The State of Water and Cell Morphology In Deep Frozen Populus

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Hirsh, Allen Gene
Solomos, Theophanes
By using differential scanning calorimetry, electron microscopy, light microscopy, and freezing survival experiments, it is shown that superhardy Populus balsamifera v. Virginiana (Sarg.) is capable of withstanding liquid nitrogen (LN 2) temperatures because of the formation during cooling, at a temperature of about -30°C and cooling rates less than 30°C/hr, of aqueous glasses in the intracellular solutions. In more detail, the major findings concerning the state of intracellular water are: (1) the bulk of the intracellular contents go through an equilibrium glass transition at about -28°C during slow (<5°C/hr) cooling; (2) smaller additional amounts of intracellular material go through equilibrium glass transitions at about -47°C and -70°C; (3) as a result of the resistance to homogeneous nucleation of these glass forming intracellular solutions when they are in equilibrium with extracellular ice at<.-20°c, cooling/warming at any combination of rates from 3°C/hr to 1200°C/min between -20°c and -196°C is non-injurious to fully hardened wood; (4) death associated with quench cooling in LN2 from -15°C is correlated with the devitrification, (cold crystallization) near -90°C upon warming of the very low temperature glass forming component, followed by further devitrification of the higher temperature components, especially between -30°c and -20°c; and (5) the vacuolar compartment appears least resistant to devitrification and capable of thereby causing death even when the cytoplasm resists devitrification. In addition, it was found that when fully superhardy wood is cooled slowly (3°C/hr) after being imbibed with water (doubling total water content) massive intracellular freezing occurs. Despite the fact that total tissue water of tender Populus (summer wood) is 2x that of the artifically water loaded hardy wood on a gram H20/gram dry weight basis, tender wood cooled at 3°C/hr to -50°c does not display intracellular freezing. It is killed by -2°C. It is shown that in both tender and hardy wood <10% of water is extracellular. Thus a significant excess of extracellular water appears to cause intracellular freezing and this may be a major reason for the large water loss seen in the fall 'hardening off' of most temperature zone woody plants. It is also shown that during slow cooling, the plasma membranes of both hardy and tender Populus cells stick to and collapse the cell wall, but that these membranes stay smooth in the case of superhardy cells and wrinkle markedly in the case of tender cells. Membrane-associated particles appeared to clump in the membranes of slowly cooled tender cells but not in the slowly cooled hardy cells.