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Publikation

Soboleva, A.; Frolova, N.; Bureiko, K.; Shumilina, J.; Balcke, G. U.; Zhukov, V. A.; Tikhonovich, I. A.; Frolov, A.; Dynamics of Reactive Carbonyl Species in Pea Root Nodules in Response to Polyethylene Glycol (PEG)-Induced Osmotic Stress Int. J. Mol. Sci. 23, 2726, (2022) DOI: 10.3390/ijms23052726

Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants’ response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.
Publikation

Leonova, T.; Shumilina, J.; Kim, A.; Frolova, N.; Wessjohann, L.; Bilova, T.; Frolov, A.; Agar-based polyethylene glycol (PEG) infusion model for pea (Pisum sativum L.) — perspectives of translation to legume crop plants Biol. Commun. 67, 236-244, (2022) DOI: 10.21638/spbu03.2022.309

Due to the oncoming climate changes water deficit represents one of the most important abiotic stressors which dramatically affects crop productivity worldwide. Because of their importance as the principal source of food protein, legumes attract a special interest of plant scientists. Moreover, legumes are involved in symbiotic association with rhizobial bacteria, which is morphologically localized to root nodules. These structures are critical for fixation of atmospheric nitrogen and highly sensitive to drought. Therefore, new drought-tolerant legume cultivars need to be developed to meet the growing food demand. However, this requires a comprehensive knowledge of the molecular mechanisms behind the plant stress response. To access these mechanisms, adequate and reliable drought stress models need to be established. The agar-based polyethylene glycol (PEG) infusion model allows a physiologically relevant reduction of soil water potential (Ψw), although it is restricted to seedlings and does not give access to proteomics and metabolomics studies. Earlier, we successfully overcame this limitation and optimized this model for mature Arabidopsis plants. Here we make the next step forward and address its application to one of the major crop legumes — pea. Using a broad panel of physiological and biochemical markers, we comprehensively prove the applicability of this setup to legumes. The patterns of drought-related physiological changes are well-interpretable and generally resemble the stress response of plants grown in soil-based stop-watering models. Thus, the proposed model can be efficiently used in the study of stress-related metabolic adjustment in green parts, roots and root nodules of juvenile and flowering plants.
Publikation

Gladchuk, A.; Shumilina, J.; Kusnetsova, A.; Bureiko, K.; Billig, S.; Tsarev, A.; Alexandrova, I.; Leonova, L.; Zhukov, V. A.; Tikhonovich, I. A.; Birkemeyer, C.; Podolskaya, E.; Frolov, A.; High-Throughput Fingerprinting of Rhizobial Free Fatty Acids by Chemical Thin-Film Deposition and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Methods Protoc. 3, 36, (2020) DOI: 10.3390/mps3020036

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.
Publikation

Shumilina, J.; Kusnetsova, A.; Tsarev, A.; Janse van Rensburg, H. C.; Medvedev, S.; Demidchik, V.; Van den Ende, W.; Frolov, A.; Glycation of Plant Proteins: Regulatory Roles and Interplay with Sugar Signalling? Int. J. Mol. Sci. 20, 2366, (2019) DOI: 10.3390/ijms20092366

Glycation can be defined as an array of non-enzymatic post-translational modifications of proteins formed by their interaction with reducing carbohydrates and carbonyl products of their degradation. Initial steps of this process rely on reducing sugars and result in the formation of early glycation products—Amadori and Heyns compounds via Schiff base intermediates, whereas their oxidative degradation or reactions of proteins with α-dicarbonyl compounds yield a heterogeneous group of advanced glycation end products (AGEs). These compounds accompany thermal processing of protein-containing foods and are known to impact on ageing, pathogenesis of diabetes mellitus and Alzheimer’s disease in mammals. Surprisingly, despite high tissue carbohydrate contents, glycation of plant proteins was addressed only recently and its physiological role in plants is still not understood. Therefore, here we summarize and critically discuss the first steps done in the field of plant protein glycation during the last decade. We consider the main features of plant glycated proteome and discuss them in the context of characteristic metabolic background. Further, we address the possible role of protein glycation in plants and consider its probable contribution to protein degradation, methylglyoxal and sugar signalling, as well as interplay with antioxidant defense.
Bücher und Buchkapitel

