Publications by authors named "Radosław Mazur"

25 Publications

  • Page 1 of 1

Bean and Pea Plastoglobules Change in Response to Chilling Stress.

Int J Mol Sci 2021 Nov 2;22(21). Epub 2021 Nov 2.

Department of Plant Anatomy and Cytology, Faculty of Biology, Institute of Experimental Plant Biology and Biotechnology, University of Warsaw, I. Miecznikowa 1, PL-02096 Warsaw, Poland.

Plastoglobules (PGs) might be characterised as microdomains of the thylakoid membrane that serve as a platform to recruit proteins and metabolites in their spatial proximity in order to facilitate metabolic channelling or signal transduction. This study provides new insight into changes in PGs isolated from two plant species with different responses to chilling stress, namely chilling-tolerant pea () and chilling-sensitive bean (). Using multiple analytical methods, such as high-performance liquid chromatography and visualisation techniques including transmission electron microscopy and atomic force microscopy, we determined changes in PGs' biochemical and biophysical characteristics as a function of chilling stress. Some of the observed alterations occurred in both studied plant species, such as increased particle size and plastoquinone-9 content, while others were more typical of a particular type of response to chilling stress. Additionally, PGs of first green leaves were examined to highlight differences at this stage of development. Observed changes appear to be a dynamic response to the demands of photosynthetic membranes under stress conditions.
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http://dx.doi.org/10.3390/ijms222111895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8584975PMC
November 2021

How to Measure Grana - Ultrastructural Features of Thylakoid Membranes of Plant Chloroplasts.

Front Plant Sci 2021 6;12:756009. Epub 2021 Oct 6.

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

Granum is a basic structural unit of the thylakoid membrane network of plant chloroplasts. It is composed of multiple flattened membranes forming a stacked arrangement of a cylindrical shape. Grana membranes are composed of lipids and tightly packed pigment-protein complexes whose primary role is the catalysis of photosynthetic light reactions. These membranes are highly dynamic structures capable of adapting to changing environmental conditions by fine-tuning photochemical efficiency, manifested by the structural reorganization of grana stacks. Due to a nanometer length scale of the structural granum features, the application of high-resolution electron microscopic techniques is essential for a detailed analysis of the granum architecture. This mini-review overviews recent approaches to quantitative grana structure analyses from electron microscopy data, highlighting the basic manual measurements and semi-automated workflows. We outline and define structural parameters used by different authors, for instance, granum height and diameter, thylakoid thickness, end-membrane length, Stacking Repeat Distance, and Granum Lateral Irregularity. This article also presents insights into efficient and effective measurements of grana stacks visualized on 2D micrographs. The information on how to correctly interpret obtained data, taking into account the 3D nature of grana stacks projected onto 2D space of electron micrograph, is also given. Grana ultrastructural observations reveal key features of this intriguing membrane arrangement, broadening our knowledge of the thylakoid network's remarkable plasticity.
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http://dx.doi.org/10.3389/fpls.2021.756009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8527009PMC
October 2021

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery.

Plant Physiol 2021 Dec;187(4):2785-2802

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.
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http://dx.doi.org/10.1093/plphys/kiab438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8644180PMC
December 2021

Development of a Novel Nanoarchitecture of the Robust Photosystem I from a Volcanic Microalga on Single Layer Graphene for Improved Photocurrent Generation.

Int J Mol Sci 2021 Aug 5;22(16). Epub 2021 Aug 5.

Solar Fuels Laboratory, Center of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland.

Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His-tag was genetically engineered at the -terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm for the nickel- and 17.3 µA·cm for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing.
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http://dx.doi.org/10.3390/ijms22168396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8395140PMC
August 2021

Mechanisms shaping the synergism of zeaxanthin and PsbS in photoprotective energy dissipation in the photosynthetic apparatus of plants.

Plant J 2021 07 25;107(2):418-433. Epub 2021 May 25.

Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, Lublin, 20-031, Poland.

