolov, A. Global proteomic analysis of advanced glycation end products in the Arabidopsis proteome provides evidence for age-related glycation hotspots. J Biol Chem. 292 , 15758-15776, (2017) DOI: 10.1074/jbc.M117.794537

Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, and accumulate in tissues, being well-known markers of ageing, impacting age-related tissue stiffing and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundances of 96 AGE sites in 71 proteins were significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hotspots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in 3 ageing-specific and 8 differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hotspots might be defined by protein structure that indicates, at least partly, site-specific character of glycation. Data are available via ProteomeXchange with identifier PXD006434 
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Gantner, J., Ilse, T., Ordon, J., Kretschmer, C., Gruetzner, R., Loefke, C., Dagdas, Y., Buerstenbinder, K., Marillonnet, S. & Stuttmann, J. Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system. bioRxiv (2017) DOI: 10.1101/237768

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a "peripheral infrastructure" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

Publikationen in Druck

Gantner, J., Ilse, T., Ordon, J., Kretschmer, C., Gruetzner, R., Loefke, C., Dagdas, Y., Buerstenbinder, K., Marillonnet, S. & Stuttmann, J. Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system.  bioRxiv (2017) DOI: 10.1101/237768

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a "peripheral infrastructure" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.
Publikation

Blüher, D., Laha, D., Thieme, S., Hofer, A., Eschen-Lippold, L., Masch, A., Balcke, G., Pavlovic, I., Nagel, O., Schonsky, A., Hinkelmann, R., Wörner, J., Parvin, N., Greiner, R., Weber, S., Tissier, A., Schutkowski, M., Lee, J., Jessen, H., Schaaf, G. & Bonas, U. A 1-phytase type III effector interferes with plant hormone signaling. Nature Commun. 8(1), 2159, (2017) DOI: 10.1038/s41467-017-02195-8

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen’s benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.
Publikationen in Druck

Kowarschik, K., Hoehenwarter, W., Marillonnet, S. & Trujillo, M.  UbiGate: a synthetic biology toolbox to analyse ubiquitination. New Phytol. (2017) DOI: 10.1111/nph.14900

   Ubiquitination is mediated by an enzymatic cascade that results in the modification of substrate proteins, redefining their fate. This post-translational modification is involved in most cellular processes, yet its analysis faces manifold obstacles due to its complex and ubiquitous nature. Reconstitution of the ubiquitination cascade in bacterial systems circumvents several of these problems and was shown to faithfully recapitulate the process.
    Here, we present UbiGate − a synthetic biology toolbox, together with an inducible bacterial expression system – to enable the straightforward reconstitution of the ubiquitination cascades of different organisms in Escherichia coli by ‘Golden Gate’ cloning.
    This inclusive toolbox uses a hierarchical modular cloning system to assemble complex DNA molecules encoding the multiple genetic elements of the ubiquitination cascade in a predefined order, to generate polycistronic operons for expression.
    We demonstrate the efficiency of UbiGate in generating a variety of expression elements to reconstitute autoubiquitination by different E3 ligases and the modification of their substrates, as well as its usefulness for dissecting the process in a time- and cost-effective manner.
Publikation

Dreher, D., Yadav, H., Zander, S. & Hause, B. Is there genetic variation in mycorrhization of Medicago truncatula?  PeerJ 5, e3713, (2017) DOI: 10.7717/peerj.3713

Differences in the plant’s response among ecotypes or accessions are often used to identify molecular markers for the respective process. In order to analyze genetic diversity of Medicago truncatula in respect to interaction with the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, mycorrhizal colonization was evaluated in 32 lines of the nested core collection representing the genetic diversity of the SARDI collection. All studied lines and the reference line Jemalong A17 were inoculated with R. irregularis and the mycorrhization rate was determined at three time points after inoculation. There were, however, no reliable and consistent differences in mycorrhization rates among all lines. To circumvent possible overlay of potential differences by use of the highly effective inoculum, native sandy soil was used in an independent experiment. Here, significant differences in mycorrhization rates among few of the lines were detectable, but the overall high variability in the mycorrhization rate hindered clear conclusions. To narrow down the number of lines to be tested in more detail, root system architecture (RSA) of in vitro-grown seedlings of all lines under two different phosphate (Pi) supply condition was determined in terms of primary root length and number of lateral roots. Under high Pi supply (100 µM), only minor differences were observed, whereas in response to Pi-limitation (3 µM) several lines exhibited a drastically changed number of lateral roots. Five lines showing the highest alterations or deviations in RSA were selected and inoculated with R. irregularis using two different Pi-fertilization regimes with either 13 mM or 3 mM Pi. Mycorrhization rate of these lines was checked in detail by molecular markers, such as transcript levels of RiTubulin and MtPT4. Under high phosphate supply, the ecotypes L000368 and L000555 exhibited slightly increased fungal colonization and more functional arbuscules, respectively. To address the question, whether capability for mycorrhizal colonization might be correlated to general invasion by microorganisms, selected lines were checked for infection by the root rot causing pathogen, Aphanoymces euteiches. The mycorrhizal colonization phenotype, however, did not correlate with the resistance phenotype upon infection with two strains of A. euteiches as L000368 showed partial resistance and L000555 exhibited high susceptibility as determined by quantification of A. euteiches rRNA within infected roots. Although there is genetic diversity in respect to pathogen infection, genetic diversity in mycorrhizal colonization of M. truncatula is rather low and it will be rather difficult to use it as a trait to access genetic markers. 
Publikationen in Druck

