Membrane protein provision controls prothylakoid biogenesis in tobacco etioplasts.

The cytochrome b559 heterodimer is a conserved component of photosystem II whose physiological role in photosynthetic electron transfer is enigmatic. A particularly puzzling aspect of cytochrome b559 has been its presence in etiolated seedlings, where photosystem II is absent. Whether or not the cytochrome has a specific function in etioplasts is unknown. Here, we have attempted to address the function of cytochrome b559 by generating transplastomic tobacco (Nicotiana tabacum) plants that overexpress psbE and psbF, the plastid genes encoding the two cytochrome b559 apoproteins. We show that strong overaccumulation of the PsbE apoprotein can be achieved in etioplasts by suitable manipulations of the promoter and the translation signals, while the cytochrome b559 level is only moderately elevated. The surplus PsbE protein causes striking ultrastructural alterations in etioplasts; most notably, it causes a condensed prolamellar body and a massive proliferation of prothylakoids, with multiple membrane layers coiled into spiral-like structures. Analysis of plastid lipids revealed that increased PsbE biosynthesis strongly stimulated plastid lipid biosynthesis, suggesting that membrane protein abundance controls prothylakoid membrane biogenesis. Our data provide evidence for a structural role of PsbE in prolamellar body formation and prothylakoid biogenesis, and indicate that thylakoid membrane protein abundance regulates lipid biosynthesis in etioplasts.

Light-independent pathway of STN7 kinase activation under low temperature stress in runner bean (Phaseolus coccineus L.).

BACKGROUND: The phosphorylation of the Light-Harvesting Complex of photosystem II (LHCII) driven by STATE TRANSITION 7 (STN7) kinase is a part of one of the crucial regulatory mechanisms of photosynthetic light reactions operating in fluctuating environmental conditions, light in particular. There are evidenced that STN7 can also be activated without light as well as in dark-chilling conditions. However, the biochemical mechanism standing behind this complex metabolic pathway has not been deciphered yet. RESULTS: In this work, we showed that dark-chilling induces light-independent LHCII phosphorylation in runner bean (Phaseolus coccineus L.). In dark-chilling conditions, we registered an increased reduction of the PQ pool which led to activation of STN7 kinase, subsequent LHCII phosphorylation, and possible LHCII relocation inside the thylakoid membrane. We also presented the formation of a complex composed of phosphorylated LHCII and photosystem I typically formed upon light-induced phosphorylation. Moreover, we indicated that the observed steps were preceded by the activation of the oxidative pentose phosphate pathway (OPPP) enzymes and starch accumulation. CONCLUSIONS: Our results suggest a direct connection between photosynthetic complexes reorganization and dark-chilling-induced activation of the thioredoxin system. The proposed possible pathway starts from the activation of OPPP enzymes and further NADPH-dependent thioredoxin reductase C (NTRC) activation. In the next steps, NTRC simultaneously activates ADP-glucose pyrophosphorylase and thylakoid membrane-located NAD(P)H dehydrogenase-like complex. These results in starch synthesis and electron transfer to the plastoquinone (PQ) pool, respectively. Reduced PQ pool activates STN7 kinase which phosphorylates LHCII. In this work, we present a new perspective on the mechanisms involving photosynthetic complexes while efficiently operating in the darkness. Although we describe the studied pathway in detail, taking into account also the time course of the following steps, the biological significance of this phenomenon remains puzzling.

Curvature thylakoid 1 proteins modulate prolamellar body morphology and promote organized thylakoid biogenesis in Arabidopsis thaliana.

The term "de-etiolation" refers to the light-dependent differentiation of etioplasts to chloroplasts in angiosperms. The underlying process involves reorganization of prolamellar bodies (PLBs) and prothylakoids into thylakoids, with concurrent changes in protein, lipid, and pigment composition, which together lead to the assembly of active photosynthetic complexes. Despite the highly conserved structure of PLBs among land plants, the processes that mediate PLB maintenance and their disassembly during de-etiolation are poorly understood. Among chloroplast thylakoid membrane-localized proteins, to date, only Curvature thylakoid 1 (CURT1) proteins were shown to exhibit intrinsic membrane-bending capacity. Here, we show that CURT1 proteins, which play a critical role in grana margin architecture and thylakoid plasticity, also participate in de-etiolation and modulate PLB geometry and density. Lack of CURT1 proteins severely perturbs PLB organization and vesicle fusion, leading to reduced accumulation of the light-dependent enzyme protochlorophyllide oxidoreductase (LPOR) and a delay in the onset of photosynthesis. In contrast, overexpression of CURT1A induces excessive bending of PLB membranes, which upon illumination show retarded disassembly and concomitant overaccumulation of LPOR, though without affecting greening or the establishment of photosynthesis. We conclude that CURT1 proteins contribute to the maintenance of the paracrystalline PLB morphology and are necessary for efficient and organized thylakoid membrane maturation during de-etiolation.

SPIRE-a software tool for bicontinuous phase recognition: application for plastid cubic membranes.