Safe operation of photosynthesis is vital to plants and is ensured by the activity of processes protecting chloroplasts against photo-damage. The harmless dissipation of excess excitation energy is considered to be the primary photoprotective mechanism and is most effective in the combined presence of PsbS protein and zeaxanthin, a xanthophyll accumulated in strong light as a result of the xanthophyll cycle. Here we address the problem of specific molecular mechanisms underlying the synergistic effect of zeaxanthin and PsbS. The experiments were conducted with Arabidopsis thaliana, using wild-type plants, mutants lacking PsbS (npq4), and mutants affected in the xanthophyll cycle (npq1), with the application of molecular spectroscopy and imaging techniques. The results lead to the conclusion that PsbS interferes with the formation of densely packed aggregates of thylakoid membrane proteins, thus allowing easy exchange and incorporation of xanthophyll cycle pigments into such structures. It was found that xanthophylls trapped within supramolecular structures, most likely in the interfacial protein region, determine their photophysical properties. The structures formed in the presence of violaxanthin are characterized by minimized dissipation of excitation energy. In contrast, the structures formed in the presence of zeaxanthin show enhanced excitation quenching, thus protecting the system against photo-damage.
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http://dx.doi.org/10.1111/tpj.15297DOI Listing
July 2021

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana.

Plant Physiol 2021 02;185(1):210-227

Department of Plant Anatomy and Cytology, Institute of Plant Experimental Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw 02-096, Poland.

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.
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http://dx.doi.org/10.1093/plphys/kiaa009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133577PMC
February 2021

Spatial Nano-Morphology of the Prolamellar Body in Etiolated Plants With Disturbed Pigment and Polyprenol Composition.

Front Cell Dev Biol 2020 8;8:586628. Epub 2020 Oct 8.

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

The prolamellar body (PLB) is a periodic bicontinuous membrane structure based on tubular tetrahedral units. PLBs are present in plant etioplasts and, upon illumination, directly transform into the lamellar thylakoid networks within chloroplasts. Efficient tubular-lamellar rearrangement and later formation of the photosynthetically active thylakoid membranes are crucial steps in the development of plant autotrophy. PLB membranes are mainly composed of galactolipids, carotenoids, and protochlorophyllide (Pchlide), the chlorophyll precursor, bound in a complex with NADPH and Pchlide oxidoreductase. Although the PLB structure has been studied for over 50 years, the direct role of particular membrane components in the formation of the PLB paracrystalline network remains elusive. Moreover, despite the numerous literature data regarding the PLB geometry, their reliable comparative analysis is complicated due to variable experimental conditions. Therefore, we performed comprehensive ultrastructural and low-temperature fluorescence analysis of wild type (Arabidopsis) seedlings grown in different conditions typical for studies on etiolated seedlings. We established that the addition of sucrose to the growing media significantly affected the size and compactness of the PLB. The etiolation period was also an important factor influencing the PLB structural parameters and the ratio of free to complex-bound Pchlide. Thus, a reliable PLB structural and spectral analysis requires particular attention to the applied experimental conditions. We investigated the influence of the pigment and polyprenol components of the etioplast membranes on the formation of the PLB spatial structure. The PLB 3D structure in several Arabidopsis mutants (, , , , ) with disturbed levels of particular pigments and polyprenols using electron tomography technique was studied. We found that the PLB nano-morphology was mainly affected in the and mutants. An increased level of Pchlide () resulted in the substantial shift of the structural balance between outer and inner PLB water channels and overall PLB compactness compared to wild type plants. The decrease in the relative content of β-branch xanthophylls in plants was manifested by local disturbances in the paracrystalline structure of the PLB network. Therefore, proper levels of particular etioplast pigments are essential for the formation of stable and regular PLB structure.
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http://dx.doi.org/10.3389/fcell.2020.586628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578251PMC
October 2020

Regulatory mechanisms of photosynthesis light reactions in higher plants

Postepy Biochem 2020 06 27;66(2):134-142. Epub 2020 Jun 27.

Zakład Regulacji Metabolizmu, Instytut Biochemii, Wydział Biologii, Uniwersytet Warszawski.

