Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier ³¹P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our ³¹P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by ³¹P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.

The largest stable photosystem II (PSII) supercomplex in land plants (C₂S₂M₂) consists of a core complex dimer (C₂), two strongly (S₂) and two moderately (M₂) bound light-harvesting protein (LHCB) trimers attached to C₂ via monomeric antenna proteins LHCB4–6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C₂S₂M₂ supercomplex. Here, we performed structure–function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the “spruce-type” PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The “spruce-type” PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C₂S₂ semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C₂S₂ arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.