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High soil salinity is a global problem in agriculture that directly affects seed germination and the development of the seedlings sown deep in the soil. To study how salinity affected plastid ultrastructure, leaf segments of 11-day-old light- and dark-grown (etiolated) wheat (Triticum aestivum L. cv. Mv Béres) seedlings were floated on Hoagland solution, 600 mM KCl:NaCl (1:1) salt or isosmotic polyethylene glycol solution for 4 h in the dark. Light-grown seedlings were also treated in the light. The same treatments were also performed on etio-chloroplasts of etiolated seedlings greened for different time periods. Salt stress induced slight to strong changes in the relative chlorophyll content, photosynthetic activity, and organization of thylakoid complexes. Measurements of malondialdehyde contents and high-temperature thermoluminescence indicated significantly increased oxidative stress and lipid peroxidation under salt treatment, except for light-grown leaves treated in the dark. In chloroplasts of leaf segments treated in the light, slight shrinkage of grana (determined by transmission electron microscopy and small-angle neutron scattering) was observed, while a swelling of the (pro)thylakoid lumen was observed in etioplasts. Salt-induced swelling disappeared after the onset of photosynthesis after 4 h of greening. Osmotic stress caused no significant alterations in plastid structure and only mild changes in their activities, indicating that the swelling of the (pro)thylakoid lumen and the physiological effects of salinity are rather associated with the ionic component of salt stress. Our data indicate that etioplasts of dark-germinated wheat seedlings are the most sensitive to salt stress, especially at the early stages of their greening., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (H II ) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31 P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases-with component spectra very similar to those derived from mathematical deconvolution of the 31 P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the H II phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.

In this chapter we focus our attention on the enigmatic structural and functional roles of the major, non-bilayer lipid monogalactosyl-diacylglycerol (MGDG) in the thylakoid membrane. We give an overview on the state of the art on the role of MGDG and non-bilayer lipid phases in the xanthophyll cycles in different organisms. We also discuss data on the roles of MGDG and other lipid molecules found in crystal structures of different photosynthetic protein complexes and in lipid-protein assemblies, as well as in the self-assembly of the multilamellar membrane system. Comparison and critical evaluation of different membrane models--that take into account and capitalize on the special properties of non-bilayer lipids and/or non-bilayer lipid phases, and thus to smaller or larger extents deviate from the 'standard' Singer-Nicolson model--will conclude this review. With this chapter the authors hope to further stimulate the discussion about, what we think, is perhaps the most exciting question of membrane biophysics: the why and wherefore of non-bilayer lipids and lipid phases in, or in association with, bilayer biological membranes.

The light reactions of oxygenic photosynthesis take place in the thylakoid membranes, flattened vesicles, which contain the two photosystems and also embed the cytochrome b6f complex and the ATP synthase. In general, the thylakoid membranes are assembled into multilamellar membrane systems, which warrant an optimal light capturing efficiency. In nature, they show astounding variations, primarily due to large variations in their protein composition, which is controlled by multilevel regulatory mechanisms during long-term acclimation and short-term adaptation processes and also influenced by biotic or abiotic stresses - indicating a substantial degree of flexibility in the membrane ultrastructure. The better understanding of the dynamic features of this membrane system requires the use of non-invasive techniques, such as small angle neutron scattering (SANS), which is capable of providing accurate, statistically and spatially averaged information on the repeat distances of periodically organized thylakoid membranes under physiologically relevant conditions with time resolutions of seconds and minutes. In this review, after a short section on the basic properties of neutrons, we outline the fundamental principles of SANS measurements, its strengths and weaknesses in comparison to complementary structure investigation techniques. Then we overview recent results on isolated plant thylakoid membranes, and on living cyanobacterial and algal cells as well as on whole leaves. Special attention is paid to light-induced reversible ultrastructural changes in vivo, which, in cyanobacterial and diatom cells, were uncovered with the aid of SANS measurements; we also discuss the role of membrane reorganizations in light adaptation and photoprotection mechanisms., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the light-harvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼ 200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼ 20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼ 20%) is not commensurate to the decrease in PSII antenna size (∼ 70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences.

Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well-established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well-known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de-epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen-evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor-side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV-B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes., (Copyright © Physiologia Plantarum 2012.)

