Efficiency of cold hardiness induction by desiccation stress in four winter cereals

A number of defined desiccation treatments without low temperature exposure were able to induce freezing tolerance in 20 cultivars of winter cereals. A maximal degree of freezing tolerance was induced in epicotyls at 24°C in 24 hours at 40% relative humidity in rye and wheat, 7 days at 54% RH in barley, and 4 days at 70% RH in oats. Freezing tolerance was not correlated to water content of the plants after desiccation treatment but was related to the genetic capacity of the cultivars to frost harden. Levels of freezing tolerance induced by desiccation were similar to those induced by cold acclimation in rye and wheat, but considerably less in barley and oats. This is associated with a more rapid desiccation injury in barley and oats, precluding the completion of the hardening process.     

Twenty-four-hour induction of freezing and drought tolerance in plumules of winter rye seedlings by desiccation stress at room temperature in the dark

Exposure of seedlings of winter rye (Secale cereale L., cv. Puma) for 2 weeks or 24 hours to desiccation stress (40% relative humidity) at room temperature (21°C) in the dark induced degrees of freezing and drought tolerance in the plumules comparable to those produced by cold conditioning for 2 weeks at 3°C. The induction was associated with repression of growth and could not be produced in plumules excised from the seedlings indicating a requirement for translocation of nutrients from the endosperm. Rapid increase in osmotic pressure, soluble proteins, and phospholipids in plumules in association with the development of freezing and drought tolerance and the requirement of endosperm suggested diversion of nutrient from use in extension growth, to use in augmentation of protoplasm in plumule cells. Since cold acclimation slowed or arrested growth and is associated with augmentation of protoplasm, it is suggested that the common element in the induction of freezing tolerance by cold and drought is the necessity for producing a condition of augmented protoplasm and membranes in cells thus reinforcing a similar conclusion reached from seasonal studies on woody plants.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

Responses of antioxidant enzymes to cold and high light are not correlated to freezing tolerance in natural accessions of Arabidopsis thaliana

Low temperatures and high light cause imbalances in primary and secondary reactions of photosynthesis, and thus can result in oxidative stress. Plants employ a range of low‐molecular weight antioxidants and antioxidant enzymes to prevent oxidative damage, and antioxidant defence is considered an important component of stress tolerance. To figure out whether oxidative stress and antioxidant defence are key factors defining the different cold acclimation capacities of natural accessions of the model plant Arabidopsis thaliana, we investigated hydrogen peroxide (H₂O₂) production, antioxidant enzyme activity and lipid peroxidation during a time course of cold treatment and exposure to high light in four differentially cold‐tolerant natural accessions of Arabidopsis (C24, Nd, Rsch, Te) that span the European distribution range of the species. All accessions except Rsch (from Russia) had elevated H₂O₂ in the cold, indicating that production of reactive oxygen species is part of the cold response in Arabidopsis. Glutathione reductase activity increased in all but Rsch, while ascorbate peroxidase and superoxide dismutase were unchanged and catalase decreased in all but Rsch. Under high light, the Scandinavian accession Te had elevated levels of H₂O₂. Te appeared most sensitive to oxidative stress, having higher malondialdehyde (MDA) levels in the cold and under high light, while only high light caused elevated MDA in the other accessions. Although the most freezing‐tolerant, Te had the highest sensitivity to oxidative stress. No correlation was found between freezing tolerance and activity of antioxidant enzymes in the four accessions investigated, arguing against a key role for antioxidant defence in the differential cold acclimation capacities of Arabidopsis accessions.     

Comparison of thylakoid structure and organization in sun and shade Haberlea rhodopensis populations under desiccation and rehydration

The resurrection plant, Haberlea rhodopensis can survive nearly total desiccation only in its usual low irradiation environment. However, populations with similar capacity to recover were discovered recently in several sunny habitats. To reveal what kind of morphological, structural and thylakoid-level alterations play a role in the acclimation of this low-light adapted species to high-light environment and how do they contribute to the desiccation tolerance mechanisms, the structure of the photosynthetic apparatus, the most sensitive component of the chlorophyll-retaining resurrection plants, was analyzed by transmission electron microscopy, steady state low-temperature fluorescence and two-dimensional Blue-Native/SDS PAGE under desiccation and rehydration.     

