31P-NMR observation of the temperature and glycerol induced non-lamellar phase formation in wheat thylakoid membranes

³¹P-NMR spectra at 162 MHz were used to monitor phase changes of wheat thylakoid membranes as a function of temperature. At room temperature the³¹P-NMR line was a superposition of anisotropic component characteristic of phospholipid lamellar phase and isotropic line due to inorganic phosphorus or small membrane vesicles arising as an effect of preparation. For temperatures higher than +35 °C an increase of the isotropic component occurs, which is irreversible as the sample is cooled. For the temperatures between +55 °C and +60 °C the presence of the hexagonal phase cylinders is suggested, as monitored by phosphorus lineshape. However, the addition of glycerol stimulates a formation of the isotropic phase. The effect of reconstitution of freeze-dried thylakoid membranes by addition of water or water-glycerol medium to the sample was examined. As lyophilizate was gradually diluted, the increase of isotropic line component was observed. For thylakoid membranes suspended in D₂O at the highest dilution examined, the line contribution due to small membrane fragments is not greater than 50%, but in presence of glycerol, this contribution could reach 70%. This suggests that the presence of glycerol increases the formation of the small membrane particles as the thylakoid membrane is reconstituted from lyophilizate. The wheat thylakoid membranes reconstituted from lyophilizate show, in comparison to native membranes, the increased contribution of small membrane vesicles. Moreover, the³¹P -NMR spectra suggest the appearance of the hexagonal phase cylinders even at +50 °C.Abbreviations DGDG digalactosyldiacylglycerol - DLPC dilinoleoyl phosphatidylcholine - DLPE dilinoleoyl phosphatidylethanolamine - EDTA ethylenediamine-tetraacetic acid - MGDG monogalactosyldiacylglycerol - NMR nuclear magnetic resonance - PC phosphatidylcholine - PG phosphatidylglycerol - PSII photosystem II - TGDG trigalactosyldiacylglycerol - Tris Tris-(hydroxymethyl)-aminomethan - S/N signal to noise ratio