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

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.

• Keywords
• Dehydration, Inverted hexagonal phase, MGDG, Non-bilayer lipid, Thylakoid membranes,
• MeSH
• Photosynthesis MeSH
• Galactolipids metabolism   chemistry   MeSH
• Lipid Bilayers metabolism   chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Temperature MeSH
• Thylakoids * metabolism   chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Galactolipids MeSH
• Lipid Bilayers MeSH
• monogalactosyldiacylglycerol MeSH Browser