The Effect of Abscisic Acid on the Freezing Tolerance of Callus Cultures of Lotus corniculatus L

The effects of growth temperature (2°C and 24°C), abscisic acid (ABA) concentration, duration of exposure to ABA, and light were assessed for their ability to induce acclimation to freezing temperatures in callus cultures of Lotus corniculatus L. cv Leo, a perennial forage legume. The maximal expression of freezing tolerance was achieved on B₅ media containing 10⁻⁵ molar ABA, at 24°C for 7 or 14 days. Under these culture conditions, the freezing tolerance of the callus approximated that observed in field grown plants. In contrast, low temperatures (2°C) induced only a limited degree of freezing tolerance in these cultures. Viability was assessed by tetrazolium reduction and by regrowth of the callus. The two assays often differed in their estimates of absolute freezing tolerance. Regression analysis of the temperature profile suggested that there may be two or more distinct populations of cells differing in freezing tolerance, which may have contributed to the variability between viability assays.     

Metabolic profiling of the resurrection plant Haberlea rhodopensis during desiccation and recovery

Desiccation tolerance is among the most important parameters for crop improvement under changing environments. Resurrection plants are useful models for both theoretical and practical studies. We performed metabolite profiling via gas chromatography coupled with mass spectrometry (GC‐MS) and high‐performance liquid chromatography (HPLC) and analyzed the antioxidant capacity of the endemic resurrection plant Haberlea rhodopensis at desiccation and recovery. More than 100 compounds were evaluated. Stress response included changes in both primary and secondary metabolic pathways. The high amounts of the specific glycoside myconoside and some phenolic acids – e.g. syringic and dihydrocaffeic acid under normal conditions tend to show their importance for the priming of H. rhodopensis to withstand severe desiccation and oxidative stress. The accumulation of sucrose (resulting from starch breakdown), total phenols, β‐aminoisobutyric acid, β‐sitosterol and α‐tocopherol increased up to several times at later stages of desiccation. Extracts of H. rhodopensis showed high antioxidant capacity at stress and normal conditions. Myconoside was with the highest antioxidant properties among tested phenolic compounds. Probably, the evolution of resurrection plants under various local environments has resulted in unique desiccation tolerance with specific metabolic background. In our case, it includes the accumulation of a relatively rare compound (myconoside) that contributes alone and together with other common metabolites. Further systems biology studies on the involvement of carbohydrates, phenolic acids and glycosides in the desiccation tolerance and antioxidant capacity of H. rhodopensis will definitely help in achieving the final goal – improving crop drought tolerance.     

Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins

In many woody plants photoperiod signals the initiation of dormancy and cold acclimation. The photoperiod-specific physiological and molecular mechanisms have remained uncharacterised. The role of abscisic acid (ABA) and dehydrins in photope-riod-induced dormancy and freezing tolerance was investigated in birch, Betula pubescens Ehrh. The experiments were designed to investigate if development of dormancy and freezing tolerance under long-day (LD) and short-day (SD) conditions could be affected by manipulation of the endogenous ABA content, and if accumulation of dehydrin-like proteins was correlated with SD and/or the water content of the buds. Experimentally, the internal ABA content was increased by ABA application and by water stress treatment under LD, and decreased by blocking the synthesis of ABA with fluridone under SD. Additionally, high humidity (95% RH) was applied to establish if accidental water stress was involved in SD. ABA content was monitored by gas chromatography-mass spectrometry with selective ion monitoring (SIM). Short days induced a transient increase in ABA content, which was absent in 95% RH, whereas fluridone treatment decreased ABA. Short days induced a typical pattern of bud desiccation and growth cessation regardless of the treatment, and improved freezing tolerance except in the fluridone treatment. ABA content of the buds was significantly increased after spraying ABA on leaves and after water stress, treatments that did not induce cessation of growth and dormancy, but improved freezing tolerance. In addition to several constitutively produced dehydrins, two SD-specific proteins of molecular masses 34 and 36 kDa were found. Photoperiod- and experimentally-induced alterations in ABA contents affected freezing tolerance but not cessation of growth and dormancy. Therefore, involvement of ABA in the photoperiodic control of cold acclimation is more direct than in growth cessation and dormancy. As the typical desiccation pattern of the buds was found in all SD plants, and was not directly related to ABA content or to freezing tolerance, this pattern characterises the onset of photo-period-induced growth cessation and dormancy. The results provide evidence for the existence of various constitutively and two photoperiod-induced dehydrins in buds of birch, and reveal characteristics of dormancy and freezing tolerance that may facilitate further investigations of photoperiodic control of growth in trees.     

