Role of isotropic lipid phase in the fusion of photosystem II membranes.

It has been thoroughly documented, by using 31P-NMR spectroscopy, that plant thylakoid membranes (TMs), in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (HII) phase. However, our knowledge concerning the structural and functional roles of the non-bilayer phases is still rudimentary. The objective of the present study is to elucidate the origin of I phases which have been hypothesized to arise, in part, from the fusion of TMs (Garab et al. 2022 Progr Lipid Res 101,163). We take advantage of the selectivity of wheat germ lipase (WGL) in eliminating the I phases of TMs (Dlouhý et al. 2022 Cells 11: 2681), and the tendency of the so-called BBY particles, stacked photosystem II (PSII) enriched membrane pairs of 300-500 nm in diameter, to form large laterally fused sheets (Dunahay et al. 1984 BBA 764: 179). Our 31P-NMR spectroscopy data show that BBY membranes contain L and I phases. Similar to TMs, WGL selectively eliminated the I phases, which at the same time exerted no effect on the molecular organization and functional activity of PSII membranes. As revealed by sucrose-density centrifugation, magnetic linear dichroism spectroscopy and scanning electron microscopy, WGL disassembled the large laterally fused sheets. These data provide direct experimental evidence on the involvement of I phase(s) in the fusion of stacked PSII membrane pairs, and strongly suggest the role of non-bilayer lipids in the self-assembly of the TM system.

Structural Entities Associated with Different Lipid Phases of Plant Thylakoid Membranes-Selective Susceptibilities to Different Lipases and Proteases.

It is well established that plant thylakoid membranes (TMs), in addition to a bilayer, contain two isotropic lipid phases and an inverted hexagonal (HII) phase. To elucidate the origin of non-bilayer lipid phases, we recorded the 31P-NMR spectra of isolated spinach plastoglobuli and TMs and tested their susceptibilities to lipases and proteases; the structural and functional characteristics of TMs were monitored using biophysical techniques and CN-PAGE. Phospholipase-A1 gradually destroyed all 31P-NMR-detectable lipid phases of isolated TMs, but the weak signal of isolated plastoglobuli was not affected. Parallel with the destabilization of their lamellar phase, TMs lost their impermeability; other effects, mainly on Photosystem-II, lagged behind the destruction of the original phases. Wheat-germ lipase selectively eliminated the isotropic phases but exerted little or no effect on the structural and functional parameters of TMs-indicating that the isotropic phases are located outside the protein-rich regions and might be involved in membrane fusion. Trypsin and Proteinase K selectively suppressed the HII phase-suggesting that a large fraction of TM lipids encapsulate stroma-side proteins or polypeptides. We conclude that-in line with the Dynamic Exchange Model-the non-bilayer lipid phases of TMs are found in subdomains separated from but interconnected with the bilayer accommodating the main components of the photosynthetic machinery.

Salt Stress Induces Paramylon Accumulation and Fine-Tuning of the Macro-Organization of Thylakoid Membranes in Euglena gracilis Cells.

The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR), and energy transfer between the photosystems did not change considerably in the salt-grown cells. We have observed notable increases in the carotenoid-to-Chl ratio and the accumulation of paramylon in the salt-treated cells. We propose that the accumulation of storage polysaccharides and changes in the pigment composition and thylakoid membrane organization help the adaptation of E. gracilis cells to salt stress and contribute to the maintenance of cellular processes under stress conditions.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. I. 31P-NMR Spectroscopy.

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 (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-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 31P-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 HII 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.

Lipid Polymorphism of the Subchloroplast-Granum and Stroma Thylakoid Membrane-Particles. II. Structure and Functions.

In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.

Role of Protein-Water Interface in the Stacking Interactions of Granum Thylakoid Membranes-As Revealed by the Effects of Hofmeister Salts.

