The Relationship of Carbohydrates to Cold Acclimation of Hedera helix L. cv. Thorndale

While both the total sugar content and cold hardiness greatly increased during artificial cold acclimation, no direct parallelism was demonstrated. In fact, plants hardened in the dark exhibited an increase in hardiness during the period when the total sugar content declined, furthermore, while there was evidence for an accumulation of sugars (especially sucrose) incorporation of ¹⁴C indicated that all of the isolated fractions were in a dynamic state. Dehardening, paralleled by a large increase in the starch content, was more rapid in the light than in the dark. Furthermore, in stems the sugar content increased after 3 days of dehardening but declined after 7 days. This increase may represent the release of sugars from a previously unextractable form such as a glycoprotein complex. Starvation experiments indicate that photosynthates produced during the cold acclimation period are preferentially used during cold acclimation rather than reserve carbohydrates. This was also indicated by the smaller amount of starch hydrolysis in plants hardened in the light. Thus, while there appears to be a role for carbohydrates in the cold acclimation process, the lack of parallelism between sugar content and hardiness may be interpreted as indicating 1) cold acclimation is not merely an accumulation of sugars or an osmotic effect per se, and 2) under normal conditions, the level of carbohydrates is not limiting the rate or degree of cold acclimation.     

The Role of Light in Cold Acclimation of Hedera helix L. var. Thorndale

The role of light in cold acclimation of Hedera helix L. var. Thorndale appears to differ from that reported for winter annuals. Although light greatly enhances the degree of hardiness attained, cold acclimation is not obligatorily linked to a light requirement. Photoperiods, varying from 8 to 24 hours, received during the cold acclimation period were equally effective in promoting maximum hardiness. Relatively low light intensities and short photoperiods stimulated maximum hardiness, and proportional increases in hardiness in response to increased photoperiods were demonstrated only in stems of prestarved plants. Exclusion of CO₂ and high concentrations of photosynthetic inhibitors decreased hardiness, but in no instance was hardiness reduced to the level of the dark control. The data are only compatible with a photosynthetic role of light if it is assumed that only a small portion of the total photosynthates are required to elicit maximum hardiness. Alternatively, the light stimulation which was elicited by low light intensities, short photoperiods, in the absence of CO₂, and in the presence of photosynthetic inhibitors, may be a light signal similar to a phytochrome response.     

Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Chill-responsive dehydrins in blueberry: Are they associated with cold hardiness or dormancy transitions?

To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse-grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one-half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy-neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one-dimensional SDS-PAGE. Dehydrin-like proteins were identified by immunoblotting, using anti-dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin-like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.     

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Is tissue culture a viable system with which to examine environmental and hormonal regulation of cold acclimation in woody plants?

In vitro-grown saskatoon berry (Amelanchier alnifolia Nutt.) plantlets were exposed to various hormonal treatments, dormancy-inducing and cold acclimation conditions to determine if this in vitro system would be viable for dormancy/hardiness studies in woody plants. Low temperature induced significant hardiness levels in plantlets to ?27°C after 6 weeks at 4°C but did not approach liquid nitrogen levels of fully hardened, field-grown buds. Control plantlets were consistently killed at ?5°C throughout this period. Significant hardiness was attained under both short and long day/low temperature conditions; however, hardiness was reduced under continuous light or dark treatments. A pre-exposure to the typical short photoperiod regime of woody plants did not significantly increase the rate of acclimation in these plantlets. The presence/absence of phytohormones in the media have a pronounced influence on the ability of plantlets to cold acclimate. Hormone-free media increased hardiness to ?10.5°C after 2 weeks in treatment. Addition of abscisic acid (ABA) increased cold hardiness levels (?12°C) while addition of benzylaminopurine (BAP) to this hormone-free media decreased hardiness to ?5.3°C. A combination of BAP and ABA treatments produced LT₅₀ values intermediate between individual applications of either hormone. Conversely, α-naphthaleneacetic acid (NAA) could not counteract the ABA-induced hardiness. ABA treatments alone were not able to harden plantlets to the extent attained under low temperature acclimation conditions. Further, ABA could not maintain the hardiness levels of cold-acclimating treatments and plantlets de-acclimated to ?9°C in BAP + ABA media. Subculturing in itself significantly elevated cold hardiness in plantlets to ?9°C on BAP + NAA media within 3 days after subculture and thereafter plantlets dehardened to ?5°C. While tissue culture has value in specific cases, caution should be taken when using tissue-cultured plantlets as a system to evaluate environmental regulation of cold acclimation in woody plants, in part, due to the influence of phytohormones in the media.     

