Publications by authors named "Monika Ostaszewska-Bugajska"

8 Publications

  • Page 1 of 1

Markers for Mitochondrial ROS Status.

Methods Mol Biol 2022 ;2363:199-213

Department of Plant Bioenergetics, Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

Mitochondria actively participate in oxygenic metabolism and are one of the major sources of reactive oxygen species (ROS) production in plant cells. However, instead of measuring ROS concentrations in organelles it is more worthwhile to observe active ROS generation or downstream oxidation products, because the steady state level of ROS is easily buffered. Here, we describe how to measure the in vitro production of superoxide anion radicals (O·) by mitochondria and the release of O· into the cytosol. A method to determine glutathione, which is the most abundant mitochondrial low-mass antioxidant, is presented since changes in the redox state of glutathione can be indicative of the oxidative action of ROS. The identification of oxidative damage to mitochondrial components is the ultimate symptom that ROS homeostasis is not under control. We present how to determine the extent of oxidation of membrane lipids and the carbonylation of mitochondrial proteins. In summary, oxidative stress symptoms have to be analyzed at different levels, including ROS production, scavenging capacity, and signs of destruction, which only together can be considered markers of mitochondrial ROS status.
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http://dx.doi.org/10.1007/978-1-0716-1653-6_15DOI Listing
January 2022

Quantification of Methylglyoxal Levels in Cowpea Leaves in Response to Cowpea Aphid Infestation.

Bio Protoc 2020 Oct 20;10(20):e3795. Epub 2020 Oct 20.

Graduate program in Biochemistry and Molecular Biology, University of California, Riverside, USA.

Aphids are a serious pest of crops across the world. Aphids feed by inserting their flexible hypodermal needlelike mouthparts, or stylets, into their host plant tissues. They navigate their way to the phloem where they feed on its sap causing little mechanical damage to the plant. Additionally, while feeding, aphids secrete proteinaceous effectors in their saliva to alter plant metabolism and disrupt plant defenses to gain an advantage over the plant. Even with these arsenals to overcome plant responses, plants have evolved ways to detect and counter with defense responses to curtail aphid infestation. One of such response of cowpea to cowpea aphid infestation, is accumulation of the metabolite methylglyoxal. Methylglyoxal is an α,β-dicarbonyl ketoaldehyde that is toxic at high concentrations. Methylglyoxal levels increase modestly after exposure to a number of different abiotic and biotic stresses and has been shown to act as an emerging defense signaling molecule at low levels. Here we describe a protocol to measure methylglyoxal in cowpea leaves after cowpea aphid infestation, by utilizing a perchloric acid extraction process. The extracted supernatant was neutralized with potassium carbonate, and methylglyoxal was quantified through its reaction with N-acetyl-L-cysteine to form N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine, a product that is quantified spectrophotometrically.
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http://dx.doi.org/10.21769/BioProtoc.3795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842737PMC
October 2020

Efficient Photosynthetic Functioning of Through Electron Dissipation in Chloroplasts and Electron Export to Mitochondria Under Ammonium Nutrition.

Front Plant Sci 2020 26;11:103. Epub 2020 Feb 26.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

An improvement in photosynthetic rate promotes the growth of crop plants. The sink-regulation of photosynthesis is crucial in optimizing nitrogen fixation and integrating it with carbon balance. Studies on these processes are essential in understanding growth inhibition in plants with ammonium ( ) syndrome. Hence, we sought to investigate the effects of using nitrogen sources with different states of reduction (during assimilation of versus ) on the photosynthetic performance of . Our results demonstrated that photosynthetic functioning during long-term nutrition was not disturbed and that no indication of photoinhibition of PSII was detected, revealing the robustness of the photosynthetic apparatus during stressful conditions. Based on our findings, we propose multiple strategies to sustain photosynthetic activity during limited reductant utilization for assimilation. One mechanism to prevent chloroplast electron transport chain overreduction during nutrition is for cyclic electron flow together with plastid terminal oxidase activity. Moreover, redox state in chloroplasts was optimized by a dedicated type II NAD(P)H dehydrogenase. In order to reduce the amount of energy that reaches the photosynthetic reaction centers and to facilitate photosynthetic protection during nutrition, non-photochemical quenching (NPQ) and ample xanthophyll cycle pigments efficiently dissipate excess excitation. Additionally, high redox load may be dissipated in other metabolic reactions outside of chloroplasts due to the direct export of nucleotides through the malate/oxaloacetate valve. Mitochondrial alternative pathways can downstream support the overreduction of chloroplasts. This mechanism correlated with the improved growth of with the overexpression of the alternative oxidase 1a (AOX1a) during nutrition. Most remarkably, our findings demonstrated the capacity of chloroplasts to tolerate syndrome instead of providing redox poise to the cells.
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http://dx.doi.org/10.3389/fpls.2020.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054346PMC
February 2020