The light phase of photosynthesis is a key energy process in higher plants. Its purpose is to convert light energy into chemical one stored in ATP and NADPH molecules, which are then used to assimilate CO2 and in numerous metabolic processes. Maintaining optimal photosynthesis performance requires strict regulation of thylakoid membranes organization and rapid response to changing environmental conditions. The main factor affecting photosynthesis is light, which, if applied in excessive amounts, leads to a slowdown in the process. Therefore, plants have developed many protective mechanisms regulating the light reactions of photosynthesis and operating at the level of light energy absorption, electron transport, and the distribution and use of reducing power. These include, among others: (i) non-photochemical energy quenching regulating the amount of excitation energy delivered to the photosystems; (ii) ‘state transition’ process redistributing excitation energy between photosystems; (iii) redundant electron transport pathways responsible for maintaining redox balance in chloroplasts. All these mechanisms, in combination with antioxidant systems, are designed to maintain the function of the photosynthetic apparatus in adverse growth conditions.
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http://dx.doi.org/10.18388/pb.2020_325DOI Listing
June 2020

Specific Composition of Lipid Phases Allows Retaining an Optimal Thylakoid Membrane Fluidity in Plant Response to Low-Temperature Treatment.

Front Plant Sci 2020 5;11:723. Epub 2020 Jun 5.

Department of Metabolic Regulation, Faculty of Biology, Institute of Biochemistry, University of Warsaw, Warsaw, Poland.

Thylakoid membranes isolated from leaves of two plant species, the chilling tolerant (CT) pea and chilling sensitive (CS) runner bean, were assessed for the composition of lipids, carotenoids as well as for the arrangement of photosynthetic complexes. The response to stress conditions was investigated in dark-chilled and subsequently photo-activated detached leaves of pea and bean. Thylakoids of both species have a similar level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), but different sulfoquinovosyldiacylglycerol to phosphatidylglycerol (PG) ratio. In pea thylakoid fraction, the MGDG, DGDG and PG, have a higher double bond index (DBI), whereas bean thylakoids contain higher levels of high melting point PG. Furthermore, the lutein to the β-carotene ratio is higher in bean thylakoids. Smaller protein/lipid ratio in pea than in bean thylakoids suggests different lipid-protein interactions in both species. The differences between species are also reflected by the course of temperature-dependent plots of chlorophyll fluorescence pointing various temperatures of the lipid phase transitions of pea and bean thylakoids. Our results showed higher fluidity of the thylakoid membrane network in pea than in bean in optimal temperature conditions. Dark-chilling decreases the photochemical activity and induces significant degradation of MGDG in bean but not in pea leaves. Similarly, substantial changes in the arrangement of photosynthetic complexes with increase in LHCII phosphorylation and disturbances of the thylakoid structure take place in bean thylakoids only. Changes in the physical properties of bean thylakoids are manifested by the conversion of a three-phase temperature-dependent plot to a one-phase plot. Subsequent photo-activation of chilled bean leaves caused a partial restoration of the photochemistry and of membrane physical properties, but not of the photosynthetic complexes arrangement nor the thylakoid network structure. Summarizing, the composition of the thylakoid lipid matrix of CT pea allows retaining the optimal fluidity of its chloroplast membranes under low temperatures. In contrast, the fluidity of CS bean thylakoids is drastically changed, leading to the reorganization of the supramolecular structure of the photosynthetic complexes and finally results in structural remodeling of the CS bean thylakoid network.
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http://dx.doi.org/10.3389/fpls.2020.00723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291772PMC
June 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

Galactolipid deficiency disturbs spatial arrangement of the thylakoid network in Arabidopsis thaliana plants.

J Exp Bot 2019 09;70(18):4689-4704

Department of Plant Anatomy and Cytology, Faculty of Biology, University of Warsaw, Miecznikowa, Warsaw, Poland.

