Thylakoid membranes (TMs) of oxygenic photosynthetic organisms are flat membrane vesicles, which form highly organised, interconnected membrane networks. In vascular plants, they are differentiated into stacked and unstacked regions, the grana and stroma lamellae, respectively; they are densely packed with protein complexes performing the light reactions of photosynthesis and generating a proton motive force (pmf). The maintenance of pmf and its utilisation for ATP synthesis requires sealing the TMs at their highly curved regions (CRs). These regions are devoid of chlorophyll-containing proteins but contain the curvature-inducing CURVATURE THYLAKOID1 (CURT1) proteins and are enriched in lipids. Because of the highly curved nature of this region, at the margins of grana and stroma TMs, the molecular organisation of lipid molecules is likely to possess distinct features compared to those in the major TM domains. To clarify this question, we isolated CR fractions from Spinacia oleracea and, using BN-PAGE and western blot analysis, verified that they are enriched in CURT1 proteins and in lipids. The lipid phase behaviour of these fractions was fingerprinted with 31P-NMR spectroscopy, which revealed that the bulk lipid molecules assume a non-bilayer, isotropic lipid phase. This finding underpins the importance of the main, non-bilayer lipid species, monogalactosyldiacylglycerol, of TMs in their self-assembly and functional activity.

• Keywords
• 31P‐NMR, CURT1 protein, granum margin, non‐bilayer lipid phase, thylakoid membrane,
• MeSH
• Lipids * chemistry   MeSH
• Plant Proteins metabolism   MeSH
• Spinacia oleracea * metabolism   MeSH
• Thylakoids * metabolism   chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Lipids * MeSH
• Plant Proteins MeSH

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
• Models, Biological MeSH
• Photosynthesis MeSH
• Galactolipids metabolism   MeSH
• Lipid Bilayers metabolism   MeSH
• Plants * metabolism   MeSH
• Thylakoids * metabolism   chemistry   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Galactolipids MeSH
• Lipid Bilayers 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.

• Keywords
• 31P-NMR spectroscopy; BBY membrane, Linear dichroism spectroscopy, Membrane fusion; non-bilayer lipids, Wheat germ lipase,
• MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Membrane Fusion physiology   MeSH
• Lipids chemistry   MeSH
• Magnetic Resonance Spectroscopy MeSH
• Thylakoids * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Photosystem II Protein Complex * MeSH
• Lipids MeSH

• Publication type
• Editorial MeSH