Earlier experiments, using 31P‐NMR and time‐resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non‐bilayer (or non‐lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in 31P‐NMR signatures. However, changes in the lipid‐phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording 31P‐NMR spectra on isolated thylakoid membranes that were suspended in sorbitol‐ or NaCl‐based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol‐based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash‐induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a‐fluorescence transients; all variations were much less pronounced in the NaCl‐based medium. These observations suggest that non‐bilayer lipids and non‐lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes. [ABSTRACT FROM AUTHOR]

Abstract: Chloroplast thylakoid membranes accommodate densely packed protein complexes in ordered, often semi-crystalline arrays and are assembled into highly organized multilamellar systems, an organization warranting a substantial degree of stability. At the same time, they exhibit remarkable structural flexibility, which appears to play important – yet not fully understood – roles in different short-term adaptation mechanisms in response to rapidly changing environmental conditions. In this review I will focus on dynamic features of the hierarchically organized photosynthetic machineries at different levels of structural complexity: (i) isolated light harvesting complexes, (ii) molecular macroassemblies and supercomplexes, (iii) thylakoid membranes and (iv) their multilamellar membrane systems. Special attention will be paid to the most abundant systems, the major light harvesting antenna complex, LHCII, and to grana. Two physical mechanisms, which are less frequently treated in the literature, will receive special attention: (i) thermo-optic mechanism —elementary structural changes elicited by ultrafast local heat transients due to the dissipation of photon energy, which operates both in isolated antenna assemblies and the native thylakoid membranes, regulates important enzymatic functions and appears to play role in light adaptation and photoprotection mechanisms; and (ii) the mechanism by which non-bilayer lipids and lipid phases play key role in the functioning of xanthophyll cycle de-epoxidases and are proposed to regulate the protein-to-lipid ratio in thylakoid membranes and contribute to membrane dynamics. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components. [Copyright &y& Elsevier]

The 'standard' fluid-mosaic membrane model can provide a framework for the operation of the photosynthetic and respiratory electron transport systems, the generation of the proton motive force (pmf) and its utilization for ATP synthesis according to the chemiosmotic theory. However, this model, with the bilayer organization of all lipid molecules, assigns no function to non-bilayer lipids – while in recent years it became clear that the two fundamental energy transducing membranes of the biosphere, chloroplast thylakoid membranes (TMs) and inner mitochondrial membranes (IMMs), contain large amounts of non-bilayer (non-lamellar) lipid phases. In this review, we summarize our understanding on the role of non-lamellar phases in TMs and IMMs: (i) We propose that for these membrane vesicles the dynamic exchange model (DEM) provides a more suitable framework than the 'standard' model; DEM complements the 'standard' model by assuming the co-existence of bilayer and non-bilayer phases and their interactions, which contribute to the structural dynamics of the membrane systems and safe-guard the membranes' high protein:lipid ratios. (ii) Non-bilayer phases play pivotal roles in membrane fusion and intermembrane lipid exchanges – essential processes in the self-assembly of these highly folded intricate membranes. (iii) The photoprotective, lipocalin-like lumenal enzyme, violaxanthin de-epoxidase, in its active state requires the presence of non-bilayer lipid phase. (iv) Cardiotoxins, water-soluble polypeptides, induce non-bilayer phases in mitochondria. (v) ATP synthesis, in mammalian heart IMMs, is positively correlated with the amount of non-bilayer packed lipids with restricted mobility. (vi) The hypothesized sub-compartments, due to non-lamellar phases, are proposed to enhance the utilization of pmf and might contribute to the recently documented functional independence of individual cristae within the same mitochondrion. Further research is needed to identify and characterize the structural entities associated with the observed non-bilayer phases; and albeit fundamental questions remain to be elucidated, non-lamellar lipid phases should be considered on a par with the bilayer phase, with which they co-exist in functional TMs and IMMs. • Functional thylakoid and mitochondrial membranes display marked lipid polymorphisms. • Fusion, dynamics and plasticity of membranes depend largely on non-bilayer phases. • Non-bilayer propensity of lipids safe-guards the membranes' high protein:lipid ratio. • Photoprotective activity of water-soluble thylakoid enzyme needs non-bilayer phase. • Mitochondrial ATP synthesis is associated with the presence of a non-bilayer phase. [ABSTRACT FROM AUTHOR]

The complex structure of higher plant chloroplasts has fascinated researchers for many years. Although the spatial relationship between granum and stroma thylakoids has been known for more than 20 years, most textbooks and research papers continue to include erroneous 3D models and simplified schemes. Here we present a simple computer model, based on electron micrographs from serial section of granum–stroma assemblies, showing the striking 3D structure of the stroma membrane wound around the granum. This model also provides an insight into some previously unknown functions of this intriguing multilamellar membrane system. However, many areas, such as self-assembly, structural flexibility and evolutionary niche, still remain to be explored. [Copyright &y& Elsevier]