The thylakoid membranes of vascular plants are differentiated into stacked granum and unstacked stroma regions. The formation of grana is triggered by the macrodomain formation of photosystem II and light-harvesting complex II (PSII-LHCII) and thus their lateral segregation from the photosystem I-light-harvesting complex I (PSI-LHCI) super-complexes and the ATP-synthase; which is then stabilized by stacking interactions of the adjacent PSII-LHCII enriched regions of the thylakoid membranes. The self-assembly and dynamics of this highly organized membrane system and the nature of forces acting between the PSII-LHCII macrodomains are not well understood. By using circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS) and transmission electron microscopy (TEM), we investigated the effects of Hofmeister salts on the organization of pigment-protein complexes and on the ultrastructure of thylakoid membranes. We found that the kosmotropic agent (NH4)2SO4 and the Hofmeister-neutral NaCl, up to 2 M concentrations, hardly affected the macro-organization of the protein complexes and the membrane ultrastructure. In contrast, chaotropic salts, NaClO4, and NaSCN destroyed the mesoscopic structures, the multilamellar organization of the thylakoid membranes and the chiral macrodomains of the protein complexes but without noticeably affecting the short-range, pigment-pigment excitonic interactions. Comparison of the concentration- and time-dependences of SANS, TEM and CD parameters revealed the main steps of the disassembly of grana in the presence of chaotropes. It begins with a rapid diminishment of the long-range periodic order of the grana membranes, apparently due to an increased stacking disorder of the thylakoid membranes, as reflected by SANS experiments. SANS measurements also allowed discrimination between the cationic and anionic effects-in stacking and disorder, respectively. This step is followed by a somewhat slower disorganization of the TEM ultrastructure, due to the gradual loss of stacked membrane pairs. Occurring last is the stepwise decrease and disappearance of the long-range chiral order of the protein complexes, the rate of which was faster in LHCII-deficient membranes. These data are interpreted in terms of a theory, from our laboratory, according to which Hofmeister salts primarily affect the hydrophylic-hydrophobic interactions of proteins, and the stroma-exposed regions of the intrinsic membrane proteins, in particular-pointing to the role of protein-water interface in the stacking interactions of granum thylakoid membranes.

Thylakoid membrane reorganizations revealed by small-angle neutron scattering of Monstera deliciosa leaves associated with non-photochemical quenching.

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants and algae. Although the process is extensively studied, little is known about its relationship with ultrastructural changes of the thylakoid membranes. In order to better understand this relationship, we studied the effects of illumination on the organization of thylakoid membranes in Monstera deliciosa leaves. This evergreen species is known to exhibit very large NPQ and to possess giant grana with dozens of stacked thylakoids. It is thus ideally suited for small-angle neutron scattering measurements (SANS)-a non-invasive technique, which is capable of providing spatially and statistically averaged information on the periodicity of the thylakoid membranes and their rapid reorganizations in vivo. We show that NPQ-inducing illumination causes a strong decrease in the periodic order of granum thylakoid membranes. Development of NPQ and light-induced ultrastructural changes, as well as the relaxation processes, follow similar kinetic patterns. Surprisingly, whereas NPQ is suppressed by diuron, it impedes only the relaxation of the structural changes and not its formation, suggesting that structural changes do not cause but enable NPQ. We also demonstrate that the diminishment of SANS peak does not originate from light-induced redistribution and reorientation of chloroplasts inside the cells.

Similarities and Differences in the Effects of Toxic Concentrations of Cadmium and Chromium on the Structure and Functions of Thylakoid Membranes in Chlorella variabilis.

Trace metal contaminations in natural waters, wetlands, and wastewaters pose serious threats to aquatic ecosystems-mainly via targeting microalgae. In this work, we investigated the effects of toxic amounts of chromium and cadmium ions on the structure and function of the photosynthetic machinery of Chlorella variabilis cells. To halt the propagation of cells, we used high concentrations of Cd and Cr, 50-50 mg L-1, in the forms of CdCl2 x 2.5 H2O and K2Cr2O7, respectively. Both treatments led to similar, about 50% gradual diminishment of the chlorophyll contents of the cells in 48 h, which was, however, accompanied by a small (~10%) but statistically significant enrichment (Cd) and loss (Cr) of ß-carotene. Both Cd and Cr inhibited the activity of photosystem II (PSII)-but with more severe inhibitions with Cr. On the contrary, the PsbA (D1) protein of PSII and the PsbO protein of the oxygen-evolving complex were retained more in Cr-treated cells than in the presence of Cd. These data and the higher susceptibility of P700 redox transients in Cr-treated cells suggest that, unlike with Cd, PSII is not the main target in the photochemical apparatus. These differences at the level of photochemistry also brought about dissimilarities at higher levels of the structural complexity of the photosynthetic apparatus. Circular dichroism (CD) spectroscopy measurements revealed moderate perturbations in the macro-organization of the protein complexes-with more pronounced decline in Cd-treated cells than in the cells with Cr. Also, as reflected by transmission electron microscopy and small-angle neutron scattering, the thylakoid membranes suffered shrinking and were largely fragmented in Cd-treated cells, whereas no changes could be discerned with Cr. The preservation of integrity of membranes in Cr-treated cells was most probably aided by high proportion of the de-epoxidized xanthophylls, which were absent with Cd. It can thus be concluded that beside strong similarities of the toxic effects of Cr and Cd, the response of the photosynthetic machinery of C. variabilis to these two trace metal ions substantially differ from each other-strongly suggesting different inhibitory and protective mechanisms following the primary toxic events.