The chloroplast thylakoid network is a dynamic structure which, through possible rearrangements, plays a crucial role in regulation of photosynthesis. Although the importance of the main components of the thylakoid membrane matrix, galactolipids, in the formation of the network of internal plastid membrane was found before, the structural role of monogalactosyldiacylglycerol (MGDG) and digalactosylidacylglycerol (DGDG) is still largely unknown. We elucidated detailed structural modifications of the thylakoid membrane system in Arabidopsis thaliana MGDG- and DGDG-deficient mutants. An altered MGDG/DGDG ratio was structurally reflected by formation of smaller grana, local changes in grana stacking repeat distance, and significant changes in the spatial organization of the thylakoid network compared with wild-type plants. The decrease of the MGDG level impaired the formation of the typical helical grana structure and resulted in a 'helical-dichotomic' arrangement. DGDG deficiency did not affect spatial grana organization but changed the shape of the thylakoid membrane network in situ from lens like into a flattened shape. Such structural disturbances were accompanied by altered composition of carotenoid and chlorophyll-protein complexes, which eventually led to the decreased photosynthetic efficiency of MGDG- and DGDG-deficient plants.
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http://dx.doi.org/10.1093/jxb/erz219DOI Listing
September 2019

Dark-chilling and subsequent photo-activation modulate expression and induce reversible association of chloroplast lipoxygenase with thylakoid membrane in runner bean (Phaseolus coccineus L.).

Plant Physiol Biochem 2018 Jan 26;122:102-112. Epub 2017 Nov 26.

Department of Metabolic Regulation, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids. This reaction is the first step in biosynthesis of oxylipins, which play important and diverse roles in stress response. In this study, we identified four LOX genes (PcLOXA, B, C, D) in chilling-sensitive runner bean (Phaseolus coccineus L.) plant and analyzed their expression patterns during long term dark-chilling (4 °C) stress and during day/night (21ºC/4 °C) temperature fluctuations. Three of the four identified LOX genes, namely PcLOXA, PcLOXB and PcLOXD, were induced by wounding stress, while only the PcLOXA was induced by dark-chilling of both detached (wounded) leaves and whole plants. We identified PcLOXA as a chloroplast-targeted LOX protein and investigated its expression during chilling stress in terms of abundance, localization inside chloroplasts and interactions with the thylakoid membranes. The analysis by immunogold electron microscopy has shown that more than 60% of detectable PcLOXA protein was associated with thylakoids, and dark-chilling of leaves resulted in increased amounts of this protein detected within grana margins of thylakoids. This effect was reversible under subsequent photo-activation of chilled leaves. PcLOXA binding to thylakoids is not mediated by the posttranslational modification but rather is based on direct interactions of the protein with membrane lipids; the binding strength increases under dark-chilling conditions.
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http://dx.doi.org/10.1016/j.plaphy.2017.11.015DOI Listing
January 2018

Sound-field measurement with moving microphones.

J Acoust Soc Am 2017 05;141(5):3220

Institute for Signal Processing, University of Lübeck, 23562 Lübeck, Germany.

Closed-room scenarios are characterized by reverberation, which decreases the performance of applications such as hands-free teleconferencing and multichannel sound reproduction. However, exact knowledge of the sound field inside a volume of interest enables the compensation of room effects and allows for a performance improvement within a wide range of applications. The sampling of sound fields involves the measurement of spatially dependent room impulse responses, where the Nyquist-Shannon sampling theorem applies in the temporal and spatial domains. The spatial measurement often requires a huge number of sampling points and entails other difficulties, such as the need for exact calibration of a large number of microphones. In this paper, a method for measuring sound fields using moving microphones is presented. The number of microphones is customizable, allowing for a tradeoff between hardware effort and measurement time. The goal is to reconstruct room impulse responses on a regular grid from data acquired with microphones between grid positions, in general. For this, the sound field at equidistant positions is related to the measurements taken along the microphone trajectories via spatial interpolation. The benefits of using perfect sequences for excitation, a multigrid recovery, and the prospects for reconstruction by compressed sensing are presented.
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http://dx.doi.org/10.1121/1.4983093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433932PMC
May 2017

A chloroplast "wake up" mechanism: Illumination with weak light activates the photosynthetic antenna function in dark-adapted plants.

J Plant Physiol 2017 Mar 15;210:1-8. Epub 2016 Dec 15.

Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, 20-031 Lublin, Poland. Electronic address:

The efficient and fluent operation of photosynthesis in plants relies on activity of pigment-protein complexes called antenna, absorbing light and transferring excitations toward the reaction centers. Here we show, based on the results of the fluorescence lifetime imaging analyses of single chloroplasts, that pigment-protein complexes, in dark-adapted plants, are not able to act effectively as photosynthetic antennas, due to pronounced, adverse excitation quenching. It appeared that the antenna function could be activated by a short (on a minute timescale) illumination with light of relatively low intensity, substantially below the photosynthesis saturation threshold. The low-light-induced activation of the antenna function was attributed to phosphorylation of the major accessory light-harvesting complex LHCII, based on the fact that such a mechanism was not observed in the stn7 Arabidopsis thaliana mutant, with impaired LHCII phosphorylation. It is proposed that the protein phosphorylation-controlled change in the LHCII clustering ability provides mechanistic background for this regulatory process.
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http://dx.doi.org/10.1016/j.jplph.2016.12.006DOI Listing
March 2017

Dark-chilling induces substantial structural changes and modifies galactolipid and carotenoid composition during chloroplast biogenesis in cucumber (Cucumis sativus L.) cotyledons.

Plant Physiol Biochem 2017 Feb 28;111:107-118. Epub 2016 Nov 28.

Department of Plant Anatomy and Cytology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland. Electronic address:

Plants in a temperate climate are often subject to different environmental factors, chilling stress among them, which influence the growth especially during early stages of plant development. Chloroplasts are one of the first organelles affected by the chilling stress. Therefore the proper biogenesis of chloroplasts in early stages of plant growth is crucial for undertaking the photosynthetic activity. In this paper, the analysis of the cotyledon chloroplast biogenesis at different levels of plastid organization was performed in cucumber, one of the most popular chilling sensitive crops. Influence of low temperature on the ultrastructure was manifested by partial recrystallization of the prolamellar body, the formation of elongated grana thylakoids and a change of the prolamellar body structure from the compacted "closed" type to a more loose "open" type. Structural changes are strongly correlated with galactolipid and carotenoid content. Substantial changes in the galactolipid and the carotenoid composition in dark-chilled plants, especially a decrease of the monogalactosyldiacylglycerol to digalactosyldiacylglycerol ratio (MGDG/DGDG) and an increased level of lutein, responsible for a decrease in membrane fluidity, were registered together with a slower adaptation to higher light intensity and an increased level of non-photochemical reactions. Changes in the grana thylakoid fluidity, of their structure and photosynthetic efficiency in developing chloroplasts of dark-chilled plants, without significant changes in the PSI/PSII ratio, could distort the balance of photosystem rearrangements and be one of the reasons of cucumber sensitivity to chilling.
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http://dx.doi.org/10.1016/j.plaphy.2016.11.022DOI Listing
February 2017

Overlapping toxic effect of long term thallium exposure on white mustard (Sinapis alba L.) photosynthetic activity.

BMC Plant Biol 2016 09 2;16(1):191. Epub 2016 Sep 2.

Laboratory of Chromatography and Environmental Analysis, Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland.

Background: Heavy metal exposure affect plant productivity by interfering, directly and indirectly, with photosynthetic reactions. The toxic effect of heavy metals on photosynthetic reactions has been reported in wide-ranging studies, however there is paucity of data in the literature concerning thallium (Tl) toxicity. Thallium is ubiquitous natural trace element and is considered the most toxic of heavy metals; however, some plant species, such as white mustard (Sinapis alba L.) are able to accumulate thallium at very high concentrations. In this study we identified the main sites of the photosynthetic process inhibited either directly or indirectly by thallium, and elucidated possible detoxification mechanisms in S. alba.

Results: We studied the toxicity of thallium in white mustard (S. alba) growing plants and demonstrated that tolerance of plants to thallium (the root test) decreased with the increasing Tl(I) ions concentration in culture media. The root growth of plants exposed to Tl at 100 μg L(-1) for 4 weeks was similar to that in control plants, while in plants grown with Tl at 1,000 μg L(-1) root growth was strongly inhibited. In leaves, toxic effect became gradually visible in response to increasing concentration of Tl (100 - 1,000 μg L(-1)) with discoloration spreading around main vascular bundles of the leaf blade; whereas leaf margins remained green. Subsequent structural analyses using chlorophyll fluorescence, microscopy, and pigment and protein analysis have revealed different effects of varying Tl concentrations on leaf tissue. At lower concentration partial rearrangement of the photosynthetic complexes was observed without significant changes in the chloroplast structure and the pigment and protein levels. At higher concentrations, the decrease of PSI and PSII quantum yields and massive oxidation of pigments was observed in discolored leaf areas, which contained high amount of Tl. Substantial decline of the photosystem core proteins and disorder of the photosynthetic complexes were responsible for disappearance of the chloroplast grana.

