In oxygenic photosynthetic organisms, the light reactions are performed by protein complexes embedded in the lipid bilayer of thylakoid membranes (TMs). The organization of the bulk lipid molecules into bilayer structures provide optimal conditions for the build-up of the proton motive force (pmf) and its utilization for ATP synthesis. However, the lipid composition of TMs is dominated by the non-bilayer lipid species monogalactosyl diacylglycerol (MGDG), and functional plant TMs, besides the bilayer, contain large amounts of non-bilayer lipid phases. Bulk lipids have been shown to be associated with lumenal, stromal-side and marginal-region proteins and proposed to play roles in the self-assembly and photoprotection of the photosynthetic machinery. Furthermore, it has recently been pointed out that the generation and utilization of pmf for ATP synthesis according to the 'protet' or protonic charge transfer model Kell (Biochim Biophys Acta Bioenerg 1865(4):149504, 2024), requires high MGDG content Garab (Physiol Plant 177(2):e70230, 2025). In this study, to gain better insight into the structural and functional roles of MGDG, we employed all atom and coarse-grained molecular dynamics simulations to explore how temperature, hydration levels and varying MGDG concentrations affect the structural and dynamic properties of bilayer membranes constituted of plant thylakoid lipids. Our findings reveal that MGDG promotes increased membrane fluidity and dynamic fluctuations in membrane thickness. MGDG-rich stacked bilayers spontaneously formed inverted hexagonal phases; these transitions were enhanced at low hydration levels and at elevated but physiologically relevant temperatures. It can thus be inferred that MGDG plays important roles in heat and drought stress mechanisms.

• Klíčová slova
• Dehydration, Inverted hexagonal phase, MGDG, Non-bilayer lipid, Thylakoid membranes,
• MeSH
• fotosyntéza MeSH
• galaktolipidy metabolismus   chemie   MeSH
• lipidové dvojvrstvy metabolismus   chemie   MeSH
• simulace molekulární dynamiky * MeSH
• teplota MeSH
• tylakoidy * metabolismus   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• galaktolipidy MeSH
• lipidové dvojvrstvy MeSH
• monogalactosyldiacylglycerol MeSH Prohlížeč

The light reactions of oxygenic photosynthesis are performed by protein complexes embedded in the lipid bilayer of thylakoid membranes (TMs). Bilayers provide optimal conditions for the build-up of the proton motive force (pmf) and ATP synthesis. However, functional plant TMs, besides the bilayer, contain an inverted hexagonal (HII) phase and isotropic phases, a lipid polymorphism due to their major, non-bilayer lipid species, monogalactosyldiacylglycerol (MGDG). The lipid phase behavior of TMs is explained within the framework of the Dynamic Exchange Model (DEM), an extension of the fluid-mosaic model. DEM portrays the bilayer phase as inclusions between photosynthetic supercomplexes - characterized by compromised membrane impermeability and restricted sizes inflicted by the segregation propensity of lipid molecules, safe-guarding the high protein density of TMs. Isotropic phases mediate membrane fusions and are associated with the lumenal lipocalin-like enzyme, violaxanthin de-epoxidase. Stromal-side proteins surrounded by lipids give rise to the HII phase. These features instigate experimentally testable hypotheses: (i) non-bilayer phases mediate functional sub-compartmentalization of plant chloroplasts - a quasi-autonomous energization and ATP synthesis of each granum-stroma TM assembly; and (ii) the generation and utilization of pmf depend on hydrated protein networks and proton-conducting pathways along membrane surfaces - rather than on strict impermeability of the bilayer.

• MeSH
• biologické modely MeSH
• fotosyntéza MeSH
• galaktolipidy metabolismus   MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• rostliny * metabolismus   MeSH
• tylakoidy * metabolismus   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• galaktolipidy MeSH
• lipidové dvojvrstvy MeSH

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.

• Klíčová slova
• 31P-NMR spectroscopy; BBY membrane, Linear dichroism spectroscopy, Membrane fusion; non-bilayer lipids, Wheat germ lipase,
• MeSH
• fotosystém II (proteinový komplex) * metabolismus   MeSH
• fúze membrán fyziologie   MeSH
• lipidy chemie   MeSH
• magnetická rezonanční spektroskopie MeSH
• tylakoidy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fotosystém II (proteinový komplex) * MeSH
• lipidy MeSH

