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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. 
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

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.

Publikationen in Druck

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 
Publikation

Paudel, G., Bilova, T., Schmidt, R., Greifenhagen, U., Berger, R., Tarakhovskaya, E., Stöckhardt, S., Balcke, G. U., Humbeck, K., Brandt, W., Sinz, A., Vogt, T., Birkemeyer, C., Wessjohann, L. & Frolov, A Osmotic stress is accompanied by protein glycation in Arabidopsis thaliana J. Exp. Bot. 67, 6283-6295, (2016) DOI: 10.1093/jxb/erw395

Among the environmental alterations accompanying oncoming climate changes, drought is the most important factor influencing crop plant productivity. In plants, water deficit ultimately results in the development of oxidative stress and accumulation of osmolytes (e.g. amino acids and carbohydrates) in all tissues. Up-regulation of sugar biosynthesis in parallel to the increasing overproduction of reactive oxygen species (ROS) might enhance protein glycation, i.e. interaction of carbonyl compounds, reducing sugars and α-dicarbonyls with lysyl and arginyl side-chains yielding early (Amadori and Heyns compounds) and advanced glycation end-products (AGEs). Although the constitutive plant protein glycation patterns were characterized recently, the effects of environmental stress on AGE formation are unknown so far. To fill this gap, we present here a comprehensive in-depth study of the changes in Arabidopsis thaliana advanced glycated proteome related to osmotic stress. A 3 d application of osmotic stress revealed 31 stress-specifically and 12 differentially AGE-modified proteins, representing altogether 56 advanced glycation sites. Based on proteomic and metabolomic results, in combination with biochemical, enzymatic and gene expression analysis, we propose monosaccharide autoxidation as the main stress-related glycation mechanism, and glyoxal as the major glycation agent in plants subjected to drought. 

Publikation

Scheler, U., Brandt, W., Porzel, A., Rothe, K., Manzano, D., Božić, D., Papaefthimiou, D., Balcke, G. U., Henning, A., Lohse, S., Marillonnet, S., Kanellis, A. K., Ferrer, A. & Tissier, A. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast Nat Commun 7, 12942, (2016) DOI: 10.1038/ncomms12942

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities.

Publikation

Treutler, H., Tsugawa, H., Porzel, A., Gorzolka, K., Tissier, A., Neumann, S. & Balcke, G. U. Discovering regulated metabolite families in untargeted metabolomics studies. Anal Chem 88, 8082-8090, (2016) DOI: 10.1021/acs.analchem.6b01569

The identification of metabolites by mass spectrometry constitutes a major bottleneck which considerably limits the throughput of metabolomics studies in biomedical or plant research. Here, we present a novel approach to analyze metabolomics data from untargeted, data-independent LC-MS/MS measurements. By integrated analysis of MS1 abundances and MS/MS spectra, the identification of regulated metabolite families is achieved. This approach offers a global view on metabolic regulation in comparative metabolomics. We implemented our approach in the web application “MetFamily”, which is freely available at http://msbi.ipb-halle.de/MetFamily/. MetFamily provides a dynamic link between the patterns based on MS1-signal intensity and the corresponding structural similarity at the MS/MS level. Structurally related metabolites are annotated as metabolite families based on a hierarchical cluster analysis of measured MS/MS spectra. Joint examination with principal component analysis of MS1 patterns, where this annotation 

Publikation

Taylor, I., Wang, Y., Seitz, K., Baer, J., Bennewitz, S., Mooney, B. P. & Walker, J. C. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission Plos One 11, e0147203, (2016) DOI: org/10.1371/journal.pone.0147203

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.

Bücher und Buchkapitel

Bilova, T., Greifenhagen, U., Paudel, G., Lukasheva, E., Brauch, D., Osmolovskaya, N., Tarakhovskaya, E., Balcke, G. U., Tissier, A., Vogt, T., Milkowski, C., Birkemeyer, C., Wessjohann, L. & Frolov, A. Glycation of Plant Proteins under Environmental Stress — Methodological Approaches, Potential Mechanisms and Biological Role. In: Abiotic and Biotic Stress in Plants - Recent Advances and Future Perspectives (Shanker, A. K.; Shanker, C.). (2016) ISBN: 978-953-51-2250-0 DOI: 10.5772/61860

 Environmental stress is one of the major factors reducing crop productivity. Due to the oncoming climate changes, the effects of drought and high light on plants play an increasing role in modern agriculture. These changes are accompanied with a progressing contamination of soils with heavy metals. Independent of their nature, environmental alterations result in development of oxidative stress, i.e. increase of reactive oxygen species (ROS) contents, and metabolic adjustment, i.e. accumulation of soluble primary metabolites (amino acids and sugars). However, a simultaneous increase of ROS and sugar concentrations ultimately results in protein glycation, i.e. non-enzymatic interaction of reducing sugars or their degradation products (α-dicarbonyls) with proteins. The eventually resulting advanced glycation end-products (AGEs) are known to be toxic and pro-inflammatory in mammals. Recently, their presence was unambiguously demonstrated in vivo in stressed Arabidopsis thaliana plants. Currently, information on protein targets, modification sites therein, mediators and mechanisms of plant glycation are being intensively studied. In this chapter, we comprehensively review the methodological approaches for plant glycation research and discuss potential mechanisms of AGE formation under stress conditions. On the basis of these patterns and additional in vitro experiments, the pathways and mechanisms of plant glycation can be proposed.

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