Numerous studies have been published that attempted to correlate fructan concentrations with freezing and drought tolerance. Studies investigating the effect of fructan on liposomes indicated that a direct interaction between membranes and fructan was possible. This new area of research began to move fructan and its association with stress beyond mere correlation by confirming that fructan has the capacity to stabilize membranes during drying by inserting at least part of the polysaccharide into the lipid headgroup region of the membrane. This helps prevent leakage when water is removed from the system either during freezing or drought. When plants were transformed with the ability to synthesize fructan, a concomitant increase in drought and/or freezing tolerance was confirmed. These experiments indicate that besides an indirect effect of supplying tissues with hexose sugars, fructan has a direct protective effect that can be demonstrated by both model systems and genetic transformation.

Changes in apoplastic carbohydrate concentrations and activities of carbohydrate-degrading enzymes were determined in crown tissues of oat (Avena sativa L., cv Wintok) during cold hardening. During second-phase hardening (؊3°C for 3 d) levels of fructan, sucrose, glucose, and fructose in the apoplast increased significantly above that in nonhardened and first-phase-hardened plants. The extent of the increase in apoplastic fructan during second-phase hardening varied with the degree of fructan polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP > 7). Activities of invertase and fructan exohydrolase in the crown apoplast increased approximately 4-fold over nonhardened and first-phase-hardened plants. Apoplastic fluid extracted from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by malate dehydrogenase activity. The significance of these results in relation to increases in freezing tolerance from second-phase hardening is discussed.Cold-hardening winter cereals such as rye (Secale cereale), wheat (Triticum aestivum), barley (Hordeum vulgare), and oat (Avena sativa) is generally accomplished by exposure to temperatures just above freezing. As early as 1935, Tumenov (cited by Trunova, 1965) reported that an additional phase of hardening by exposure of cold-hardened plants to nonlethal, below-freezing temperatures resulted in significant increases in cold hardiness. Tumenov called this 2PH. With a 2PH treatment, Trunova (1965) and Siminovich (cited by Steponkus, 1978) induced an increase in freezing tolerance of wheat significantly beyond that achieved from cold hardening above freezing (1PH). Olien (1984) reported similar results in rye and barley, and reported a decrease in the LT 50 of oats from Ϫ13°C with 1PH only to Ϫ18°C after a 3-d 2PH period at Ϫ3°C.After 2PH Trunova (1965) found decreased levels of fructan and increased levels of Glc, Fru, and Suc in whole crown tissue of wheat. He suggested that the sugar increase during 2PH was providing osmotic protection to cells in the over-wintering organ (crown) of the plant. Olien (1984), using a perfusion technique, reported an increase in apoplastic sugars during 2PH and suggested that these sugars helped prevent adhesion of ice to critical cellular tissue during freezing.In the present study we wanted to determine the percent distribution of fructan exohydrolase and invertase in the apoplast and symplast of oat crowns, and what the effect of 1PH and 2PH would be on their activities in the respective locations. Additionally, we wanted to determine if fructan was present in the apoplast and the effect of cold hardening on its presence. Finally, we wanted to re-examine the effect of cold hardening on simple sugar levels in the apoplast using a different technique than that used by Olien (1984). MATERIALS AND METHODS Plant CultureSeeds of the oat (Avena sativa L.) cv Wintok were planted in Scotts Metromix 220 1 (Scotts-Sierra Horticultural Products Co...

Cold-acclimated plants acquire an additional 3-5 degrees C increase in freezing tolerance when exposed to -3 degrees C for 12-18 h before a freezing test (LT50) is applied. The -3 degrees C treatment replicates soil freezing that can occur in the days or weeks leading to overwintering by freezing-tolerant plants. This additional freezing tolerance is called subzero acclimation (SZA) to differentiate it from cold acclimation (CA) that is acquired at above-freezing temperatures. Using wheat as a model, results have been obtained indicating that SZA is accompanied by changes in physiology, cellular structure, the transcriptome, and the proteome. Using a variety of assays, including DNA arrays, reverse transcription-polymerase chain reaction (RT-PCR), 2D gels with mass spectroscopic identification of proteins, and electron microscopy, changes were observed to occur as a consequence of SZA and the acquisition of added freezing tolerance. In contrast to CA, SZA induced the movement of intracellular water to the extracellular space. Many unknown and stress-related genes were upregulated by SZA including some with obvious roles in SZA. Many genes related to photosynthesis and plastids were downregulated. Changes resulting from SZA often appeared to be a loss of rather than an appearance of new proteins. From a cytological perspective, SZA resulted in alterations of organelle structure including the Golgi. The results indicate that the enhanced freezing tolerance of SZA is correlated with a wide diversity of changes, indicating that the additional freezing tolerance is the result of complex biological processes.