Dual Action of the PN159/KLAL/MAP Peptide: Increase of Drug Penetration across Caco-2 Intestinal Barrier Model by Modulation of Tight Junctions and Plasma Membrane Permeability.

The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood⁻brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.

Anisotropic Circular Dichroism of Light-Harvesting Complex II in Oriented Lipid Bilayers: Theory Meets Experiment.

Anisotropic circular dichroism (ACD) spectroscopy of macroscopically aligned molecules reveals additional information about their excited states that is lost in the CD of randomly oriented solutions. ACD spectra of light-harvesting complex II (LHCII)-the main peripheral antenna of photosystem II in plants-in oriented lipid bilayers were recorded from the far-UV to the visible wavelength region. ACD spectra show a drastically enhanced magnitude and level of detail compared to the isotropic CD spectra, resolving a greater number of bands and weak optical transitions. Exciton calculations show that the spectral features in the chlorophyll Q y region are well-reproduced by an existing Hamiltonian for LHCII, providing further evidence for the identity of energy sinks at chlorophylls a603 and a610 in the stromal layer and chlorophylls a604 and a613 in the luminal layer. We propose ACD spectroscopy to be a valuable tool linking the three-dimensional structure and the photophysical properties of pigment-protein complexes.

Lipid-polymorphism of plant thylakoid membranes. Enhanced non-bilayer lipid phases associated with increased membrane permeability.

Earlier experiments, using 31 P-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 31 P-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 31 P-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.

Effects of selenate and red Se-nanoparticles on the photosynthetic apparatus of Nicotiana tabacum.

Selenium (Se) is a natural trace element, which shifts its action in a relatively narrow concentration range from nutritional role to toxicity. Although it has been well established that in plants chloroplasts are among the primary targets, the mechanism of toxicity on photosynthesis is not well understood. Here, we compared selenate and red-allotrope elemental selenium nanoparticles (red nanoSe) in in vitro tobacco cultures to investigate their effects on the structure and functions of the photosynthetic machinery. Selenate at 10 mg/L concentration retarded plant growth; it also led to a decreased chlorophyll content, accompanied with an increase in the carotenoid-to-chlorophyll ratio. Structural examinations of the photosynthetic machinery, using electron microscopy, small-angle neutron scattering and circular dichroism spectroscopy, revealed significant perturbation in the macro-organization of the pigment-protein complexes and sizeable shrinkage in the repeat distance of granum thylakoid membranes. As shown by chlorophyll a fluorescence transient measurements, these changes in the ultrastructure were associated with a significantly diminished photosystem II activity and a reduced performance of the photosynthetic electron transport, and an enhanced capability of non-photochemical quenching. These changes in the structure and function of the photosynthetic apparatus explain, at least in part, the retarded growth of plantlets in the presence of 10 mg/L selenate. In contrast, red nanoSe, even at 100 mg/L and selenate at 1 mg/L, exerted no negative effect on the growth of plantlets and affected only marginally the thylakoid membrane ultrastructure and the photosynthetic functions.

Selenate tolerance and selenium hyperaccumulation in the monocot giant reed (Arundo donax), a biomass crop plant with phytoremediation potential.