Bjornson, M., Balcke, G. U., Xiao, Y., de Souza, A., Wang, J.-Z., , Zhabinskaya, D., Tagkopoulos, I., Tissier, A. & Dehesh, K. Integrated omics analyses of retrograde signaling mutant delineate interrelated stress-response strata.  Plant J. (2017) DOI: 10.1111/tpj.13547

To maintain homeostasis in the face of intrinsic and extrinsic insults, cells have evolved elaborate quality control networks to resolve damage at multiple levels. Interorganellar communication is a key requirement for this maintenance, however the underlying mechanisms of this communication have remained an enigma. Here we integrate the outcome of transcriptomic, proteomic, and metabolomics analyses of genotypes including ceh1, a mutant with constitutively elevated levels of both the stress-specific plastidial retrograde signaling metabolite methyl-erythritol cyclodiphosphate (MEcPP) and the defense hormone salicylic acid (SA), as well as the high MEcPP but SA deficient genotype ceh1/eds16, along with corresponding controls. Integration of multi-omic analyses enabled us to delineate the function of MEcPP from SA, and expose the compartmentalized role of this retrograde signaling metabolite in induction of distinct but interdependent signaling cascades instrumental in adaptive responses. Specifically, here we identify strata of MEcPP-sensitive stress-response cascades, among which we focus on selected pathways including organelle-specific regulation of jasmonate biosynthesis; simultaneous induction of synthesis and breakdown of SA; and MEcPP-mediated alteration of cellular redox status in particular glutathione redox balance. Collectively, these integrated multi-omic analyses provided a vehicle to gain an in-depth knowledge of genome-metabolism interactions, and to further probe the extent of these interactions and delineate their functional contributions. Through this approach we were able to pinpoint stress-mediated transcriptional and metabolic signatures and identify the downstream processes modulated by the independent or overlapping functions of MEcPP and SA in adaptive responses.

Publikation

Frolov, A., Bilova, T., Paudel, G., Berger, R., Balcke, G. U., Birkemeyer, C. & Wessjohann, L. A. Early responses of mature Arabidopsis thaliana plants to reduced water potential in the agar-based polyethylene glycol infusion drought model. J Plant Physiol. 208, 70-83, (2017) DOI: 10.1016/j.jplph.2016.09.013

Drought is one of the most important environmental stressors resulting in increasing losses of crop plant productivity all over the world. Therefore, development of new approaches to increase the stress tolerance of crop plants is strongly desired. This requires precise and adequate modeling of drought stress. As this type of stress manifests itself as a steady decrease in the substrate water potential (ψw), agar plates infused with polyethylene glycol (PEG) are the perfect experimental tool: they are easy in preparation and provide a constantly reduced ψw, which is not possible in soil models. However, currently, this model is applicable only to seedlings and cannot be used for evaluation of stress responses in mature plants, which are obviously the most appropriate objects for drought tolerance research. To overcome this limitation, here we introduce a PEG-based agar infusion model suitable for 6–8-week-old A. thaliana plants, and characterize, to the best of our knowledge for the first time, the early drought stress responses of adult plants grown on PEG-infused agar. We describe essential alterations in the primary metabolome (sugars and related compounds, amino acids and polyamines) accompanied by qualitative and quantitative changes in protein patterns: up to 87 unique stress-related proteins were annotated under drought stress conditions, whereas further 84 proteins showed a change in abundance. The obtained proteome patterns differed slightly from those reported for seedlings and soil-based models.

Publikation

Dhakarey, R., Raorane, M. L., Treumann, A., Peethambaran, P. K., Schendel, R. R., Sahi, V. P., Hause, B., Bunzel, M., Henry, A., Kohli, A. & Riemann, M. Physiological and proteomic analysis of the rice mutant cpm2 suggests a negative regulatory role of Jasmonic acid in drought tolerance. Front Plant Sci 8, 1903, (2017) DOI: 10.3389/fpls.2017.01903

It is widely known that numerous adaptive responses of drought-stressed plants are stimulated by chemical messengers known as phytohormones. Jasmonic acid (JA) is one such phytohormone. But there are very few reports revealing its direct implication in drought related responses or its cross-talk with other phytohormones. In this study, we compared the morpho-physiological traits and the root proteome of a wild type (WT) rice plant with its JA biosynthesis mutant coleoptile photomorphogenesis 2 (cpm2), disrupted in the allene oxide cyclase (AOC) gene, for insights into the role of JA under drought. The mutant had higher stomatal conductance, higher water use efficiency and higher shoot ABA levels under severe drought as compared to the WT. Notably, roots of cpm2 were better developed compared to the WT under both, control and drought stress conditions. Root proteome was analyzed using the Tandem Mass Tag strategy to better understand this difference at the molecular level. Expectedly, AOC was unique but notably highly abundant under drought in the WT. Identification of other differentially abundant proteins (DAPs) suggested increased energy metabolism (i.e., increased mobilization of resources) and reactive oxygen species scavenging in cpm2 under drought. Additionally, various proteins involved in secondary metabolism, cell growth and cell wall synthesis were also more abundant in cpm2 roots. Proteome-guided transcript, metabolite, and histological analyses provided further insights into the favorable adaptations and responses, most likely orchestrated by the lack of JA, in the cpm2 roots. Our results in cpm2 are discussed in the light of JA crosstalk to other phytohormones. These results together pave the path for understanding the precise role of JA during drought stress in rice.
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