Accumulation of theanderose in association with development of freezing tolerance in the moss Physcomitrella patens

Mosses are known to have the ability to develop high degrees of resistance to desiccation and freezing stress at cellular levels. However, underlying cellular mechanisms leading to the development of stress resistance in mosses are not understood. We previously showed that freezing tolerance in protonema cells of the moss Physcomitrella patens was rapidly increased by exogenous application of the stress hormone abscisic acid (ABA) [Minami, A., Nagao, M., Arakawa, K., Fujikawa, S., Takezawa, D., 2003a. Abscisic acid-induced freezing tolerance in the moss Physcomitrella patens is accompanied by increased expression of stress-related genes. J. Plant Physiol. 160, 475-483]. Herein it is shown that protonema cells with acquired freezing tolerance specifically accumulate low-molecular-weight soluble sugars. Analysis of the most abundant trisaccharide revealed that the cells accumulated theanderose (G6-alpha-glucosyl sucrose) in close association with enhancement of freezing tolerance by ABA treatment. The accumulation of theanderose was inhibited by cycloheximide, an inhibitor of nuclear-encoded protein synthesis, coinciding with a remarkable decrease in freezing tolerance. Furthermore, theanderose accumulation was promoted by cold acclimation and treatment with hyperosmotic solutes, both of which had been shown to enhance cellular freezing tolerance. These results reveal a novel role for theanderose, whose biological function has been obscure, in high freezing tolerance in moss cells.     

Exogenous 4-hydroxybenzoic acid and salicylic acid modulate the effect of short-term drought and freezing stress on wheat plants

Exogenous salicylic acid has been shown to confer tolerance against biotic and abiotic stresses. In the present work the ability of its analogue, 4-hydroxybenzoic acid to increase abiotic stress tolerance was demonstrated: it improved the drought tolerance of the winter wheat (Triticum aestivum L.) cv. Cheyenne and the freezing tolerance of the spring wheat cv. Chinese Spring. Salicylic acid, however, reduced the freezing tolerance of Cheyenne and the drought tolerance of Chinese Spring, in spite of an increase in the guaiacol peroxidase and ascorbate peroxidase activity. The induction of cross tolerance between drought and freezing stress was observed: drought acclimation increased the freezing tolerance of Cheyenne plants and cold acclimation enhanced the drought tolerance. The induction of drought tolerance in Cheyenne was correlated with an increase in catalase activity.     

Temporal cell wall changes during cold acclimation and deacclimation and their potential involvement in freezing tolerance and growth

Plants adapt to freezing stress through cold acclimation, which is induced by nonfreezing low temperatures and accompanied by growth arrest. A later increase in temperature after cold acclimation leads to rapid loss of freezing tolerance and growth resumption, a process called deacclimation. Appropriate regulation of the trade-off between freezing tolerance and growth is necessary for efficient plant development in a changing environment. The cell wall, which mainly consists of polysaccharide polymers, is involved in both freezing tolerance and growth. Still, it is unclear how the balance between freezing tolerance and growth is affected during cold acclimation and deacclimation by the changes in cell wall structure and what role is played by its monosaccharide composition. Therefore, to elucidate the regulatory mechanisms controlling freezing tolerance and growth during cold acclimation and deacclimation, we investigated cell wall changes in detail by sequential fractionation and monosaccharide composition analysis in the model plant Arabidopsis thaliana, for which a plethora of information and mutant lines are available. We found that arabinogalactan proteins and pectic galactan changed in close coordination with changes in freezing tolerance and growth during cold acclimation and deacclimation. On the other hand, arabinan and xyloglucan did not return to nonacclimation levels after deacclimation but stabilized at cold acclimation levels. This indicates that deacclimation does not completely restore cell wall composition to the nonacclimated state but rather changes it to a specific novel composition that is probably a consequence of the loss of freezing tolerance and provides conditions for growth resumption.     

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35 degrees C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 microg trehalose/mg dry weight, respectively) during acclimation at 5 and 35 degrees C, respectively. Warm and cold acclimation enhanced heat (40 degrees C for 8h) and freezing (-20 degrees C for 4h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.