Build-up of the energized state of thylakoid membranes and the synthesis of ATP are warranted by organizing their bulk lipids into a bilayer. However, the major lipid species of these membranes, monogalactosyldiacylglycerol, is a non-bilayer lipid. It has also been documented that fully functional thylakoid membranes, in addition to the bilayer, contain an inverted hexagonal (HII) phase and two isotropic phases. To shed light on the origin of these non-lamellar phases, we performed 31P-NMR spectroscopy experiments on sub-chloroplast particles of spinach: stacked, granum and unstacked, stroma thylakoid membranes. These membranes exhibited similar lipid polymorphism as the whole thylakoids. Saturation transfer experiments, applying saturating pulses at characteristic frequencies at 5 °C, provided evidence for distinct lipid phases-with component spectra very similar to those derived from mathematical deconvolution of the 31P-NMR spectra. Wheat-germ lipase treatment of samples selectively eliminated the phases exhibiting sharp isotropic peaks, suggesting easier accessibility of these lipids compared to the bilayer and the HII phases. Gradually increasing lipid exchanges were observed between the bilayer and the two isotropic phases upon gradually elevating the temperature from 5 to 35 °C, suggesting close connections between these lipid phases. Data concerning the identity and structural and functional roles of different lipid phases will be presented in the accompanying paper.

EN FIST Center of Excellence SI 1000 Ljubljana Slovenia

Faculty of Chemistry and Chemical Technology University of Ljubljana SI 1000 Ljubljana Slovenia

Group of Biophysics Department of Physics Faculty of Science University of Ostrava 710 00 Ostrava Czech Republic

Laboratory of Ecological Plant Physiology Domain of Environmental Effects on Terrestrial Ecosystems Global Change Research Institute of the Czech Academy of Sciences 603 00 Brno Czech Republic

Photosynthetic Membranes Group Plant Light Perception and Utilization Research Unit Institute of Plant Biology Biological Research Centre Eötvös Loránd Research Network 6726 Szeged Hungary

Slovenian NMR Center National Institute of Chemistry SI 1000 Ljubljana Slovenia

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