Lipid assemblies mediating the fusion of photosystem-II enriched membranes characterized by DPH (1,6-diphenyl-1,3,5-hexatriene) fluorescence and fluorescence anisotropy lifetimes

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.