• Publikační typ
• úvodníky MeSH

The investigation of spatial heterogeneity within the thylakoid membrane (TM) proteins has gained increasing attention in photosynthetic research. The recent advances in live-cell imaging have allowed the identification of heterogeneous organisation of photosystems in small cyanobacterial cells. These sub-micrometre TM regions, termed microdomains in cyanobacteria, exhibit functional similarities with granal (Photosystem II dominant) and stromal (Photosystem I dominant) regions observed in TM of higher plants. This study delves into microdomain heterogeneity using super-resolution Airyscan-based microscopy enhancing resolution to approximately ~125 nm in x-y dimension. The new data reveal membrane areas rich in Photosystem I within the inner TM rings. Moreover, we identified analogous dynamics in the mobility of Photosystem II and phycobilisomes; countering earlier models that postulated differing mobility of these complexes. These novel findings thus hold significance for our understanding of photosynthesis regulation, particularly during state transitions.

• Klíčová slova
• Airyscan, FRAP, cyanobacteria, microdomain, photosystem, protein mobility, super-resolution microscopy, thylakoid membrane heterogeneity,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• 31P-NMR spectroscopy, lipid polymorphism, lipocalins, membrane fusion, membrane models, non-bilayer lipids, plastoglobuli, structural and functional plasticity, thylakoid membrane,
• MeSH
• lipasa metabolismus   MeSH
• lipidové dvojvrstvy * metabolismus   MeSH
• magnetická rezonanční spektroskopie MeSH
• proteasy metabolismus   MeSH
• tylakoidy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipasa MeSH
• lipidové dvojvrstvy * MeSH
• proteasy MeSH

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.

• Klíčová slova
• SAXS, bilayer, chlorophyll fluorescence, cryo-electron-tomography, electron microscopy, membrane energization, membrane networks, non-bilayer lipid phases, violaxanthin de-epoxidase,
• MeSH
• cirkulární dichroismus metody   MeSH
• elektronová mikroskopie metody   MeSH
• fotosyntéza genetika   MeSH
• lipidy genetika   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• tylakoidy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidy MeSH

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.

• Klíčová slova
• 31P-NMR, DEM—dynamic exchange model, HII phase, bilayer membrane, grana, isotropic phase, non-bilayer lipids, non-lamellar lipid phases, structural flexibility, thylakoid membranes,
• MeSH
• chloroplasty chemie   MeSH
• galaktolipidy chemie   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• membránové lipidy chemie   MeSH
• teplota MeSH
• tylakoidy chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• galaktolipidy MeSH
• membránové lipidy MeSH
• monogalactosyldiacylglycerol MeSH Prohlížeč

The present review is an attempt to conceptualize a contemporary understanding about the roles that cardiolipin, a mitochondrial specific conical phospholipid, and non-bilayer structures, predominantly found in the inner mitochondrial membrane (IMM), play in mitochondrial bioenergetics. This review outlines the link between changes in mitochondrial cardiolipin concentration and changes in mitochondrial bioenergetics, including changes in the IMM curvature and surface area, cristae density and architecture, efficiency of electron transport chain (ETC), interaction of ETC proteins, oligomerization of respiratory complexes, and mitochondrial ATP production. A relationship between cardiolipin decline in IMM and mitochondrial dysfunction leading to various diseases, including cardiovascular diseases, is thoroughly presented. Particular attention is paid to the targeting of cardiolipin by Szeto-Schiller tetrapeptides, which leads to rejuvenation of important mitochondrial activities in dysfunctional and aging mitochondria. The role of cardiolipin in triggering non-bilayer structures and the functional roles of non-bilayer structures in energy-converting membranes are reviewed. The latest studies on non-bilayer structures induced by cobra venom peptides are examined in model and mitochondrial membranes, including studies on how non-bilayer structures modulate mitochondrial activities. A mechanism by which non-bilayer compartments are formed in the apex of cristae and by which non-bilayer compartments facilitate ATP synthase dimerization and ATP production is also presented.

• Klíčová slova
• ATP synthase, cardiolipin, cardiovascular disease, electron-transport chain, inner mitochondrial membrane, non-bilayer structures,
• MeSH
• energetický metabolismus * MeSH
• kardiolipiny chemie   metabolismus   MeSH
• kardiovaskulární nemoci metabolismus   MeSH
• lidé MeSH
• lipidové dvojvrstvy metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   patologie   ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• kardiolipiny MeSH
• lipidové dvojvrstvy MeSH

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment-protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound 'free' proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 - 2.95 µm2s-1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50-500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII-light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein-protein interactions in the mobility restrictions for large thylakoid protein complexes.

• Klíčová slova
• FCS, cyanobacteria, photosynthesis, proteins mobility, thylakoids,
• Publikační typ
• časopisecké články MeSH