The response of giant reed (Arundo donax L.) to selenium (Se), added as selenate, was studied. The development, stress response, uptake, translocation, and accumulation of Se were documented in three giant reed ecotypes STM (Hungary), BL (USA), and ESP (Spain), representing different climatic zones. Plantlets regenerated from sterile tissue cultures were grown under greenhouse conditions in sand supplemented with 0, 2.5, 5, and 10 mg Se kg-1 added as sodium selenate. Total Se content was measured in different plant parts using hydride generation atomic fluorescence spectroscopy. All plants developed normally in the 0-5.0 mg Se kg-1 concentration range regardless of ecotype, but no growth occurred at 10.0 mg Se kg-1. There were no signs of chlorosis or necrosis, and the photosynthetic machinery was not affected as evidenced by no marked differences in the structure of thylakoid membranes. There was no change in the maximum quantum yield of photosystem II (Fv/Fm ratio) in the three ecotypes under Se stress, except for a significant negative effect in the ESP ecotype in the 5.0 mg Se kg-1 treatment. Glutathione peroxidase (GPx) activity increased as the Se concentration increased in the growth medium. GPx activity was higher in the shoot system than the root system in all Se treatments. All ecotypes showed great capacity of take up, translocate and accumulate selenium in their stem and leaf. Relative Se accumulation is best described as leaf ˃˃ stem ˃ root. The ESP ecotype accumulated 1783 µg g-1 in leaf, followed by BL with 1769 µg g-1, and STM with 1606 µg g-1 in the 5.0 mg Se kg-1 treatment. All ecotypes showed high values of translocation and bioaccumulation factors, particularly the ESP ecotype (10.1 and 689, respectively, at the highest tolerated Se supplementation level). Based on these findings, Arundo donax has been identified as the first monocot hyperaccumulator of selenium, because Se concentration in the leaves of all three ecotypes, and also in the stem of the ESP ecotype, is higher than 0.1% (dry weight basis) under the conditions tested. Tolerance up to 5.0 mg Se kg-1 and the Se hyperaccumulation capacity make giant reed a promising tool for Se phytoremediation.

Biochemical and pharmacological characterization of three opioid-nociceptin hybrid peptide ligands reveals substantially differing modes of their actions.

In an attempt to design opioid-nociceptin hybrid peptides, three novel bivalent ligands, H-YGGFGGGRYYRIK-NH2, H-YGGFRYYRIK-NH2 and Ac-RYYRIKGGGYGGFL-OH were synthesized and studied by biochemical, pharmacological, biophysical and molecular modelling tools. These chimeric molecules consist of YGGF sequence, a crucial motif in the N-terminus of natural opioid peptides, and Ac-RYYRIK-NH2, which was isolated from a combinatorial peptide library as an antagonist or partial agonist that inhibits the biological activity of the endogenously occurring heptadecapeptide nociceptin. Solution structures for the peptides were studied by analysing their circular dichroism spectra. Receptor binding affinities were measured by equilibrium competition experiments using four highly selective radioligands. G-protein activating properties of the multitarget peptides were estimated in [35S]GTPγS binding tests. The three compounds were also measured in electrically stimulated mouse vas deferens (MVD) bioassay. H-YGGFGGGRYYRIK-NH2 (BA55), carrying N-terminal opioid and C-terminal nociceptin-like sequences interconnected with GGG tripeptide spacer displayed a tendency of having either unordered or ß-sheet structures, was moderately potent in MVD and possessed a NOP/KOP receptor preference. A similar peptide without spacer H-YGGFRYYRIK-NH2 (BA62) exhibited the weakest effect in MVD, more α-helical periodicity was present in its structure and it exhibited the most efficacious agonist actions in the G-protein stimulation assays. The third hybrid peptide Ac-RYYRIKGGGYGGFL-OH (BA61) unexpectedly displayed opioid receptor affinities, because the opioid message motif is hidden within the C-terminus. The designed chimeric peptide ligands presented in this study accommodate well into a group of multitarget opioid compounds that include opioid-non-opioid peptide dimer analogues, dual non-peptide dimers and mixed peptide- non-peptide bifunctional ligands.

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes - As revealed by small-angle neutron scattering.

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity - similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.

A voltage-dependent chloride channel fine-tunes photosynthesis in plants.