Nitrogen Source Dependent Changes in Central Sugar Metabolism Maintain Cell Wall Assembly in Mitochondrial Complex I-Defective and Secondarily Affect Programmed Cell Death.

Int J Mol Sci 2018 Jul 28;19(8). Epub 2018 Jul 28.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.

For optimal plant growth, carbon and nitrogen availability needs to be tightly coordinated. Mitochondrial perturbations related to a defect in complex I in the () mutant, carrying a point mutation in the 8-kD Fe-S subunit of NDUFS4 protein, alter aspects of fundamental carbon metabolism, which is manifested as stunted growth. During nitrate nutrition, plants showed a dominant sugar flux toward nitrogen assimilation and energy production, whereas cellulose integration in the cell wall was restricted. However, when cultured on NH₄⁺ as the sole nitrogen source, which typically induces developmental disorders in plants (i.e., the ammonium toxicity syndrome), showed improved growth as compared to NO₃ nourishing. Higher energy availability in plants was correlated with restored cell wall assembly during NH₄⁺ growth. To determine the relationship between mitochondrial complex I disassembly and cell wall-related processes, aspects of cell wall integrity and sugar and reactive oxygen species signaling were analyzed in plants. The responses of plants to NH₄⁺ treatment were consistent with the inhibition of a form of programmed cell death. Resistance of plants to NH₄⁺ toxicity coincided with an absence of necrotic lesion in plant leaves.
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http://dx.doi.org/10.3390/ijms19082206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121878PMC
July 2018

Enhanced Formation of Methylglyoxal-Derived Advanced Glycation End Products in Under Ammonium Nutrition.

Front Plant Sci 2018 24;9:667. Epub 2018 May 24.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland.

Nitrate (NO) and ammonium (NH) are prevalent nitrogen (N) sources for plants. Although NH should be the preferred form of N from the energetic point of view, ammonium nutrition often exhibits adverse effects on plant physiological functions and induces an important growth-limiting stress referred as ammonium syndrome. The effective incorporation of NH into amino acid structures requires high activity of the mitochondrial tricarboxylic acid cycle and the glycolytic pathway. An unavoidable consequence of glycolytic metabolism is the production of methylglyoxal (MG), which is very toxic and inhibits cell growth in all types of organisms. Here, we aimed to investigate MG metabolism in plants grown on NH as a sole N source. We found that changes in activities of glycolytic enzymes enhanced MG production and that markedly elevated MG levels superseded the detoxification capability of the glyoxalase pathway. Consequently, the excessive accumulation of MG was directly involved in the induction of dicarbonyl stress by introducing MG-derived advanced glycation end products (MAGEs) to proteins. The severe damage to proteins was not within the repair capacity of proteolytic enzymes. Collectively, our results suggest the impact of MG (mediated by MAGEs formation in proteins) in the contribution to NH toxicity symptoms in .
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http://dx.doi.org/10.3389/fpls.2018.00667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976750PMC
May 2018

Suppression of External NADPH Dehydrogenase-NDB1 in Confers Improved Tolerance to Ammonium Toxicity via Efficient Glutathione/Redox Metabolism.

Int J Mol Sci 2018 May 9;19(5). Epub 2018 May 9.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, I. Miecznikowa 1, 02-096 Warsaw, Poland.