Plasma Membrane Alterations in Callus Tissues of Tuber-bearing Solanum Species during Cold Acclimation

Plasma membrane alterations in two tuber-bearing potato species during a 20-day cold acclimation period were investigated. Leaf-callus tissues of the frost-resistant Solanum acaule Hawkes `Oka 3878' and the frost-susceptible, commonly grown Solanum tuberosum `Red Pontiac,' were used. The former is a species that can be hardened after subjecting to the low temperature, and the latter does not harden. Samples for the electron microscopy were prepared from callus cultures after hardening at 2 C in the dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acaule increased in frost hardiness from −6 to − 9 C (killing temperature), whereas frost hardiness of S. tuberosum remained unchanged (killed at −3 C). Actually, after 15 days acclimation, a −9 C frost hardiness level in S. acaule callus cultures had been achieved.     

Physiology of drought tolerance and cold hardiness of the Mediterranean tiger moth Cymbalophora pudica during summer diapause

Prepupae of the arctiid moth Cymbalophora pudica spend spring and summer months in a summer diapause (aestivation), the duration of which is photoperiodically controlled. Cold hardiness, drought tolerance and some physiological and biochemical parameters were measured in aestivating prepupae. Large amounts of metabolic reserves, in the form of lipids and glycogen, accumulated prior to aestivation. Glycogen served as the main metabolic fuel for aestivating prepupae. Metabolic rate decreased rapidly after the onset of the inactive prepupal stage and remained low (5-15% of the level in active larva) during aestivation. A spontaneous increase of the respiration rate occurred before pupation. Neither low mol. wt sugars or alcohols (polyols) accumulated nor the haemolymph osmotic pressure changed during aestivation. Drought tolerance of aestivating prepupae was high (no decrease in survival after exposure to r.h.<10% at a temperature of 23 degrees C for a substantial part of diapause) owing to their extensive capacity to stabilize the relative body water content irrespective of the r.h. of surrounding air. Cold hardiness was low (>90% decrease in survival after exposure to -7 degrees C for 24h). Cold and drought acclimations did not lead to significant changes in the measured physiological and biochemical parameters but cold (not drought) acclimation caused a significant increase in cold hardiness. Neither drought tolerance nor the increase in cold hardiness after cold acclimation appear to be related to presence/accumulation of polyols in aestivating C. pudica prepupae.     

Habitat temperature and the temporal scaling of cold hardening in the high Arctic collembolan, Hypogastrura tullbergi (Schäffer)

Abstract.  1. Cold tolerance is a fundamental adaptation of insects to high latitudes. Flexibility in the cold hardening process, in turn, provides a useful indicator of the extent to which polar insects can respond to spatial and temporal variability in habitat temperature.
2. A scaling approach was adopted to investigate flexibility in the cold tolerance of the high Arctic collembolan, Hypogastrura tullbergi , over different time-scales. The cold hardiness of animals was compared from diurnal warming and cooling phases in the field, and controlled acclimation and cooling treatments in the laboratory. Plasticity in acclimation responses was examined using three parameters: low temperature survival, cold shock survival, and supercooling points (SCPs).
3. Over time-scales of 24–48 h, both field animals from warm diurnal phases and laboratory cultures from a 'warm' acclimation regime (18 °C) consistently showed greater or equivalent cold hardiness to animals from cool diurnal phases and acclimation regimes (3 °C).
4. No significant evidence was found of low temperature acclimation after either hours or days of low temperature exposure. The cold hardiness of H. tullbergi remained 'seasonal' in character and mortality throughout was indicative of the summer state of acclimatization.
5. These data suggest that H. tullbergi employs an 'all or nothing' cryoprotective strategy, cold hardening at seasonal but not diel-temporal scales.
6. It is hypothesised that rapid cold hardening offers little advantage to these high Arctic arthropods because sub-zero habitat temperatures during the summer on West Spitsbergen are rare and behavioural migration into soil profiles offers sufficient buffering against low summer temperatures.     

