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Publications - Cell and Metabolic Biology

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Publications

Frolov, A.; Mamontova, T.; Ihling, C.; Lukasheva, E.; Bankin, M.; Chantseva, V.; Vikhnina, M.; Soboleva, A.; Shumilina, J.; Mavropolo-Stolyarenko, G.; Grishina, T.; Osmolovskaya, N.; Zhukov, V.; Hoehenwarter, W.; Sinz, A.; Tikhononovich, I.; Wessjohann, L.; Bilova, T.; Smolikova, G.; Medvedev, S.; Mining seed proteome: from protein dynamics to modification profiles Biol. Commun. 63, 43-58, (2018) DOI: 10.21638/spbu03.2018.106

In the modern world, crop plants represent a major source of daily consumed foods. Among them, cereals and legumes — i.e. the crops accumulating oils, carbohydrates and proteins in their seeds — dominate in European agriculture, tremendously impacting global protein consumption and biodiesel production. Therefore, the seeds of crop plants attract the special attention of biologists, biochemists, nutritional physiologists and food chemists. Seed development and germination, as well as age- and stress-related changes in their viability and nutritional properties, can be addressed by a variety of physiological and biochemical methods. In this context, the methods of functional genomics can be applied to address characteristic changes in seed metabolism, which can give access to stress-resistant genotypes. Among these methods, proteomics is one of the most effective tools, allowing mining metabolism changes on the protein level. Here we discuss the main methodological approaches of seed proteomics in the context of physiological changes related to environmental stress and ageing. We provide a comprehensive comparison of gel- and chromatographybased approaches with a special emphasis on advantages and disadvantages of both strategies in characterization of the seed proteome.
Publications

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.
Books and chapters

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.
Publications

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).
Publications

Staniek, A.; Bouwmeester, H.; Fraser, P. D.; Kayser, O.; Martens, S.; Tissier, A.; van der Krol, S.; Wessjohann, L.; Warzecha, H.; Natural products - learning chemistry from plants Biotechnol. J. 9, 326-336, (2014) DOI: 10.1002/biot.201300059

Plant natural products (PNPs) are unique in that they represent a vast array of different structural features, ranging from relatively simple molecules to very complex ones. Given the fact that many plant secondary metabolites exhibit profound biological activity, they are frequently used as fragrances and flavors, medicines, as well as industrial chemicals. As the intricate structures of PNPs often cannot be mimicked by chemical synthesis, the original plant providers constitute the sole source for their industrial, large‐scale production. However, sufficient supply is not guaranteed for all molecules of interest, making the development of alternative production systems a priority. Modern techniques, such as genome mining and thorough biochemical analysis, have helped us gain preliminary understanding of the enzymatic formation of the valuable ingredients in planta. Herein, we review recent advances in the application of biocatalytical processes, facilitating generation of complex PNPs through utilization of plant‐derived specific enzymes and combinatorial biochemistry. We further evaluate the options of employing heterologous organisms harboring PNP biosynthetic pathways for the production of secondary metabolites of interest.
Publications

Staniek, A.; Bouwmeester, H.; Fraser, P. D.; Kayser, O.; Martens, S.; Tissier, A.; van der Krol, S.; Wessjohann, L.; Warzecha, H.; Natural products - modifying metabolite pathways in plants Biotechnol. J. 8, 1159-1171, (2013) DOI: 10.1002/biot.201300224

The diversity of plant natural product (PNP) molecular structures is reflected in the variety of biochemical and genetic pathways that lead to their formation and accumulation. Plant secondary metabolites are important commodities, and include fragrances, colorants, and medicines. Increasing the extractable amount of PNP through plant breeding, or more recently by means of metabolic engineering, is a priority. The prerequisite for any attempt at metabolic engineering is a detailed knowledge of the underlying biosynthetic and regulatory pathways in plants. Over the past few decades, an enormous body of information about the biochemistry and genetics of biosynthetic pathways involved in PNPs production has been generated. In this review, we focus on the three large classes of plant secondary metabolites: terpenoids (or isoprenoids), phenylpropanoids, and alkaloids. All three provide excellent examples of the tremendous efforts undertaken to boost our understanding of biosynthetic pathways, resulting in the first successes in plant metabolic engineering. We further consider what essential information is still missing, and how future research directions could help achieve the rational design of plants as chemical factories for high‐value products.
Publications

Wessjohann, L.; Vogt, T.; Kufka, J.; Klein, R.; Prenyl- und Methyltransferasen in Natur und Synthese BIOspektrum 18, 22-25, (2012) DOI: 10.1007/s12268-012-0137-4

Late stage enzymatic prenylation and methylation are means to diversify (natural) compounds and to specify their functions. In eukaryotes and microbes, these steps are performed by large enzyme families, the prenyl and methyl transferases, which modify various types of small molecules, like isoprenoids, phenolics or alkaloids, but also DNA and proteins. We investigate the theoretical basis of these processes and possible commercial applications in synthetic chemistry.
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