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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 

Bilova, T., Lukasheva, E., Brauch, D., Greifenhagen, U., Paudel, G., Tarakhovskaya, E., Frolova, N., Mittasch, J., Balcke, G. U., Tissier, A., Osmolovskaya, N., Vogt, T., Wessjohann, L. A., Birkemeyer, C., Milkowski, C. & Frolov A Snapshot of the Plant Glycated Proteome: structural, functional, and mechanistic aspects J. Biol. Chem. 291, 7621-7636, (2016) DOI: 10.1074/jbc.M115.678581

Glycation is the reaction of carbonyl compounds (reducing sugars and α-dicarbonyls) with amino acids, lipids, and proteins, yielding early and advanced glycation end products (AGEs). The AGEs can be formed via degradation of early glycation intermediates (glycoxidation) and by interaction with the products of monosaccharide autoxidation (autoxidative glycosylation). Although formation of these potentially deleterious compounds is well characterized in animal systems and thermally treated foods, only a little information about advanced glycation in plants is available. Thus, the knowledge of the plant AGE patterns and the underlying pathways of their formation are completely missing. To fill this gap, we describe the AGE-modified proteome of Brassica napus and characterize individual sites of advanced glycation by the methods of liquid chromatography-based bottom-up proteomics. The modification patterns were complex but reproducible: 789 AGE-modified peptides in 772 proteins were detected in two independent experiments. In contrast, only 168 polypeptides contained early glycated lysines, which did not resemble the sites of advanced glycation. Similar observations were made with Arabidopsis thaliana. The absence of the early glycated precursors of the AGE-modified protein residues indicated autoxidative glycosylation, but not glycoxidation, as the major pathway of AGE formation. To prove this assumption and to identify the potential modifying agents, we estimated the reactivity and glycative potential of plant-derived sugars using a model peptide approach and liquid chromatography-mass spectrometry-based techniques. Evaluation of these data sets together with the assessed tissue carbohydrate contents revealed dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, ribulose, erythrose, and sucrose as potential precursors of plant AGEs.

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.


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. 


Fellenberg, C. & Vogt, T. Evolutionarily conserved phenylpropanoid pattern on angiosperm pollen Trends in Plant Science 20, 212-218, (2015) DOI: 10.1016/j.tplants.2015.01.011

The male gametophyte of higher plants appears as a solid box containing the essentials to transmit genetic material to the next generation. These consist of haploid generative cells that are required for reproduction, and an invasive vegetative cell producing the pollen tube, both mechanically protected by a rigid polymer, the pollen wall, and surrounded by a hydrophobic pollen coat. This coat mediates the direct contact to the biotic and abiotic environments. It contains a mixture of compounds required not only for fertilization but also for protection against biotic and abiotic stressors. Among its metabolites, the structural characteristics of two types of phenylpropanoids, hydroxycinnamic acid amides and flavonol glycosides, are highly conserved in Angiosperm pollen. Structural and functional aspects of these compounds will be discussed.


Brandt, W., Manke, K. & Vogt, T. A catalytic triad – Lys-Asn-Asp – Is essential for the catalysis of the methyl transfer in plant cation-dependent O-methyltransferases Phytochemistry 113, 130-139, (2015) DOI: 10.1016/j.phytochem.2014.12.018

Crystal structure data of cation-dependent catechol O-methyltransferases (COMTs) from mammals and related caffeoyl coenzyme A OMTs (CCoAOMTs) from plants have suggested operative molecular mechanisms. These include bivalent cations that facilitate deprotonation of vicinal aromatic dihydroxy systems and illustrate a conserved arrangement of hydroxyl and carboxyl ligands consistent with the requirements of a metal-activated catalytic mechanism. The general concept of metal-dependent deprotonation via a complexed aspartate is only one part of a more pronounced proton relay, as shown by semiempirical and DFT quantum mechanical calculations and experimental validations. A previously undetected catalytic triad, consisting of Lys157-Asn181-Asp228 residues is required for complete methyl transfer in case of a cation-dependent phenylpropanoid and flavonoid OMT, as described in this report. This triad appears essential for efficient methyl transfer to catechol-like hydroxyl group in phenolics. The observation is consistent with a catalytic lysine in the case of mammalian COMTs, but jettisons existing assumptions on the initial abstraction of the meta-hydroxyl proton to the metal stabilizing Asp154 (PFOMT) or comparable Asp-carboxyl groups in type of cation-dependent enzymes in plants. The triad is conserved among all characterized plant CCoAOMT-like enzymes, which are required not only for methylation of soluble phenylpropanoids like coumarins or monolignol monomers, but is also present in the similar microbial and mammalian cation-dependent enzymes which methylate a comparable set of substrates.

Bücher und Buchkapitel

Tissier, A., Ziegler, J. & Vogt T. Specialized plant metabolites: Diversity and biosynthesis . In: Ecological Biochemistry: environmental and Interspecies Interactions (Krauß, G. J.; Nies, D. H.). 14-37, (2014) ISBN: 978-3-527-31650-2 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.

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.


Fellenberg, C., Ziegler, J., Handrick, V. & Vogt, T. Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamens. Front Plant Sci. doi: 10.3389/fpls.2012.00180 3, 180, (2012)

Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers.


Bektas, I., Fellenberg, C. & Paulsen, H. Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique. Planta 236, 251-259, (2012)

Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.

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