Conclusions: Based on the presented results we postulate two phases of thallium toxicity on photosynthesis: the non-destructive phase at early stages of toxicant accumulation and the destructive phase that is restricted to the discolored leaf areas containing high toxicant content. There was no distinct border between the two phases of thallium toxicity in leaves and the degree of toxicity was proportional to the migration rate of the toxicant outside the vascular bundles. The three-fold (nearly linear) increase of Tl(I) concentration was observed in damaged tissue and the damage appears to be associated with the presence of the oxidized form of thallium - Tl(III).
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http://dx.doi.org/10.1186/s12870-016-0883-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009500PMC
September 2016

Tetraphenylporphyrin as a protein label for triple detection analytical systems.

Heliyon 2015 Dec 22;1(4):e00053. Epub 2015 Dec 22.

Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology Noakowskiego 3, 00-664 Warsaw, Poland.

Porphyrins and metalloporphyrins are promising new protein labels that can be detected using multiple techniques; improving the reliability of the analysis and broadening the range of the linear response. Here, we investigate the potential of 5,10,15,20-tetraphenyl-21H,23H-porphyrin (Tpp) as a hybrid protein label. The electrochemical and optical properties of porphyrin conjugated with bovine serum albumin (BSA), chicken egg albumin (CEA) and immunoglobulin G (IgG) were determined and optimal conditions for Tpp-protein conjugation established. Model conjugates of carboxylated Tpp with BSA and short peptides were characterized using differential pulse voltammetry, UV-Vis spectrophotometry and spectrofluorimetry. These results reveal that Tpp is a promising molecule to be used in a triple detection protein labelling system.
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http://dx.doi.org/10.1016/j.heliyon.2015.e00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945755PMC
December 2015

Three-Dimensional Visualization of the Tubular-Lamellar Transformation of the Internal Plastid Membrane Network during Runner Bean Chloroplast Biogenesis.

Plant Cell 2016 04 21;28(4):875-91. Epub 2016 Mar 21.

Department of Plant Anatomy and Cytology, Faculty of Biology, University of Warsaw, 02-096 Warsaw, Poland

Chloroplast biogenesis is a complex process that is integrated with plant development, leading to fully differentiated and functionally mature plastids. In this work, we used electron tomography and confocal microscopy to reconstruct the process of structural membrane transformation during the etioplast-to-chloroplast transition in runner bean (Phaseolus coccineus). During chloroplast development, the regular tubular network of paracrystalline prolamellar bodies (PLBs) and the flattened porous membranes of prothylakoids develop into the chloroplast thylakoids. Three-dimensional reconstruction is required to provide us with a more complete understanding of this transformation. We provide spatial models of the bean chloroplast biogenesis that allow such reconstruction of the internal membranes of the developing chloroplast and visualize the transformation from the tubular arrangement to the linear system of parallel lamellae. We prove that the tubular structure of the PLB transforms directly to flat slats, without dispersion to vesicles. We demonstrate that the grana/stroma thylakoid connections have a helical character starting from the early stages of appressed membrane formation. Moreover, we point out the importance of particular chlorophyll-protein complex components in the membrane stacking during the biogenesis. The main stages of chloroplast internal membrane biogenesis are presented in a movie that shows the time development of the chloroplast biogenesis as a dynamic model of this process.
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http://dx.doi.org/10.1105/tpc.15.01053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863387PMC
April 2016

Molecular architecture of plant thylakoids under physiological and light stress conditions: a study of lipid-light-harvesting complex II model membranes.

Plant Cell 2013 Jun 28;25(6):2155-70. Epub 2013 Jun 28.

Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, 20-031 Lublin, Poland.