Modern light microscopy imaging techniques have substantially advanced our knowledge about the ultrastructure of plant cells and their organelles. Laser-scanning microscopy and digital light microscopy imaging techniques, in general—in addition to their high sensitivity, fast data acquisition, and great versatility of 2D–4D image analyses—also opened the technical possibilities to combine microscopy imaging with spectroscopic measurements. In this review, we focus our attention on differential polarization (DP) imaging techniques and on their applications on plant cell walls and chloroplasts, and show how these techniques provided unique and quantitative information on the anisotropic molecular organization of plant cell constituents: (i) We briefly describe how laser-scanning microscopes (LSMs) and the enhanced-resolution Re-scan Confocal Microscope (RCM of Confocal.nl Ltd. Amsterdam, Netherlands) can be equipped with DP attachments—making them capable of measuring different polarization spectroscopy parameters, parallel with the 'conventional' intensity imaging. (ii) We show examples of different faces of the strong anisotropic molecular organization of chloroplast thylakoid membranes. (iii) We illustrate the use of DP imaging of cell walls from a variety of wood samples and demonstrate the use of quantitative analysis. (iv) Finally, we outline the perspectives of further technical developments of micro-spectropolarimetry imaging and its use in plant cell studies. [ABSTRACT FROM AUTHOR]

Spectropolarimetry of intact plant leaves allows to probe the molecular architecture of vegetation photosynthesis in a non-invasive and non-destructive way and, as such, can offer a wealth of physiological information. In addition to the molecular signals due to the photosynthetic machinery, the cell structure and its arrangement within a leaf can create and modify polarization signals. Using Mueller matrix polarimetry with rotating retarder modulation, we have visualized spatial variations in polarization in transmission around the chlorophyll a absorbance band from 650 nm to 710 nm. We show linear and circular polarization measurements of maple leaves and cultivated maize leaves and discuss the corresponding Mueller matrices and the Mueller matrix decompositions, which show distinct features in diattenuation, polarizance, retardance and depolarization. Importantly, while normal leaf tissue shows a typical split signal with both a negative and a positive peak in the induced fractional circular polarization and circular dichroism, the signals close to the veins only display a negative band. The results are similar to the negative band as reported earlier for single macrodomains. We discuss the possible role of the chloroplast orientation around the veins as a cause of this phenomenon. Systematic artefacts are ruled out as three independent measurements by different instruments gave similar results. These results provide better insight into circular polarization measurements on whole leaves and options for vegetation remote sensing using circular polarization. [ABSTRACT FROM AUTHOR]

Abstract: The multilamellar organization of freshly isolated spinach and pea chloroplast thylakoid membranes was studied using small-angle neutron scattering. A broad peak at ~0.02Å−1 is ascribed to diffraction from domains of ordered, unappressed stroma lamellae, revealing a repeat distance of 294ű7Å in spinach and 345ű11Å in pea. The peak position and hence the repeat distance of stroma lamellae is strongly dependent on the osmolarity and the ionic strength of the suspension medium, as demonstrated by varying the sorbitol and the Mg++-concentration in the sample. For pea thylakoid membranes, we show that the repeat distance decreases when illuminating the sample with white light, in accordance with our earlier results on spinach, also regarding the observation that addition of an uncoupler prohibits the light-induced structural changes, a strong indication that these changes are driven by the transmembrane proton gradient. We show that the magnitude of the shrinkage is strongly dependent on light intensity and that the repeat distance characteristic of the dark state after illumination is different from the initial dark state. Prolonged strong illumination leads to irreversible changes and swelling as reflected in increased repeat distances. The observed reorganizations are discussed within the frames of the current structural models of the granum-stroma thylakoid membrane assembly and the regulatory mechanisms in response to variations in the environmental conditions in vivo. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. [Copyright &y& Elsevier]

Abstract: The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above −30 °C and that a phase transition occurs only above 45 °C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases – upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 °C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 °C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane. [Copyright &y& Elsevier]

Abstract: Absorption spectrophotometry, a standard tool for quantitative analysis, suffers from two major drawbacks: lack of sensitivity and vulnerability to scattering. It has been pointed out earlier that the solution to these problems lies in using a reflecting cavity as a sample holder. Due to multiple reflections at the cavity wall, the effective pathlength becomes considerably larger than the diameter of the cavity, and scattering losses are eliminated because scattered light is prevented from escaping the detector. Though much effort has been spent in analysing and improving the performance of such a device, often called an integrating cavity absorption meter (ICAM), a simple strategy for deducing the absorbance of the sample is still lacking. It is shown here that the absorbance A′ measured by using an ICAM exhibits a sublinear increase with the solute concentration C. The physical reason for this departure from linearity is explained, and a straightforward procedure for converting A′ to the true absorbance A (proportional to C) is established. The reliability of the procedure is demonstrated by comparing the ICAM absorption spectrum of dilute dye solutions with the spectra of more concentrated solutions recorded in a conventional spectrophotometer. The ability of the device to cope with scattering was tested by filling the ICAM with a suspension of chloroplasts, and the spectrum was found, as expected, to be free from scattering artefacts. [Copyright &y& Elsevier]