Bicontinuous membranes in cell organelles epitomize nature's ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterized by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. Their structure sizes, (around 50-500 nm), and fluid nature make transmission electron microscopy (TEM) the analysis method of choice to decipher their nanostructural features. Here we present a tool, Surface Projection Image Recognition Environment (SPIRE), to identify bicontinuous structures from TEM sections through interactive identification by comparison to mathematical "nodal surface" models. The prolamellar body (PLB) of plant etioplasts is a bicontinuous membrane structure with a key physiological role in chloroplast biogenesis. However, the determination of its spatial structural features has been held back by the lack of tools enabling the identification and quantitative analysis of symmetric membrane conformations. Using our SPIRE tool, we achieved a robust identification of the bicontinuous diamond surface as the dominant PLB geometry in angiosperm etioplasts in contrast to earlier long-standing assertions in the literature. Our data also provide insights into membrane storage capacities of PLBs with different volume proportions and hint at the limited role of a plastid ribosome localization directly inside the PLB grid for its proper functioning. This represents an important step in understanding their as yet elusive structure-function relationship.

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

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.

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

Beyond the darkness: recent lessons from etiolation and de-etiolation studies.

The state of etiolation is generally defined by the presence of non-green plastids (etioplasts) in plant tissues that would normally contain chloroplasts. In the commonly used dark-grown seedling system, etiolation is coupled with a type of growth called skotomorphogenesis. Upon illumination, de-etiolation occurs, marked by the transition from etioplast to chloroplast, and, at the seedling level, a switch to photomorphogenic growth. Etiolation and de-etiolation systems are therefore important for understanding both the acquisition of photosynthetic capacity during chloroplast biogenesis and plant responses to light-the most relevant signal in the life and growth of the organism. In this review, we discuss recent discoveries (within the past 2-3 years) in the field of etiolation and de-etiolation, with a particular focus on post-transcriptional processes and ultrastructural changes. We further discuss ambiguities in definitions of the term 'etiolation', and benefits and biases of common etiolation/de-etiolation systems. Finally, we raise several open questions and future research possibilities.

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

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.

Three-Dimensional Visualization of the Tubular-Lamellar Transformation of the Internal Plastid Membrane Network 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.

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

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).

Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species.

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis.

Curvature in Biological Systems: Its Quantification, Emergence, and Implications across the Scales.

Surface curvature both emerges from, and influences the behavior of, living objects at length scales ranging from cell membranes to single cells to tissues and organs. The relevance of surface curvature in biology is supported by numerous experimental and theoretical investigations in recent years. In this review, first, a brief introduction to the key ideas of surface curvature in the context of biological systems is given and the challenges that arise when measuring surface curvature are discussed. Giving an overview of the emergence of curvature in biological systems, its significance at different length scales becomes apparent. On the other hand, summarizing current findings also shows that both single cells and entire cell sheets, tissues or organisms respond to curvature by modulating their shape and their migration behavior. Finally, the interplay between the distribution of morphogens or micro-organisms and the emergence of curvature across length scales is addressed with examples demonstrating these key mechanistic principles of morphogenesis. Overall, this review highlights that curved interfaces are not merely a passive by-product of the chemical, biological, and mechanical processes but that curvature acts also as a signal that co-determines these processes.

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

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.

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

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 thaliana (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 (ccr1-1, lut5-1, szl1-1npq1-2, aba1-6, pif1, cpt7) 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 pif1 and aba1-6 mutants. An increased level of Pchlide (pif1) 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 ß-branch xanthophylls in aba1-6 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.

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

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 ß-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.

Electrochemical characterization of LHCII on graphite electrodes - Potential-dependent photoactivation and arrangement of complexes.

Light-dependent electrochemical properties of the light harvesting complexes of Photosystem II (LHCII) and the corresponding interactions with screen-printed graphite electrodes (GEs) are determined. No exogenous soluble redox mediators are used. LHCII isolated from spinach leaves are immobilized on GE by physical adsorption and through interactions with glutaraldehyde. Importantly, the insertion of LHCII into the pores of a GE is achieved by subjecting the electrode to specific potentials. Both trimeric and aggregated forms of LHCII located within the graphite layer retain their native structures. Voltammetric current peaks centred at ca. -230 and + 50 mV vs. Ag/AgCl (+94 and + 374 mV vs. NHE) limit the investigation of the reduction and oxidation processes of immobilized LHCII. An anodic photocurrent is generated in the LHCII-GE proportional to light intensity and can reach a value of 150 nA/cm2. Light-dependent charge separation in LHCII followed by electron transfer to the GE occurs only at potentials of above -200 mV vs. Ag/AgCl (+124 mV vs. NHE). Our results illustrate the importance of the structural proximity of LHCII and GE for photocurrent generation.

Ultrastructural modeling of small angle scattering from photosynthetic membranes.

The last decade has seen a range of studies using non-invasive neutron and X-ray techniques to probe the ultrastructure of a variety of photosynthetic membrane systems. A common denominator in this work is the lack of an explicitly formulated underlying structural model, ultimately leading to ambiguity in the data interpretation. Here we formulate and implement a full mathematical model of the scattering from a stacked double bilayer membrane system taking instrumental resolution and polydispersity into account. We validate our model by direct simulation of scattering patterns from 3D structural models. Most importantly, we demonstrate that the full scattering curves from three structurally typical cyanobacterial thylakoid membrane systems measured in vivo can all be described within this framework. The model provides realistic estimates of key structural parameters in the thylakoid membrane, in particular the overall stacking distance and how this is divided between membranes, lumen and cytoplasmic liquid. Finally, from fitted scattering length densities it becomes clear that the protein content in the inner lumen has to be lower than in the outer cytoplasmic liquid and we extract the first quantitative measure of the luminal protein content in a living cyanobacteria.

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

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.

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

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.