Osmolovskaya, N.; Shumilina, J.; Bureiko, K.; Chantseva, V.; Bilova, T.; Kuchaeva, L.; Laman, N.; Wessjohann, L. A.; Frolov, A.; Ion Homeostasis Response to Nutrient-Deficiency Stress in Plants (Vikas, B. & Fasullo, M., eds.). 1-23, (2019) ISBN: 978-1-78985-311-7 DOI: 10.5772/intechopen.89398

A crucial feature of plant performance is its strong dependence on the availability of essential mineral nutrients, affecting multiple vital functions. Indeed, mineral-nutrient deficiency is one of the major stress factors affecting plant growth and development. Thereby, nitrogen and potassium represent the most abundant mineral contributors, critical for plant survival. While studying plant responses to nutrient deficiency, one should keep in mind that mineral nutrients, along with their specific metabolic roles, are directly involved in maintaining cell ion homeostasis, which relies on a finely tuned equilibrium between cytosolic and vacuolar ion pools. Therefore, in this chapter we briefly summarize the role of the ion homeostasis system in cell responses to environmental deficiency of nitrate and potassium ions. Special attention is paid to the implementation of plant responses via NO3− and K+ root transport and regulation of ion distribution in cell compartments. These responses are strongly dependent on plant species, as well as severity and duration of nutrient deficiency.
Preprints

Osmolovskaya, N.; Shumilina, J.; Kim, A.; Didio, A.; Grishina, T.; Bilova, T.; Keltsieva, O. A.; Zhukov, V.; Tikhonovich, I.; Tarakhovskaya, E.; Wessjohann, L. A.; Frolov, A.; Methodology of Drought Stress Research: Experimental Setup and Physiological Characterization Preprints (2018) DOI: 10.20944/preprints201812.0145.v1

Drought is one of the major stress factors affecting growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants responses to water deficit by multiple physiological and metabolic adaptations at the molecular, cellular and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponic or agar culture. These experimental setups give access to different aspects of plant response to drought, like decrease of tissue water potential, reduction of stomata conductance and photosynthesis efficiency, accumulation of low-molecular weight solutes (metabolic adjustment) and drought protective proteins. Till now, this pattern of markers was successfully extended to the methods of enzyme chemistry, molecular biology and omics techniques. Thus, conventional tests can be efficiently complemented by determination of phytohormone and reactive oxygen species (ROS) contents, activities of antioxidant enzymes, as well as comprehensive profiling of transcriptome, proteome and metabolome.
Publikation

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.
Publikation

Osmolovskaya, N.; Shumilina, J.; Kim, A.; Didio, A.; Grishina, T.; Bilova, T.; Keltsieva, O. A.; Zhukov, V.; Tikhonovich, I.; Tarakhovskaya, E.; Frolov, A.; Wessjohann, L. A.; Methodology of Drought Stress Research: Experimental Setup and Physiological Characterization Int. J. Mol. Sci. 19, 4089, (2018) DOI: 10.3390/ijms19124089

Drought is one of the major stress factors affecting the growth and development of plants. In this context, drought-related losses of crop plant productivity impede sustainable agriculture all over the world. In general, plants respond to water deficits by multiple physiological and metabolic adaptations at the molecular, cellular, and organism levels. To understand the underlying mechanisms of drought tolerance, adequate stress models and arrays of reliable stress markers are required. Therefore, in this review we comprehensively address currently available models of drought stress, based on culturing plants in soil, hydroponically, or in agar culture, and critically discuss advantages and limitations of each design. We also address the methodology of drought stress characterization and discuss it in the context of real experimental approaches. Further, we highlight the trends of methodological developments in drought stress research, i.e., complementing conventional tests with quantification of phytohormones and reactive oxygen species (ROS), measuring antioxidant enzyme activities, and comprehensively profiling transcriptome, proteome, and metabolome.

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