Factors contributing to ice nucleation and sequential freezing of leaves in wheat.

MAIN CONCLUSION: Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions. Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.

3D volumes constructed from pixel-based images by digitally clearing plant and animal tissue.

Construction of three-dimensional volumes from a series of two-dimensional images has been restricted by the limited capacity to decrease the opacity of tissue. The use of commercial software that allows colour-keying and manipulation of two-dimensional images in true three-dimensional space allowed us to construct three-dimensional volumes from pixel-based images of stained plant and animal tissue without generating vector information. We present three-dimensional volumes of (1) the crown of an oat plant showing internal responses to a freezing treatment, (2) a sample of a hepatocellular carcinoma from a woodchuck liver that had been heat-treated with computer-guided radiofrequency ablation to induce necrosis in the central portion of the tumour, and (3) several features of a sample of mouse lung. The technique is well suited to images from large sections (greater than 1 mm) generated from paraffin-embedded tissues. It is widely applicable, having potential to recover three-dimensional information at virtually any resolution inherent in images generated by light microscopy, computer tomography, magnetic resonance imaging or electron microscopy.

Carbohydrate partitioning between upper and lower regions of the crown in oat and rye during cold acclimation and freezing.

Carbohydrates have long been recognized as an important aspect of freezing tolerance in plants but the association between these two factors is often ambiguous. To help clarify the relationship, the allocation of carbohydrates between specific tissues within the over wintering organ (crown) of winter cereals was measured. A winter-hardy and non-winter-hardy oat (Avena sativa L.), and a rye (Secale cereale L.) cultivar were grown and frozen under controlled conditions. Crown tissue was fractionated into an upper portion, called the apical region, and a lower portion, called the lower crown. These tissues were ground in liquid N and extracted with water. Extracts were analyzed by HPLC for the simple sugars, sucrose, glucose, fructose, and for fructan of various size classes. After 3 weeks of cold acclimation at 3 degrees C, carbohydrates accounted for approximately 40% of the dry weight of oats and 60% of the dry weight of rye. The apical region, which is the tissue within the crown that acclimates to the greatest extent, was generally 10% higher in total carbohydrates than the lower crown. During a mild freeze, various carbohydrates were allocated differently between specific tissues in the three genotypes. When frozen, fructan generally decreased to a greater extent in the lower crown than in the apical region but sugars increased more in the apical region than in the lower crown. Results suggest that to understand how carbohydrates relate to freezing tolerance, regions of the crown that endure freezing stress differently should be compared.

Thermal effect of CO(2) on apoplastic ice in rye and oat during freezing.

Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at -3 degrees C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to -194 microW in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from -194 to 229 microW within 1 h, suggesting that water had frozen in the plants. We propose that CO(2) from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO(2) came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, non-biological system using a glycol/water mixture that was partially frozen at -3 degrees C.

Barley yellow dwarf virus: effects on carbohydrate metabolism in oat (Avena sativa) during cold hardening.

Barley yellow dwarf virus (BYDV) causes significant losses in yield and in overwintering ability of winter cereals. Mechanisms by which the physiology of plants is affected by the virus are not clear. To see how carbohydrates in the crown of winter cereals were affected by BYDV, fructan isomers of degree of polymerization (DP) 3-5, fructan DP>6 and the simple sugars, glucose, fructose and sucrose, were measured before and during cold hardening in three oat (Avena sativa L.) cultivars, 'Wintok', 'Coast Black' and 'Fulghum'. On a fresh weight basis fructan DP>6 decreased by 50% in infected 'Wintok' and 'Coast Black' and by 25% in 'Fulghum'. Two DP3, one DP4 and one DP5 isomer were significantly higher than non-infected controls. The percentages of simple sugars in infected crowns were significantly higher than controls in all three cultivars in every week except the first week of hardening. Crude enzyme extracts from BYDV infected plants incubated with sucrose suggested higher invertase and lower sucrose-sucrosyl transferase activity. When incubated with 1-kestose and neokestin, no significant difference was found in fructose fructosyl transferase or in hydrolase activity. The activity of unidentified enzymes catalysing the synthesis of larger (DP>5) fructan was altered by BYDV. The decrease of carbohydrates in the crown induced indirectly by BYDV may alter the plant's capacity to regenerate tillers in the spring. The ability of plants to prevent or tolerate carbohydrate fluctuations induced by BYDV infection may be an important genetically regulated characteristic for developing virus-resistant cultivars.

Purification and characterization of an oat fructan exohydrolase that preferentially hydrolyzes beta-2,6-fructans.

Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of beta-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal beta-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal beta-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates. The Km for neokestin (beta-2,6-linked fructans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v), and the Vmax was 0.041 mumol min-1 mL-1. The Km for hydrolysis of 6G,6-kestotetraose was 5.6% (w/v), and the Vmax was 0.138 mumol min-1 mL-1. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.

Fructan precipitation from a water/ethanol extract of oats and barley.

Fructan was precipitated from a water and ethanol extract of oat (Avena sativa L.) and barley (Hordeum vulgare L.). The degree of polymerization and response on a differential refractometer, based on peak area and height, was compared to fructan collected from a lead-based HPLC column and to commercially available inulin. Statistically significant differences are discussed.