Maciej Garstka

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

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.

Conformational rearrangements in light-harvesting complex II accompanying light-induced chlorophyll a fluorescence quenching

Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 micromol m(-2) s(-1)) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV-Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600-1700 cm(-1)). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm(-1), corresponding to a protein in the alpha-helical structure at the expense of the band centered at 1621 cm(-1), characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.

Molecular Architecture of Plant Thylakoids under Physiological and Light Stress Conditions: A Study of Lipid–Light-Harvesting Complex II Model Membranes

The organization of plant thylakoid membranes under physiological and light stress conditions was analyzed in studies of model membranes formed with galactolipids and LHCII. The results show adaptation of an organization pattern of lipid-protein membranes to better fulfill two opposite physiological functions: harvesting of light quanta versus quenching of excess energy. 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.

A Reaction Center-dependent Photoprotection Mechanism in a Highly Robust Photosystem II from an Extremophilic Red Alga, Cyanidioschyzon merolae

Background: PSII is a protein complex that captures sunlight to drive water oxidation. Results: Cyanidioschyzon merolae PSII is protected by reversible reaction center-based non-photochemical quenching. Conclusion: C. merolae PSII employs reaction center non-photochemical quenching as the main photoprotective mechanism. Significance: We provide the first direct evidence of the PSII reaction center as the primary locus of non-photochemical quenching in the extremophilic red algae. 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.

Monitoring the mitochondrial transmembrane potential with the JC-1 fluorochrome in programmed cell death during mesophyll leaf senescence

Summary.Analysis of the mitochondrial transmembrane potential (ΔΨm) with the help of the JC-1 fluorochrome (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide) during mesophyll leaf senescence was performed in order to determine whether a reduction of ΔΨm takes place during mesophyll senescence and whether plant mitochondria, like mammalian ones, might be involved in the induction of programmed cell death. Fluorescence analysis of mesophyll protoplasts of Pisum sativum in a confocal microscope, fluorescent spectra analysis and time dependence of fluorescence intensity of monomers and of J-aggregates revealed that JC-1 is incorporated and accumulated specifically in plant mitochondria. Analysis of ΔΨm during mesophyll protoplast senescence revealed that two subpopulations of mitochondria which differ in ΔΨm exist in all analyzed stages of leaf senescence. The first subpopulation contains mitochondria with red fluorescence of J-aggregates due to an unperturbed high ΔΨm. The second subpopulation comprises mitochondria with green fluorescence of monomers due to a low ΔΨm, proving total depolarization of mitochondrial membranes. Fluorescence analysis demonstrated that even in the latest analyzed stages of leaf senescence, mitochondria with a high ΔΨm still exist. Fluorometric measurements revealed that the fluorescence intensity of J-aggregates decreases with the age of plants, which indicates that a reduction of ΔΨm during the mesophyll senescence process takes place; however, it does not take place within the whole population of mitochondria of the same protoplast. The reason of this can be due to a dramatic reorganization of mitochondria in mesophyll cells and the appearance of large mitochondria with local heterogeneity of ΔΨm in the oldest analyzed stages. All mitochondria in every stage of senescence maintained their membrane organization even when their size, distribution, and spatial organization in protoplasts changed dramatically. We stated that the reduction of ΔΨm does not directly induce programmed cell death in mesophyll cells, as opposed to animal apoptosis.

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

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.

Chloroplast biogenesis — Correlation between structure and function

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.

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

Three-dimensional visualization of the internal plastid membrane network using electron tomography reveals a dynamic tubular-parallel transformation and a helical manner of grana formation during runner bean chloroplast biogenesis. 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.

Potato Annexin STANN1 Promotes Drought Tolerance and Mitigates Light Stress in Transgenic Solanum tuberosum L. Plants.

Annexins are a family of calcium- and membrane-binding proteins that are important for plant tolerance to adverse environmental conditions. Annexins function to counteract oxidative stress, maintain cell redox homeostasis, and enhance drought tolerance. In the present study, an endogenous annexin, STANN1, was overexpressed to determine whether crop yields could be improved in potato (Solanum tuberosum L.) during drought. Nine potential potato annexins were identified and their expression characterized in response to drought treatment. STANN1 mRNA was constitutively expressed at a high level and drought treatment strongly increased transcription levels. Therefore, STANN1 was selected for overexpression analysis. Under drought conditions, transgenic potato plants ectopically expressing STANN1 were more tolerant to water deficit in the root zone, preserved more water in green tissues, maintained chloroplast functions, and had higher accumulation of chlorophyll b and xanthophylls (especially zeaxanthin) than wild type (WT). Drought-induced reductions in the maximum efficiency and the electron transport rate of photosystem II (PSII), as well as the quantum yield of photosynthesis, were less pronounced in transgenic plants overexpressing STANN1 than in the WT. This conferred more efficient non-photochemical energy dissipation in the outer antennae of PSII and probably more efficient protection of reaction centers against photooxidative damage in transgenic plants under drought conditions. Consequently, these plants were able to maintain effective photosynthesis during drought, which resulted in greater productivity than WT plants despite water scarcity. Although the mechanisms underlying this stress protection are not yet clear, annexin-mediated photoprotection is probably linked to protection against light-induced oxidative stress.

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

BackgroundThe 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.ResultsOur 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.ConclusionsBased 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.