In this study, we analyzed multibilayer lipid-protein membranes composed of the photosynthetic light-harvesting complex II (LHCII; isolated from spinach [Spinacia oleracea]) and the plant lipids monogalcatosyldiacylglycerol and digalactosyldiacylglycerol. Two types of pigment-protein complexes were analyzed: those isolated from dark-adapted leaves (LHCII) and those from leaves preilluminated with high-intensity light (LHCII-HL). The LHCII-HL complexes were found to be partially phosphorylated and contained zeaxanthin. The results of the x-ray diffraction, infrared imaging microscopy, confocal laser scanning microscopy, and transmission electron microscopy revealed that lipid-LHCII membranes assemble into planar multibilayers, in contrast with the lipid-LHCII-HL membranes, which form less ordered structures. In both systems, the protein formed supramolecular structures. In the case of LHCII-HL, these structures spanned the multibilayer membranes and were perpendicular to the membrane plane, whereas in LHCII, the structures were lamellar and within the plane of the membranes. Lamellar aggregates of LHCII-HL have been shown, by fluorescence lifetime imaging microscopy, to be particularly active in excitation energy quenching. Both types of structures were stabilized by intermolecular hydrogen bonds. We conclude that the formation of trans-layer, rivet-like structures of LHCII is an important determinant underlying the spontaneous formation and stabilization of the thylakoid grana structures, since the lamellar aggregates are well suited to dissipate excess energy upon overexcitation.
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http://dx.doi.org/10.1105/tpc.113.113076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723618PMC
June 2013

A reaction center-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga, Cyanidioschyzon merolae.

J Biol Chem 2013 Aug 17;288(32):23529-42. Epub 2013 Jun 17.

Department of Plant Molecular Physiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extremely low pH levels and moderately high temperatures. The photosynthetic apparatus of the red alga Cyanidioschyzon merolae represents an intermediate type between cyanobacteria and higher plants, suggesting that this alga may provide the evolutionary link between prokaryotic and eukaryotic phototrophs. Although we now have a detailed structural model of photosystem II (PSII) from cyanobacteria at an atomic resolution, no corresponding structure of the eukaryotic PSII complex has been published to date. Here we report the isolation and characterization of a highly active and robust dimeric PSII complex from C. merolae. We show that this complex is highly stable across a range of extreme light, temperature, and pH conditions. By measuring fluorescence quenching properties of the isolated C. merolae PSII complex, we provide the first direct evidence of pH-dependent non-photochemical quenching in the red algal PSII reaction center. This type of quenching, together with high zeaxanthin content, appears to underlie photoprotection mechanisms that are efficiently employed by this robust natural water-splitting complex under excess irradiance. In order to provide structural details of this eukaryotic form of PSII, we have employed electron microscopy and single particle analyses to obtain a 17 Å map of the C. merolae PSII dimer in which we locate the position of the protein mass corresponding to the additional extrinsic protein stabilizing the oxygen-evolving complex, PsbQ'. We conclude that this lumenal subunit is present in the vicinity of the CP43 protein, close to the membrane plane.
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http://dx.doi.org/10.1074/jbc.M113.484659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5395030PMC
August 2013

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

BMC Plant Biol 2012 May 25;12:72. Epub 2012 May 25.

Department of Plant Anatomy and Cytology, Institute of Plant Experimental Biology and Biotechnology, Faculty of Biology, University of Warsaw, Miecznikowa 1, Warsaw, PL-02-096, Poland.

Background: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency.

Results: Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested.

Conclusions: Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.
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http://dx.doi.org/10.1186/1471-2229-12-72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499227PMC
May 2012

Chloroplast biogenesis - correlation between structure and function.

Biochim Biophys Acta 2012 Aug 20;1817(8):1380-7. Epub 2012 Mar 20.

Department of Plant Anatomy and Cytology, University of Warsaw, Warsaw, Poland.