Characteristics of Cold Acclimation and Deacclimation in Tuber-bearing Solanum Species

The effect of temperatures on cold acclimation and deacclimation in foliage tissues was studied in Solanum commersonii (Oka 4583), a tuber-bearing potato. The threshold temperature for cold acclimation was about 12 C. In a temperature range of 2 to 12 C, the increase in hardiness was dependent on the acclimating temperature; the lower the acclimating temperature, the more hardiness achieved. A day/night temperature of 2 C, regardless of photoperiod, appeared to the optimum acclimating temperature for the Solanum species studied. A subfreezing temperature hardened plants less effectively. The maximum level of hardiness could be reached after 15 days of cold acclimation. However, it took only 1 day to deacclimate the hardened plants to a preacclimation level when plants were subjected to a warm regime from cold. The degree of deacclimation was dependent on the temperature of the warm regime.     

Interaction of Temperature and Light in the Development of Freezing Tolerance in Plants

Freezing tolerance is the result of a wide range of physical and biochemical processes, such as the induction of antifreeze proteins, changes in membrane composition, the accumulation of osmoprotectants, and changes in the redox status, which allow plants to function at low temperatures. Even in frost-tolerant species, a certain period of growth at low but nonfreezing temperatures, known as frost or cold hardening, is required for the development of a high level of frost hardiness. It has long been known that frost hardening at low temperature under low light intensity is much less effective than under normal light conditions; it has also been shown that elevated light intensity at normal temperatures may partly replace the cold-hardening period. Earlier results indicated that cold acclimation reflects a response to a chloroplastic redox signal while the effects of excitation pressure extend beyond photosynthetic acclimation, influencing plant morphology and the expression of certain nuclear genes involved in cold acclimation. Recent results have shown that not only are parameters closely linked to the photosynthetic electron transport processes affected by light during hardening at low temperature, but light may also have an influence on the expression level of several other cold-related genes; several cold-acclimation processes can function efficiently only in the presence of light. The present review provides an overview of mechanisms that may explain how light improves the freezing tolerance of plants during the cold-hardening period.     

Physiology of diapause and cold hardiness in the overwintering pupae of the fall webworm Hyphantria cunea (Lepidoptera: Arctiidae) in Japan

The fall webworm Hyphantria cunea Drury, which was accidentally introduced to Japan in 1945, overwinters on the ground in pupal diapause. Diapause termination, as indicated by the respiration rate and the period required for adult emergence, began in March and ended in April. Cold hardiness (the ability to survive exposure to -15 degrees C) decreased linearly with diapause development from November to the following April under field conditions. Cold hardiness of diapause pupae (DP) decreased as the acclimation temperature decreased from 15 to -10 degrees C, whereas cold hardiness of non-diapause pupae (NDP) remained high as the acclimation temperature decreased from 5 to -5 degrees C. However, H. cunea in Japan can survive exposure to -5 degrees C for two weeks, whether it is in a diapause or non-diapause state. Trehalose was the main sugar detected in the body, but its level was less than 0.8%. Trehalose levels increased in field-collected pupae from January to March. DP accumulated less trehalose than NDP, as the acclimation temperature was decreased from 5 to -5 degrees C. The alanine content in field-collected pupae increased from November to February. Both diapause and low temperature caused an accumulation of alanine. These results suggest that under field conditions, overwintering pupae of H. cunea in Japan do not accumulate high levels of sugars and polyols and do not develop a high level of cold hardiness. Furthermore, DP do not accumulate high levels of sugars and polyols and their ability to survive exposure to -15 degrees C is not greater than that of NDP. The physiological and biochemical bases of diapause in H. cunea from Japan are discussed.     

Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji)

The lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness.     

Alfalfa water status and cold hardiness as influenced by cold acclimation and water stress

Abstract Water stress at a nonacclimating temperature (18–20°C) increased the cold hardiness of Medicagosativa L. (alfalfa) plants. This increased cold hardiness was retained when the previously water-stressed plants were cold acclimated (2–9°C) in the absence of water stress. Water stress during cold acclimation also increased cold hardiness. Alfalfa was demonstrated to suffer injury, measured as decreased growth following freezing, at sub-lethal temperatures. During cold acclimation the turgor potential (ψ) of watered plants increased, whereas the solute potential and the water content per unit dry weight decreased. The large positive psgrdap of acclimated plants indicates that the decreased water content per unit dry weight is related to an increased proportion of tissue dry matter rather than to tissue dehydration.     

Two different strategies for light utilization in photosynthesis in relation to growth and cold acclimation

Seedlings of Lodgepole pine (Pinus contorta L.) and winter wheat (Triticum aestivum L. cv. Monopol) were cold acclimated under controlled conditions to induce frost hardiness. Lodgepole pine responded to cold acclimation by partial inhibition of photosynthesis with an associated partial loss of photosystem II reaction centres, and a reduction in needle chlorophyll content. This was accompanied by a low daily carbon gain, and the development of a high and sustained capacity for non‐photochemical quenching of absorbed light. This sustained dissipation of absorbed light as heat correlated with an increased de‐epoxidation of the xanthophyll cycle pigments forming the quenching forms antheraxanthin and zeaxanthin. In addition, the PsbS protein known to bind chlorophyll and the xanthophyll cycle pigments increased strongly during cold acclimation of pine. In contrast, winter wheat maintained high photosynthetic rates, showed no loss of chlorophyll content per leaf area, and exhibited a high daily carbon gain and a minimal non‐photochemical quenching after cold acclimation. In accordance, cold acclimation of wheat neither increased the de‐epoxidation of the xanthophylls nor the content of the PsbS protein. These different responses of photosynthesis to cold acclimation are correlated with pine, reducing its need for assimilates when entering dormancy associated with termination of primary growth, whereas winter wheat maintains a high need for assimilates as it continues to grow and develop throughout the cold‐acclimation period. It appears that without evolving a sustained ability for controlled dissipation of absorbed light as heat throughout the winter, winter green conifers would not have managed to adapt and establish themselves so successfully in the cold climatic zones of the northern hemisphere.     

Regulation of a wheat actin-depolymerizing factor during cold acclimation

We have previously shown that the wheat (Triticum aestivum) TaADF gene expression level is correlated with the plants capacity to tolerate freezing. Sequence analysis revealed that this gene encodes a protein homologous to members of the actin-depolymerizing factor (ADF)/cofilin family. We report here on the characterization of the recombinant TaADF protein. Assays for ADF activity showed that TaADF is capable of sequestering actin, preventing nucleotide exchange, and inducing actin depolymerization. In vitro phosphorylation studies showed that TaADF is a substrate for a wheat 52-kD kinase. The activity of this kinase is modulated by low temperature during the acclimation period. Western-blot analyses revealed that TaADF is expressed only in cold-acclimated Gramineae species and that the accumulation level is much higher in the freezing-tolerant wheat cultivars compared with the less tolerant ones. This accumulation was found to be regulated by a factor(s) encoded by a gene(s) located on chromosome 5A, the chromosome most often found to be associated with cold hardiness. The induction of an active ADF during cold acclimation and the correlation with an increased freezing tolerance suggest that the protein may be required for the cytoskeletal rearrangements that may occur upon low temperature exposure. These remodelings might be important for the enhancement of freezing tolerance.