Combined electron-spin-resonance, X-ray-diffraction studies on phospholipid vesicles obtained from cold-hardened wheats

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Abstract
The contents of free sterols and phospholipids in leaves of wheat, Triticum aestivum L., cultivars of different frost resistances, as well as the physical state of isolated phospholipids in the presence and absence of sterols, were compared before and after hardening. There was an inverse relationship between the sterol/phospholipid ratio and frost tolerance as a consequence of both a decrease in the free sterol, and an increase in the total phospholipid content. Sterol-sterol interactions were investigated using wide angle X-ray diffraction, while the phase behaviour of phospholipid vesicles was studied using the electron-spin-resonance (ESR) technique. No sterol-sterol interactions at-10° C were detected in vesicles obtained from the hardened most cold-tolerant cultivar (Miranovskaja 808), containing sterols in a ratio (0.08) found in the original lipid extracts. In contrast, when the sterol-phospholipid ratio in the vesicles was set to the level (0.39) found in the extracts of the most sensitive cultivar, Penjamo 62, the appearance of sharp reflexion rings at 4.5·10-1, 4.8·10-1 and 5.0·10-1 nm indicated strong sterol-sterol interactions. The temperatures for the onset of phase separation for vesicles of identical sterol/phospholipid ratios found in lipid extracts of hardened Miranovskaja 808 were almost the same as those measured in purified phospholipids (-15 vs.-16° C). In contrast, the temperature for the onset of phase separation of vesicles with a sterol/phospholipid ratio characteristic of hardened Penjamo 62 was shifted upwards (from-6 to-2° C). Phase separation was not completed in the vesicles of Miranovskaja 808 in the temperature range scanned (-30° C) but was shifted from-22 to-18° C in the presence of sterols in the case of Penjamo 62. The results are discussed in terms of the composition and physical state of membranes in relation to survival at freezing temperatures.