In natural habitats, plants frequently experience rapid changes in the intensity of sunlight. To cope with these changes and maximize growth, plants adjust photosynthetic light utilization in electron transport and photoprotective mechanisms. This involves a proton motive force (PMF) across the thylakoid membrane, postulated to be affected by unknown anion (Cl(-)) channels. Here we report that a bestrophin-like protein from Arabidopsis thaliana functions as a voltage-dependent Cl(-) channel in electrophysiological experiments. AtVCCN1 localizes to the thylakoid membrane, and fine-tunes PMF by anion influx into the lumen during illumination, adjusting electron transport and the photoprotective mechanisms. The activity of AtVCCN1 accelerates the activation of photoprotective mechanisms on sudden shifts to high light. Our results reveal that AtVCCN1, a member of a conserved anion channel family, acts as an early component in the rapid adjustment of photosynthesis in variable light environments.

Fingerprinting the macro-organisation of pigment-protein complexes in plant thylakoid membranes in vivo by circular-dichroism spectroscopy.

Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment-pigment interactions and the macro-organisation (chiral macrodomains) of pigment-protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (-)674 and (+)690nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506nm band appears only in the presence of PSII-LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo.

The Arabidopsis Thylakoid Chloride Channel AtCLCe Functions in Chloride Homeostasis and Regulation of Photosynthetic Electron Transport.

Chloride ions can be translocated across cell membranes through Cl(-) channels or Cl(-)/H(+) exchangers. The thylakoid-located member of the Cl(-) channel CLC family in Arabidopsis thaliana (AtCLCe) was hypothesized to play a role in photosynthetic regulation based on the initial photosynthetic characterization of clce mutant lines. The reduced nitrate content of Arabidopsis clce mutants suggested a role in regulation of plant nitrate homeostasis. In this study, we aimed to further investigate the role of AtCLCe in the regulation of ion homeostasis and photosynthetic processes in the thylakoid membrane. We report that the size and composition of proton motive force were mildly altered in two independent Arabidopsis clce mutant lines. Most pronounced effects in the clce mutants were observed on the photosynthetic electron transport of dark-adapted plants, based on the altered shape and associated parameters of the polyphasic OJIP kinetics of chlorophyll a fluorescence induction. Other alterations were found in the kinetics of state transition and in the macro-organization of photosystem II supercomplexes, as indicated by circular dichroism measurements. Pre-treatment with KCl but not with KNO3 restored the wild-type photosynthetic phenotype. Analyses by transmission electron microscopy revealed a bow-like arrangement of the thylakoid network and a large thylakoid-free stromal region in chloroplast sections from the dark-adapted clce plants. Based on these data, we propose that AtCLCe functions in Cl(-) homeostasis after transition from light to dark, which affects chloroplast ultrastructure and regulation of photosynthetic electron transport.

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth.

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high-phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO2 fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non-photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton-motive force across thylakoids. Small-angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long-range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild-type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro-organization of complexes and induction of photoprotective mechanisms.

Heat- and light-induced detachment of the light harvesting complex from isolated photosystem I supercomplexes.

In a previous study, using photosystem I enriched stroma thylakoid membrane vesicles, we have shown that the light harvesting complexes of this photosystem are prone to heat- and light-induced, thermo-optically driven detachment from the supercomplex [43]. We have also shown that the splitting of the supercomplex occurs in a gradual and specific manner, selectively affecting the different constituents of the antenna complexes. Here we further analyse these heat- and light-induced processes in isolated Photosystem I supercomplex using circular dichroism and 77K fluorescence emission spectroscopy and immuno blotting, and obtain further details on the sequence of events of the dissociation process as well as on the thermal stability of the different components. Our absorption and circular dichroism spectroscopy and immuno blotting data show that the dissociation of LHCI from PSI-LHCI supercomplex starts above 50°C. Also, the low temperature fluorescence emission spectra depicts decrease of maximum fluorescence emission at 730nm and an increase of the intensity at 685nm, and about 10nm blue-shifts, from 730 to 720nm and from 685 to 676nm, respectively, indicating the heat (50°C) induced detachment of LHCI from PSI core complexes. The reaction centre proteins are highly stable even at high temperatures. Lhca2 is more heat stable than the other light harvesting protein complexes of PSI, whereas Lhca4 and Lhca3 are rather labile. Combined heat and light treatments significantly enhances the disorganization of PSI-LHCI supercomplexes, indicating a thermo-optic mechanism, which might have significant role under combined heat and light stress conditions.