Fructans from oat and rye: composition and effects on membrane stability during drying.

Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP>7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP>7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP>7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.

Using Arabidopsis thaliana as a model to study subzero acclimation in small grains.

The suitability of using Arabidopsis as a model plant to investigate freezing tolerance was evaluated by observing similarities to winter cereals in tissue damage following controlled freezing and determining the extent to which Arabidopsis undergoes subzero-acclimation. Plants were grown and frozen under controlled conditions and percent survival was evaluated by observing re-growth after freezing. Paraffin embedded sections of plants were triple stained and observed under light microscopy. Histological observations of plants taken 1 week after freezing showed damage analogous to winter cereals in the vascular tissue of roots and leaf axels but no damage to meristematic regions. The LT(50) of non-acclimated Arabidopsis decreased from about -6 degrees C to a minimum of about -13 degrees C after 7 days of cold-acclimation at 3 degrees C. After exposing cold-acclimated plants to -3 degrees C for 3 days (subzero-acclimation) the LT(50) was lowered an additional 3 degrees C. Defining the underlying mechanisms of subzero-acclimation in Arabidopsis may provide an experimental platform to help understand winter hardiness in economically important crop species. However, distinctive histological differences in crown anatomy between Arabidopsis and winter cereals must be taken into account to avoid misleading conclusions on the nature of winter hardiness in winter cereals.

Additional freeze hardiness in wheat acquired by exposure to -3 degreesC is associated with extensive physiological, morphological, and molecular changes.

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.