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Publikation

Tahara, K.; Nishiguchi, M.; Frolov, A.; Mittasch, J.; Milkowski, C.; Identification of UDP glucosyltransferases from the aluminum-resistant tree Eucalyptus camaldulensis forming β-glucogallin, the precursor of hydrolyzable tannins Phytochemistry 152, 154-161, (2018) DOI: 10.1016/j.phytochem.2018.05.005

In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-β-d-glucose (β-glucogallin), the precursor of hydrolyzable tannins. By homology-based cloning, seven full-length candidate cDNAs were isolated from E. camaldulensis and expressed in Escherichia coli as recombinant N-terminal His-tagged proteins. Phylogenetic analysis classified four of these as UDP glycosyltransferase (UGT) 84A subfamily proteins (UGT84A25a, -b, UGT84A26a, -b) and the other three as UGT84J subfamily proteins (UGT84J3, -4, -5). In vitro enzyme assays showed that the UGT84A proteins catalyzed esterification of UDP–glucose and gallic acid to form 1-O-galloyl-β-d-glucose, whereas the UGT84J proteins were inactive. Further analyses with UGT84A25a and −26a indicated that they also formed 1-O-glucose esters of other structurally related hydroxybenzoic and hydroxycinnamic acids with a preference for hydroxybenzoic acids. The UGT84A genes were expressed in leaves, stems, and roots of E. camaldulensis, regardless of aluminum stress. Taken together, our results suggest that the UGT84A subfamily enzymes of E. camaldulensis are responsible for constitutive production of 1-O-galloyl-β-d-glucose, which is the first step of hydrolyzable tannin biosynthesis.
Publikation

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.; 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 (Shanker, A. K. & Shanker, C., eds.). 295-316, (2016) 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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