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.

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 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.

The lipid composition of thylakoid membranes (TMs), the site of light reactions in oxygenic photosynthetic organisms, is dominated by the non-bilayer lipid species monogalactosyldiacylglycerol. It has been documented that plant TMs display strong lipid polymorphisms (Garab et al. 2022 Prog Lipid Res 86:101163). Recently, we have also shown that BBY membrane sheets, large, laterally fused photosystem-II (PSII) enriched membrane pairs, beside the lamellar phase, contain an intense isotropic phase, the lipid molecules of which mediate the fusion of membranes (Böde et al. 2024 Photosynth Res 161:127-140). To demonstrate the composite nature of BBY, we employed confocal laser scanning microscopy, using the lipid-label BODIPY-phosphatydilcholine and chlorophyll-a fluorescence emissions. To characterize the physico-chemical microenvironments of lipid molecules, we stained BBY membranes with the hydrophobic fluorescent dye DPH (1,6-diphenyl-1,3,5-hexatriene). DPH emission spectra from face- and edge-aligned BBY membranes indicated the existence of at least two distinct microenvironments. Fluorescence lifetime analyses revealed three components; the fastest one was sensitive to the enzymatic treatment with wheat germ lipase (WGL), which had earlier been shown to selectively eliminate the isotropic lipid phase of BBY and to disassemble the large sheets into its constituent membrane pairs of grana. Although the anisotropic fluorescence decay kinetics discerned no lifetime heterogeneity of the untreated DPH-stained BBY, WGL treatment led to the appearance of a second lifetime component. These data provide independent experimental evidence on the lipid polymorphism of BBY membranes and reveal that the bilayer lipids and the non-bilayer lipid arrays mediating the fusion of TMs possess distinct physico-chemical environments.

Thylakoid membranes (TMs) of vascular plants exhibit a highly organized architecture comprising two main distinct functional domains which accommodate virtually all components of the photosynthetic machinery responsible for the light reactions: the stacked, appressed granum membranes and the unstacked stroma lamellae. A domain with distinct structural, compositional, and functional characteristics, the marginal region of grana (grana margin, GM) has also been identified, which includes the highly curved region at the periphery of the granum vesicles that is enriched in CURT1 proteins. It is also well established that plant TMs, in addition to the bilayer (or lamellar, L) phase, contain at least two isotropic (I) lipid phases and an inverted hexagonal (H_II) phase. The I phases were shown to participate in membrane fusion and to be associated with the activity of the photoprotective water-soluble enzyme, the violaxanthin de-epoxidase (VDE); the H_II phase, for its trypsin sensitivity, was proposed to emerge from stroma-exposed lipid-protein assemblies. Considering the location of CURT1 in GM and that it possesses trypsin-sensitivity, it was hypothesized to participate in the formation of the H_II phase. The aim of the present study was to test this hypothesis – via fingerprinting the lipid phase behavior of GM particles isolated from spinach leaves. Our ³¹P-NMR spectroscopy reveals that the GM fraction of TMs does not contain H_II phase signal; it exhibits an isotropic signature. This non-bilayer, isotropic lipid structure, together with CURT1 proteins, thus appear to promote the formation of the highly curved region of TMs.