Chloroplast biogenesis is a multistage process leading to fully differentiated and functionally mature plastids. Complex analysis of chloroplast biogenesis was performed on the structural and functional level of its organization during the photoperiodic plant growth after initial growth of seedlings in the darkness. We correlated, at the same time intervals, the structure of etioplasts transforming into mature chloroplasts with the changes in the photosynthetic protein levels (selected core and antenna proteins of PSI and PSII) and with the function of the photosynthetic apparatus in two plant species: bean (Phaseolus vulgaris L.) and pea (Pisum sativum L). We selected these plant species since we demonstrated previously that the mature chloroplasts differ in the thylakoid organization. We showed that the protein biosynthesis as well as photosynthetic complexes formation proceeds gradually in both plants in spite of periods of darkness. We found that both steady structural differentiation of the bean chloroplast and reformation of prolamellar bodies in pea were accompanied by a gradual increase of the photochemical activity in both species. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
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http://dx.doi.org/10.1016/j.bbabio.2012.03.013DOI Listing
August 2012

Structural and functional modifications of the major light-harvesting complex II in cadmium- or copper-treated Secale cereale.

Plant Cell Physiol 2010 Aug 13;51(8):1330-40. Epub 2010 Jul 13.

Department of Plant Physiology, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, Lublin, Poland.

The effects of 50 microM cadmium (Cd) or copper (Cu) ions on the supramolecular conformation of the light-harvesting pigment-protein complex of PSII (LHCII) isolated from rye seedlings were studied. It was found that the action of these two metal ions on the LHCII structure and organization is dissimilar. The Fourier transform infrared (FTIR) measurements indicated inhibition or stimulation of formation of parallel beta-structures and aggregates in the presence of Cd or Cu ions, respectively. The Chl a fluorescence excitation spectra of LHCII extracted from Cd-treated plants showed that the decreased aggregation of complexes was correlated with a decline in efficiency of quenching of excitation energy. From the results of mass spectrometry, changes in LHCII aggregation in the presence of Cd ions might be based on decreases in the molecular mass of Lhcb1 and Lhcb2 proteins. An increase in the content of LHCII aggregates under Cu ion excess was associated with changes in the LHCII xanthophyll pigment pool. In the complexes isolated from Cu-treated plants, all-trans violaxanthin and 9'-cis neoxanthin content declined and the simultaneous appearance of the fraction of 9-cis violaxanthin was observed. 9-cis violaxanthin formation under Cu ion excess might facilitate LHCII inter-trimer interaction and, therefore, aggregation of complexes. RLS (resonance light scattering) spectra indicated that the excitonic interaction between Chl molecules and between Chls and xanthophylls was responsible for the effective dissipation of excitation energy in LHCII isolated from Cu-treated plants. Also, changes in singlet excitation energy transfer between carotenoids and Chls under the action of heavy metals were observed.
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http://dx.doi.org/10.1093/pcp/pcq093DOI Listing
August 2010

3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

Biochim Biophys Acta 2010 Oct 16;1797(10):1736-48. Epub 2010 Jul 16.

Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, University of Warsaw, Miecznikowa 1, PL-02-096 Warsaw, Poland.

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.
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http://dx.doi.org/10.1016/j.bbabio.2010.07.001DOI Listing
October 2010

Light-induced change of configuration of the LHCII-bound xanthophyll (tentatively assigned to violaxanthin): a resonance Raman study.

J Phys Chem B 2009 Feb;113(8):2506-12

Department of Biophysics, Institute of Physics, Maria Curie-Skłodowska University, 20-031 Lublin, Poland.

Raman scattering spectra of light-harvesting complex LHCII isolated from spinach were recorded with an argon laser, tuned to excite the most red-absorbing LHCII-bound xanthophylls (514.5 nm). The intensity of the nu(4) band (at ca. 950 cm-1) corresponding to the out-of-plane wagging modes of the C-H groups in the resonance Raman spectra of carotenoids appears to be inversely dependent on the probing laser power density. This observation can be interpreted in terms of excitation-induced change of configuration of the protein-bound xanthophyll owing to the fact that the intensity of this particular band is diagnostic of a chromophore twisting resulting from its binding to the protein environment. The comparison of the shape of the nu(4) band of a xanthophyll involved in the light-induced spectral changes with the shape of the nu(4) band of the xanthophylls present in LHCII, reported in the literature, lets us conclude that, most probably, violaxanthin is a pigment that undergoes light-driven changes of molecular configuration but also the involvement of lutein may not be excluded. Possible physical mechanisms responsible for the configuration changes and physiological importance of the effect observed are discussed.
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http://dx.doi.org/10.1021/jp8101755DOI Listing
February 2009
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