Environmental stresses, including ammonium (NH₄⁺) nourishment, can damage key mitochondrial components through the production of surplus reactive oxygen species (ROS) in the mitochondrial electron transport chain. However, alternative electron pathways are significant for efficient reductant dissipation in mitochondria during ammonium nutrition. The aim of this study was to define the role of external NADPH-dehydrogenase (NDB1) during oxidative metabolism of NH₄⁺-fed plants. Most plant species grown with NH₄⁺ as the sole nitrogen source experience a condition known as “ammonium toxicity syndrome”. Surprisingly, transgenic plants suppressing were more resistant to NH₄⁺ treatment. The knock-down line was characterized by milder oxidative stress symptoms in plant tissues when supplied with NH₄⁺. Mitochondrial ROS accumulation, in particular, was attenuated in the knock-down plants during NH₄⁺ treatment. Enhanced antioxidant defense, primarily concerning the glutathione pool, may prevent ROS accumulation in NH₄⁺-grown -suppressing plants. We found that induction of glutathione peroxidase-like enzymes and peroxiredoxins in the -surpressing line contributed to lower ammonium-toxicity stress. The major conclusion of this study was that NDB1 suppression in plants confers tolerance to changes in redox homeostasis that occur in response to prolonged ammonium nutrition, causing cross tolerance among plants.
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http://dx.doi.org/10.3390/ijms19051412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983774PMC
May 2018

Altered Cell Wall Plasticity Can Restrict Plant Growth under Ammonium Nutrition.

Front Plant Sci 2017 10;8:1344. Epub 2017 Aug 10.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of WarsawWarsaw, Poland.

Plants mainly utilize inorganic forms of nitrogen (N), such as nitrate (NO) and ammonium (NH). However, the composition of the N source is important, because excess of NH promotes morphological disorders. Plants cultured on NH as the sole N source exhibit serious growth inhibition, commonly referred to as "ammonium toxicity syndrome." NH-mediated suppression of growth may be attributable to both repression of cell elongation and reduction of cell division. The precondition for cell enlargement is the expansion of the cell wall, which requires the loosening of the cell wall polymers. Therefore, to understand how NH nutrition may trigger growth retardation in plants, properties of their cell walls were analyzed. We found that using NH as the sole N source has smaller cells with relatively thicker cell walls. Moreover, cellulose, which is the main load-bearing polysaccharide revealed a denser assembly of microfibrils. Consequently, the leaf blade tissue showed elevated tensile strength and indicated higher cell wall stiffness. These changes might be related to changes in polysaccharide and ion content of cell walls. Further, NH toxicity was associated with altered activities of cell wall modifying proteins. The lower activity and/or expression of pectin hydrolyzing enzymes and expansins might limit cell wall expansion. Additionally, the higher activity of cell wall peroxidases can lead to higher cross-linking of cell wall polymers. Overall, the NH-mediated inhibition of growth is related to a more rigid cell wall structure, which limits expansion of cells. The changes in cell wall composition were also indicated by decreased expression of , a receptor-like kinase involved in the control of cell wall extension.
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http://dx.doi.org/10.3389/fpls.2017.01344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554365PMC
August 2017

Antioxidative and proteolytic systems protect mitochondria from oxidative damage in S-deficient Arabidopsis thaliana.

J Plant Physiol 2015 Aug 20;186-187:25-38. Epub 2015 Aug 20.

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Poland. Electronic address:

We examined the functioning of the antioxidative defense system in Arabidopsis thaliana under sulphur (S) deficiency with an emphasis on the role of mitochondria. In tissue extracts and in isolated mitochondria from S-deficient plants, the concentration of non-protein thiols declined but protein thiols did not change. Superoxide anion and hydrogen peroxide were accumulated in leaf blades and the generation of superoxide anion by isolated mitochondria was higher. Lower abundance of reduced (GSH) plus oxidized (GSSG) glutathione in the leaf and root tissues, and leaf mitochondria from S-deficient plants was accompanied by a decrease in the level of GSH and the changes in the GSH/GSSG ratios. In the chloroplasts, the total level of glutathione decreased. Lower levels of reduced (AsA) and oxidized (DHA) ascorbate were reflected in much higher ratios of AsA/DHA. Sulphur deficiency led to an increase in the activity of cytosolic, mitochondrial and chloroplastic antioxidative enzymes, peroxidases, catalases and superoxide dismutases. The protein carbonyl level was higher in the leaves of S-deficient plants and in the chloroplasts, while in the roots, leaf and root mitochondria it remained unchanged. Protease activity in leaf extracts of S-deficient plants was higher, but in root extracts it did not differ. The proteolytic system reflected subcellular specificity. In leaf and root mitochondria the protease activity was higher, whereas in the chloroplasts it did not change. We propose that the preferential incorporation of S to protein thiols and activation of antioxidative and proteolytic systems are likely important for the survival of S-deficient plants and that the mitochondria maintain redox homeostasis.
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http://dx.doi.org/10.1016/j.jplph.2015.07.011DOI Listing
August 2015
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