Cold Acclimation of Hedera helix

The light-enhanced production and accumulation of sugars is only one step in the process of cold acclimation in Hedera helix L. var. Thorndale (English ivy). Applications of 2,4-dinitrophenol to plants with different portions exposed to light and dark indicated that the mere presence or accunmulation of the light-generated promoters did not invoke an increase in hardiness. Kinetics of cold acclimation during alternating periods of light and dark also indicate that the light stimulation of cold acclimation is only a partial component of the total process. Incubation on 50 mM solutions of sucrose can replace the light requirement. A second phase which can proceed in the dark is thought to result in the production of proteins which, due to an altered composition or configuration, have a greater capacity to bind sugars. This is evidenced by the fact that protein from cold acclimated tissue exhibited a higher sugar-binding capacity than protein from nonacclimated tissue. Furthermore, the two phases can proceed independently of each other, but only upon complementation of the products of the two phases is an increase in cold hardiness manifested. Many environmental factors influence the cold acclimation process, and of these, light appears to be as fundamental as low temperature in inducing maximal hardiness of many species (2, 7). Previously, light has been shown to greatly enhance the rate and degree of cold acclimation in Hedera helix, but it is not essential for the cold acclimation process (14). The light stimulation results in the production of translocatable promoters of hardiness and is not involved in the promotion of some predisposing condition (13). Attempts to characterize the light-generated promoters through fixation of '4C02 in an illuminated donor portion and subsequent translocation of the '4C-labeled products to a darkened receptor portion indicated that the translocatable promoter was sucrose (13). Furthermore, sucrose is the most abundant carbohydrate accumulating during cold acclimation, and hardiness of leaves of H. helix can only be significantly increased by sucrose solutions and not by equimolar solutions of glucose, galactose, or mannitol (13, 15). Although the light stimulus results in an accumulation of photosynthates in Hedera and in other species (9, 16, 17), it is doubtful that cold acclimation is an accumulation of photosynthates per se. Increases in sugar content during cold acclimation are well documented (7). And, although Heber (4) concludes that the protective influence of sugars is due to their capacity to retain or substitute water via hydrogen bonding in structures sensitive to dehydration, demonstration of a causal relationship is lacking. MATERIALS AND METHODS Method of Cold Acclimation. Unless otherwise stated, the standard procedure for artificial cold acclimation of Hedera helix L. var. Thorndale (English ivy) was as previously described (11). Plants, grown from cuttings in a greenhouse for 3 to 6 months, were placed at 5 C for a period of 6 weeks. During this time, light was provided by a combination of cool-white fluorescent lamps supplemented with incandescent lamps. An intensity of 600 ft-c at plant level was maintained during an 8-hr photoperiod. In some instances, sucrose was substituted for the light requirement. This was accomplished by severing the vines from the root system and trimming them to eight leaves per vine. The vines were then placed in a growth chamber at 26 C in water in the dark for 3 days to reduce endogenous levels of sugars and starch reserves. The stems were then placed in the appropriate solutions and maintained in a dark growth chamber at 3.5 C. Where noted, this procedure was slightly modified by using cuttings consisting of one leaf and one internode. Method of Freezing and Thawing. Samples to be frozen were first wrapped in aluminum foil and then placed in insulated boxes. The samples were then placed in a freezer at -6 C. After the temperature inside the insulated boxes reached -6 C, one box remained at this temperature for 2 hr and the remaining boxes were transferred to a freezer maintained at -12 C. The process continued with freezers maintained at 6 C increments. The temperature fall resulting from this procedure was approximately 3.5 C per hour. Air temperatures inside the insulated boxes were recorded at 2.5-min intervals. Following the 2-hr period at the desired temperature, the insulated boxes containing the frozen samples were removed to a 5 C cooler and allowed to thaw. Tissue survival was determined the following day. Method of Testing Survival. The killing temperatures of tissue samples following freezing were determined by the refined triphenyl tetrazolium chloride test (12). All killing points are the means of three replications. Kinetics of Cold Acclimation. Studies of the kinetics of cold acclimation following alternating periods of dark will indicate whether cold acclimation is solely a one step accumulation phenomenon. Proportional increases in the amount of light should invoke proportional increases in hardiness if cold acclimation is only the accumulation of cryo-protective substances produced in the light. However, this has not always been effectively proven (9). In attempting to show such a response, the usual procedure has been to vary the light intensity or duration of light, but with the latter approach difficulties arise due to photoperiodic effects. These difficulties can be overcome by