Identification of ultrastructural and biochemical cuticular markers influencing temperature of ice nucleation in selected genotypes of corn.

Corn is an economically important yet frost-sensitive crop, injured at the moment of ice nucleation. However, the influence of autumn temperatures on subsequent ice nucleation temperature is unknown. A 10-day chilling treatment under phytotron conditions ("mild", 18/6°C) or ("extreme", 10/5°C) generated no-visible damage but induced changes in the cuticle of the four genotypes in this study. The putatively more cold hardy Genotypes 884 and 959 leaves nucleated at colder temperatures compared to the more sensitive Genotypes 675 and 275. After chilling treatment, all four genotypes displayed warmer ice nucleation temperatures, with Genotype 884 expressing the largest shift to warmer nucleation temperatures. Cuticular hydrophobicity reduced while cuticular thickness remained unchanged under the chilling treatment. By contrast, under five-week field conditions, cuticle thickness increased in all genotypes, with Genotype 256 expressing a significantly thinner cuticle. FTIR spectroscopy revealed increases in the spectral regions of cuticular lipids in all genotypes after phytotron chilling treatment, while those spectral regions decreased under field conditions. A total of 142 molecular compounds were detected, with 28 compounds significantly induced under either phytotron or field conditions. Of these, seven compounds were induced under both conditions (Alkanes C31-C33, Ester C44, C46, ß-amyrin, and triterpene). While clear differential responses were observed, chilling conditions preceding a frost modified physical and biochemical properties of the leaf cuticle under both phytotron and field conditions indicating this response is dynamic and could be a factor in selecting corn genotypes better adapted to avoiding frost with lower ice nucleation temperature.

Allium fistulosum as a novel system to investigate mechanisms of freezing resistance.

Allium fistulosum was investigated as a novel model system to examine the mechanism of freezing resistance in cold hardy plants. The 250 × 50 × 90 µm average cell size and single epidermal cell layer system allowed direct observation of endoplasmic reticulum (ER), functional group localization during acclimation, freezing and thawing on an individual cell basis in live intact tissues. Cells increased freezing resistance from an LT50 of -11°C (non-acclimated) to -25°C under 2 weeks of cold acclimation. Samples were processed using Fourier transform infrared technology (FTIR) on a synchrotron light source and a focal plane array detector. In addition, confocal fluorescent microscopy combined with a cryostage using ER selective dye of ER-Tracker allowed more detailed examination of membrane responses during freezing. Cold acclimation increased the ER volume per cell, and the freeze-induced cell deformation stopped ER streaming and ER vesiculation subsequently occurred through the breakdown in the ER network. Freeze-induced ER vesicles in cold-acclimated cells were larger and more abundant than those in non-acclimated cells. According to FTIR, the carbohydrate/ester fraction and α-helical/ß-sheet secondary structure localized in the apoplast/plasma membrane region were most visibly increased during cold acclimation. Results suggest the mechanism of cold acclimation and freezing resistance in very hardy cells may be associated with both alterations in the apoplast/plasma membrane region and the ER cryodynamics. Allium fistulosum appears to be a useful system to obtain direct evidence at both intra and extracellular levels during cold acclimation and the freezing process.